Background:Electronic health record (EHR)-based phenotyping underpins genome-wide association studies, yet current ICD-code phenotypes rely heavily on manually curated lists such as Phecodes. These definitions are labour-intensive to maintain, inherently subjective, and may omit clinically relevant diagnostic codes, reducing study power. Advances in text embedding models offer an opportunity to automate and standardize ICD-based phenotype construction. Methods:We developed Phecoder, an ensemble of pre-trained text embedding models that rank ICD codes by similarity from free-text phenotype descriptions. Nine embedding models and multiple unsupervised ensemble rank-fusion methods were evaluated against 1,125 PhecodeX phenotypes. Retrieval performance was assessed using recall and average precision at top-100 (R@100, AP@100). Expert clinical review of six neuropsychiatric phenotypes was undertaken to identify relevant ICD codes absent from PhecodeX. Cohort sizes under these new definitions were compared with PhecodeX across sex and ancestry strata in the Million Veteran Program (MVP). Findings:Among individual models, Qwen3-Embedding-4B achieved the highest median recall (R@100 = 0.86). Ensemble rank-fusion further improved R@100 by 3%, and median AP@100 by 8%. Expert review confirmed that Phecoder retrieved additional clinically relevant ICD codes beyond PhecodeX across all six neuropsychiatric case studies. Median potential case expansion increased by 200%, with 700% increases for bipolar disorder and 2000% increase for eating disorders. Interpretation:Manually defining ICD phenotypes has been critiqued as subjective, potentially yielding overly restrictive definitions that miss relevant codes. To address this issue, Phecoder algorithmically identifies relevant codes for ICD-based phenotyping. Phecoder extracts relevant ICD codes to expand the potential case pool across different demographic groups. Phecoder is easily applicable to future ICD-code releases and across different ICD coding versions that are used in different countries. Taken together, Phecoder has the potential to improve reproducibility in EHR data research. Funding:This research was supported by the Department of Veterans Affairs MVP (MVP-000, MVP-076 and MVP-096). The MVP is supported by the Office of Research and Development, Department of Veterans Affairs. The authors thank the MVP staff, researchers, and volunteers, who have contributed to MVP, and especially who previously served their country in the military and now generously agreed to enroll in the study (see mvp.va.gov for more information). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. This study was supported by the Veterans Affairs Merit grants: BX006500 (to D.B.) and BX004189 (to P.R.). This work was supported by the National Institutes of Health (NIH): R01MH125246 (to P.R.), R01AG078657 (to G.V.), R01AG067025 (to P.R.), and U24AG087563 (to P.R.).

Importance:Researchers commonly use counts of diagnostic codes from EHR-linked biobanks to infer phenotypic status. However, these approaches overlook temporal changes in EHR data, such as the discontinuation or "dropout" of diagnostic codes, which may exacerbate disparities in genomics research, as EHR data quality can be confounded with demographic attributes. Objective:To address this, we propose modeling diagnostic code dropout in EHR data to inform phenotyping for schizophrenia in genomic analyses. Design:We develop and test our diagnostic dropout model by analyzing EHR data from individuals with prior schizophrenia diagnoses. We further validate model performance on a subset of patients whose diagnoses were attained through chart review. Using PRS-CS and existing GWAS summary statistics, we first extrapolate polygenic weights. Then, we apply our dropout model's outputs to construct a data-driven filter defining our target cohort for measuring polygenic score performance. Setting:Our analysis utilizes EHR and genomic data from the Million Veteran Program. Participants:To model diagnostic dropout in schizophrenia, we leverage data from 12,739 patients with a history of schizophrenia, after excluding outliers. For polygenic score analyses, we incorporate data from a potential pool of 8,385 European ancestry and 6,806 African ancestry patients with a history of schizophrenia. Main outcomes and measures:We compare the performance of our diagnostic dropout model with alternative methodologies both in predicting diagnostic dropout on a holdout set, as well as on chart review labeled data. Using the top differential diagnosis predictors in our model, we select relevant cases by filtering out patients with a prior history of mood or anxiety disorders. We then test the impact of applying different filters for measuring polygenic score performance. Results:When evaluated on chart review-labeled data, our model improves the area under the precision-recall curve (AUPRC) by 9.6% compared to competing methods. By applying our data-driven filter for schizophrenia, we achieve a 62% increase in the association effect size when transferring a European polygenic score to an African ancestry target cohort. Conclusions and Relevance:These findings highlight the potential of modeling diagnostic code dropout to enhance the phenotypic quality of EHR-linked biobank data, advancing more equitable and accurate genomics research across diverse populations.

Polygenic scores (PGS) enable the exploration of pleiotropic effects and genomic dissection of complex traits. Here, in 421,889 individuals with European ancestry from the Million Veteran Program and UK Biobank, we examine how PGS of 17 neuropsychiatric traits are related to membership in 22 broad professional categories. Overall, we find statistically significant but weak (the highest odds ratio is 1.1 per PGS standard deviation) associations between most professional categories and genetic predisposition for at least one neuropsychiatric trait. Secondary analyses in UK Biobank revealed independence of these associations from observed fluid intelligence and sex-specific effects. By leveraging aggregate population trends, we identified patterns in the public interest, such as the mediating effect of education attainment on the association of attention-deficit/hyperactivity disorder PGS with multiple professional categories. However, at the individual level, PGS explained less than 0.5% of the variance of professional membership, and almost none after we adjusted for education and socio-economic status.

EHR-linked biobanks provide longitudinal data on diverse clinical populations that can fuel clinical, genetic, and epidemiologic research. Assigning case status is an important step in conducting psychiatric research using EHR, however it is often defined using phenotypic algorithms which have been developed from majority white individuals. Bias in assigning diagnosis codes across groups could lead to poorer performance and exacerbate disparities.

Fibroblast growth factor receptor 2 (FGFR2) is almost exclusively expressed in glial cells in postnatal mouse brain, but its impact in glia for brain behavioral functioning is poorly understood. We compared behavioral effects from FGFR2 loss in both neurons and astroglial cells and from FGFR2 loss in astroglial cells by using either the pluripotent progenitor-driven hGFAP-cre or the tamoxifen-inducible astrocyte-driven GFAP-creER T2 in Fgfr2 floxed mice. When FGFR2 was eliminated in embryonic pluripotent precursors or in early postnatal astroglia, mice were hyperactive, and had small changes in working memory, sociability, and anxiety-like behavior. In contrast, FGFR2 loss in astrocytes starting at 8 weeks of age resulted only in reduced anxiety-like behavior. Therefore, early postnatal loss of FGFR2 in astroglia is critical for broad behavioral dysregulation. Neurobiological assessments demonstrated that astrocyte-neuron membrane contact was reduced and glial glutamine synthetase expression increased only by early postnatal FGFR2 loss. We conclude that altered astroglial cell function dependent on FGFR2 in the early postnatal period may result in impaired synaptic development and behavioral regulation, modeling childhood behavioral deficits like attention deficit hyperactivity disorder (ADHD).

Background: Serious mental illnesses, including schizophrenia, bipolar disorder and depression are heritable, highly multifactorial disorders and major causes of disability worldwide. Polygenic risk scores (PRS) aggregate variants identified from genome-wide association studies (GWAS) into individual-level estimates of liability, and are a promising tool for clinical risk stratification. Methods: By leveraging the VA’s extensive electronic health record (EHR) and a cohort of 9 378 individuals with confirmed diagnoses of schizophrenia or bipolar I disorder, we validated automated case-control assignments based on ICD-9/10 codes, and benchmarked the performance of current PRS for schizophrenia, bipolar disorder, and major depression in 400 000 Million Veteran Program (MVP) participants. We explored broader relationships between PRS and 1 650 disease categories via phenome-wide association studies (PheWAS). Finally, we applied genomic structural equation modeling (gSEM) to derive novel PRS indexing common and disorder-specific latent genetic factors. Findings: Among 3 953 and 5 425 individuals with diagnoses of schizophrenia or bipolar disorder type I that were confirmed by structured clinical interviews, 95% were correctly identified using ICD-9/10 codes (2 or more). Current PRS were robustly associated with case status in European (p <10-254) and African (p<10-5) participants and were higher among more frequently hospitalized patients (p<10-4). PheWAS confirmed previous associations among higher neuropsychiatric PRS and elevated risk for psychiatric and physical health problems and extended these findings to African Americans. Interpretation: Using diagnoses confirmed by in-person structured clinical interviews and current neuropsychiatric PRS, we demonstrated the validity of an EHR-based phenotyping approach in US veterans, highlighting the potential of PRS for disentangling biological and mediated pleiotropy. Funding Information: Department of Veterans Affairs Cooperative Studies Program (CSP) #572; Million Veteran Program (MVP-000, MVP-006); Office of Research and Development, Department of Veterans Affairs. Declaration of Interests: Dr. Bigdeli is the recipient of a 2019 NARSAD Young Investigator Grant (#28276). Dr. Voloudakis is supported by the National Institutes of Health (NIH) under award number K08MH122911 and is the recipient of a 2020 NARSAD Young Investigator Grant (#29350). We are grateful to Drs. R. Karlsson Linnér and T.T. Mallard for sharing their script for plotting PheWAS results, which served as the basis of the figure presented herein.Dr. Harvey has served as a consultant to multiple pharmaceutical companies and device manufacturers on phase 2 or 3 treatment development; this consulting work has been determined to be unrelated to the content of the paper. No other authors report any relevant conflicts of interest. Ethics Approval Statement: This study was approved by the VA Central Institutional Review Board (IRB), and all patients provided written informed consent.

Mosaic mutations can be used to track cell lineages in humans. We used cell cloning to analyze embryonic cell lineages in two living individuals and a postmortem human specimen. Of 10 reconstructed postzygotic divisions, none resulted in balanced contributions of daughter lineages to tissues. In both living individuals, one of two lineages from the first cleavage was dominant across tissues, with 90% frequency in blood. We propose that the efficiency of DNA repair contributes to lineage imbalance. Allocation of lineages in postmortem brain correlated with anterior-posterior axis, associating lineage history with cell fate choices in embryos. We establish a minimally invasive framework for defining cell lineages in any living individual, which paves the way for studying their relevance in health and disease.

BACKGROUND:Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells.RESULTS:Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees.CONCLUSIONS:This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

The GABAergic system is regulated by the brain-derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) pathway, but the cell-intrinsic role of TrkB signaling in parvalbumin cortical interneuron development and function is unclear. We performed conditional ablation of the TrkB receptor in parvalbumin-expressing (PV) interneurons to study whether postnatal loss of TrkB in parvalbumin cells affects their survival, connectivity, spontaneous and evoked neuronal activity and behavior. Using in vivo recordings of local field potentials, we found reduced gamma oscillations in the sensory cortex of PVcre+; TrkBF/F conditional knockout mice (TrkB cKO), along with increased firing of putative excitatory neurons. There was a significant downregulation in parvalbumin neuron number in cerebral and cerebellar cortices of TrkB cKO mice. In addition, inhibitory synaptic connections between basket cells and pyramidal neurons were profoundly reduced in the neocortex of TrkB cKO mice and there was a loss of cortical volume. TrkB cKO mice also showed profound hyperactivity, stereotypies, motor deficits and learning/memory defects. Our findings demonstrate that the targeting and/or synapse formation of PV-expressing basket cells with principal excitatory neurons require TrkB signaling in parvalbumin cells. Disruption of this signaling has major consequences for parvalbumin interneuron connectivity, network dynamics, cognitive and motor behavior.

BACKGROUND: Despite strong evidence linking fibroblast growth factor 2 (FGF2) with anxiety and depression in both rodents and humans, the molecular mechanisms linking FGF2 with anxiety are not understood.METHODS: We compare 1) mice that lack a functional Fgf2 gene (Fgf2 knockout [KO]), 2) wild-type mice, and 3) Fgf2 KO with adult rescue by FGF2 administration on measures of anxiety, depression, and motor behavior, and further investigate the mechanisms of this behavior by cellular, molecular, and neuroendocrine studies.RESULTS: We demonstrate that Fgf2 KO mice have increased anxiety, decreased hippocampal glucocorticoid receptor (GR) expression, and increased hypothalamic-pituitary-adrenal axis activity. FGF2 administration in adulthood was sufficient to rescue the entire phenotype. Blockade of GR in adult mice treated with FGF2 precluded the therapeutic effects of FGF2 on anxiety behavior, suggesting that GR is necessary for FGF2 to regulate anxiety behavior. The level of Egr-1/NGFI-A was decreased in Fgf2 KO mice and was reestablished with FGF2 treatment. By chromatin immunoprecipitation studies, we found decreased binding of EGR-1 to the GR promoter region in Fgf2 KO mice. Finally, we examined anxiety behavior in FGF receptor (FGFR) KO mice; however, FGFR1, FGFR2, and FGFR3 KO mice did not mimic the phenotype of Fgf2 KO mice, suggesting a role for other receptor subtypes (i.e., FGFR5).CONCLUSIONS: These data suggest that FGF2 levels are critically related to anxiety behavior and hypothalamicpituitary-adrenal axis activity, likely through modulation of hippocampal glucocorticoid receptor expression, an effect that is likely receptor mediated, albeit not by FGFR1, FGFR2, and FGFR3.

One major aim in quantitative and translational neuroscience is to achieve a precise and fast neuronal counting method to work on high throughput scale to obtain reliable results. Here, we tested the isotropic fractionator (IF) method for evaluating neuronal and non-neuronal cell loss in different models of central nervous system (CNS) pathologies. Sprague-Dawley rats underwent: (i) ischemic brain damage; (ii) intraperitoneal injection with kainic acid (KA) to induce epileptic seizures; and (iii) monolateral striatal injection with quinolinic acid (QA) mimicking human Huntington's disease. All specimens were processed for IF method and cell loss assessed. Hippocampus from KA-treated rats and striatum from QA-treated rats were carefully dissected using a dissection microscope and a rat brain matrix. Ischemic rat brains slices were first processed for TTC staining and then for IF. In the ischemic group the cell loss corresponded to the neuronal loss suggesting that hypoxia primarily affects neurons. Combining IF with TTC staining we could correlate the volume of lesion to the neuronal loss; by IF, we could assess that neuronal loss also occurs contralaterally to the ischemic side. In the epileptic group we observed a reduction of neuronal cells in treated rats, but also evaluated the changes in the number of non-neuronal cells in response to the hippocampal damage. In the QA model, there was a robust reduction of neuronal cells on ipsilateral striatum. This neuronal cell loss was not related to a drastic change in the total number of cells, being overcome by the increase in non-neuronal cells, thus suggesting that excitotoxic damage in the striatum strongly activates inflammation and glial proliferation. We concluded that the IF method could represent a simple and reliable quantitative technique to evaluate the effects of experimental lesions mimicking human diseases, and to consider the neuroprotective/anti-inflammatory effects of different treatments in the whole brain and also in discrete regions of interest, with the potential to investigate non-neuronal alterations. Moreover, IF could be used in addition or in substitution to classical stereological techniques or TTC staining used so far, since it is fast, precise and easily combined with complex molecular analysis.

SpringerPlus 2015, 4(Suppl 1):L1 MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3'-untranslated region (3'-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets.The long 3'-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs.Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer's disease (AD).Focusing on evolutionarily conserved and primate-specifi c miRNA controllers of cholinergic signaling ('CholinomiRs'), we fi nd modifi ed CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson's disease, with treatment-related diff erences in their levels and prominent impact on the cognitive and anti-infl ammatory consequences of cholinergic signals.Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain.Furthermore, we found that interruption of AChE mRNA's interaction with the primatespecifi c CholinomiR-608 in carriers of a SNP in the AChE's miR-608 binding site induces domino-like eff ects that reduce the levels of many other miR-608 targets.Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially aff ecting the responsiveness to AD's anti-AChE therapeutics, and show elevated trait anxiety, infl ammation and hypertension.Non-coding regions aff ecting miRNA-target interactions in neurodegenerative brains thus merit special attention.

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45 β (GADD45 β -I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK’s pathological activation. GADD45 β -I was engineered by optimizing the domain of the GADD45 β , able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45 β -I significantly reduces neuronal death in excitotoxicity induced by either N -methyl- D -aspartate exposure or by oxygen–glucose deprivation in vitro . Moreover, GADD45 β -I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45 β -I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45 β -I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.

SpringerPlus 2015, 4(Suppl 1):L1 MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3'-untranslated region (3'-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets.The long 3'-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs.Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer's disease (AD).Focusing on evolutionarily conserved and primate-specifi c miRNA controllers of cholinergic signaling ('CholinomiRs'), we fi nd modifi ed CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson's disease, with treatment-related diff erences in their levels and prominent impact on the cognitive and anti-infl ammatory consequences of cholinergic signals.Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain.Furthermore, we found that interruption of AChE mRNA's interaction with the primatespecifi c CholinomiR-608 in carriers of a SNP in the AChE's miR-608 binding site induces domino-like eff ects that reduce the levels of many other miR-608 targets.Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially aff ecting the responsiveness to AD's anti-AChE therapeutics, and show elevated trait anxiety, infl ammation and hypertension.Non-coding regions aff ecting miRNA-target interactions in neurodegenerative brains thus merit special attention.

Steroid hormones are important players to regulate adult neurogenesis in the dentate gyrus of the hippocampus, but their involvement in the regulation of the same phenomenon in the subventricular zone (SVZ) of the lateral ventricles is not completely understood. Here, in male rats, we tested the existence of activational effects of testosterone (T) on cell proliferation in the adult SVZ. To this aim, three groups of male rats: castrated, castrated and treated with T, and controls were treated with 5-bromo-20-deoxyuridine (BrdU) and killed after 24 h. The density of BrdU-labeled cells was significantly lower in castrated animals in comparison to the other two groups, thus supporting a direct correlation between SVZ proliferation and levels of circulating T. To clarify whether this effect is purely androgen-dependent, or mediated by the T metabolites, estradiol (E-2) and dihydrotestosterone (DHT), we evaluated SVZ proliferation in castrated males treated with E-2, DHT and E-2 + DHT, in comparison to T-and vehicle-treated animals, and sham-operated controls. The stereological analysis demonstrated that E-2 and T, but not DHT, increase proliferation in the SVZ of adult male rats. Quantitative evaluation of cells expressing the endogenous marker of cell proliferation phosphorylated form of Histone H3 (PHH3), or the marker of highly dividing SVZ progenitors Mash1, indicated the effect of T/E-2 is mostly restricted to SVZ proliferating progenitors. The same experimental protocol was repeated on ovariectomized female rats treated with E-2 or T. In this case, no statistically significant difference was found among groups. Overall, our results clearly show that the gonadal hormones T and E-2 represent important mediators of cell proliferation in the adult SVZ. Moreover, we show that such an effect is restricted to males, supporting adult neurogenesis in rats is a process differentially modulated in the two sexes. (C) 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

The incidence of preterm birth is on the rise. The outcome of premature birth can vary widely, spanning completely normal development to severe neurologic deficits, with most children showing mild to moderate cognitive delay and increased incidence of neuropsychiatric conditions such as anxiety, attention deficit hyperactivity, and autism spectrum disorders. Several animal models have been employed to study the consequences of prematurity, one of the most promising being chronic perinatal hypoxia in mouse, which recapitulates the cognitive impairments, partial recovery over time and enhanced recovery with environmental enrichment.

Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5—before neurogenesis and before the formation of intrahemispheric axonal connections—altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus. At the same time, we observed ectopic Er81 expression, increased proliferation of Tbr2-expressing (Tbr2+) intermediate neuronal progenitors (INPs), and elevated Tbr1+ neurogenesis in the regions that undergo gyrification, indicating region-specific actions of FGF2 on the VZ and subventricular zone (SVZ). However, the relative number of basal radial glia—recently proposed to be important in gyrification—appeared to be unchanged. These findings are consistent with the hypothesis that increased radial unit production together with rapid SVZ growth and heightened localized neurogenesis can cause cortical gyrification in lissencephalic species. These data also suggest that the position of cortical gyri can be molecularly specified in mice. In contrast, a different ligand, FGF8b, elicited surface area expansion throughout the cortical primordium but no gyrification. Our findings demonstrate that individual members of the diverse Fgf gene family differentially regulate global as well as regional cortical growth rates while maintaining cortical layer structure.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation. MSCs can express markers of the myogenic cell lineages and give rise, under specific cell culture conditions, to myotube-like structures. Nevertheless, we failed to obtain mixed myotubes both in vitro and in vivo. For in vivo transplantation, we tested a new protocol to collect human MSCs from whole bone marrow, to get larger numbers of cells. MSCs, when transplanted into the pelvic muscles close to the external urethral sphincter, survived for a long time in absence of immunosuppression, and migrated into the muscle among fibers, and towards neuromuscular endplates. Moreover, they showed low levels of cycling cells, and did not infiltrate blood vessels. We never observed formation of cell masses suggestive of tumorigenesis. Those which remained close to the injection site showed an immature phenotype, whereas those in the muscle had more elongated morphologies. Therefore, MSCs are safe and can be easily transplanted without risk of side effects in the pelvic muscles. Further studies are needed to elucidate their integration into muscle fibers, and to promote their muscular transdifferentiation either before or after transplantation.