NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients.

The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra-Bosch-Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss-of-function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria-like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long-term self-renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area-specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.

A two-hit story: Seizures and genetic mutation interaction sets phenotype severity in SCN1A epilepsies.

SCN1A (NaV1.1 sodium channel) mutations cause Dravet syndrome (DS) and GEFS+ (which is in general milder), and are risk factors in other epilepsies. Phenotypic variability limits precision medicine in epilepsy, and it is important to identify factors that set phenotype severity and their mechanisms. It is not yet clear whether SCN1A mutations are necessary for the development of severe phenotypes or just for promoting seizures. A relevant example is the pleiotropic R1648H mutation that can cause either mild GEFS+ or severe DS. We used a R1648H knock-in mouse model (Scn1aRH/+) with mild/asymptomatic phenotype to dissociate the effects of seizures and of the mutation per se. The induction of short repeated seizures, at the age of disease onset for Scn1a mouse models (P21), had no effect in WT mice, but transformed the mild/asymptomatic phenotype of Scn1aRH/+ mice into a severe DS-like phenotype, including frequent spontaneous seizures and cognitive/behavioral deficits. In these mice, we found no major modifications in cytoarchitecture or neuronal death, but increased excitability of hippocampal granule cells, consistent with a pathological remodeling. Therefore, we demonstrate for our model that an SCN1A mutation is a prerequisite for a long term deleterious effect of seizures on the brain, indicating a clear interaction between seizures and the mutation for the development of a severe phenotype generated by pathological remodeling. Applied to humans, this result suggests that genetic alterations, even if mild per se, may increase the risk of second hits to develop severe phenotypes.

Eps8 controls dendritic spine density and synaptic plasticity through its actin-capping activity.

Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects.

Action potential initiation in neocortical inhibitory interneurons.

Action potential (AP) generation in inhibitory interneurons is critical for cortical excitation-inhibition balance and information processing. However, it remains unclear what determines AP initiation in different interneurons. We focused on two predominant interneuron types in neocortex: parvalbumin (PV)- and somatostatin (SST)-expressing neurons. Patch-clamp recording from mouse prefrontal cortical slices showed that axonal but not somatic Na+ channels exhibit different voltage-dependent properties. The minimal activation voltage of axonal channels in SST was substantially higher (∼7 mV) than in PV cells, consistent with differences in AP thresholds. A more mixed distribution of high- and low-threshold channel subtypes at the axon initial segment (AIS) of SST cells may lead to these differences. Surprisingly, NaV1.2 was found accumulated at AIS of SST but not PV cells; reducing NaV1.2-mediated currents in interneurons promoted recurrent network activity. Together, our results reveal the molecular identity of axonal Na+ channels in interneurons and their contribution to AP generation and regulation of network activity.

Familial Precocious Fetal Abnormal Cortical Sulcation.

The development of the human cerebral cortex is a complex and precisely programmed process by which alterations may lead to morphological and functional neurological abnormalities. We report familial cases of prenatally diagnosed abnormal brain, characterized by aberrant symmetrical mesial oversulcation of the parietooccipital lobes, in fetuses affected by abnormal skeletal features. Fetal brain anomalies were characterized by prenatal magnetic resonance imaging at 21 weeks of gestation and histologically evaluated at 22 weeks. Histological examination added relevant information showing some focal cortical areas of micropoligyria and heterotopic extension of the cortical plate into the marginal zone beneath the cortical surface. Genetic analysis of the fetuses excluded FGFR3 mutations known to be related to skeletal dysplasia and aberrant symmetrical oversulcation in other brain areas (temporal lobes). Hence, the present report suggests the existence of a class of rare syndromes of skeleton and brain development abnormality unrelated to FGFR3 mutations or related to other not described FGFR3 gene defects. Using magnetic resonance imaging, histopathology and molecular characterization we provide an example of a translational study of a rare and unreported brain congenital malformation.

Expanding the spectrum of human ganglionic eminence region anomalies on fetal magnetic resonance imaging.

INTRODUCTION: Ganglionic eminence (GE) is a transient fetal brain structure that harvests a significant amount of precursors of cortical GABA-ergic interneurons. Prenatal magnetic resonance (MR) imaging features of GE anomalies (i.e., cavitations) have already been reported associated with severe micro-lissencephaly. The purpose of this report was to illustrate the MR imaging features of GE anomalies in conditions other than severe micro-lissencephalies. METHODS: Among all the fetuses submitted to prenatal MR imaging at our center from 2005 to 2014, we collected eight cases with GE anomalies and only limited associated brain anomalies. The median gestational age at the time of MR imaging was 21 weeks ranging from 19 to 29 weeks. Two senior pediatric neuroradiologists categorized the anomalies of the GE region in two groups: group one showing cavitation in the GE region and group two showing enlarged GE region. For each fetal case, associated cranial anomalies were also reported. RESULTS: Five out of the eight cases were included in group one and three in group two. Besides the GE region abnormality, all eight cases had additional intracranial anomalies, such as mild partial callosal agenesis, vermian hypoplasia and rotation, cerebellar hypoplasia, ventriculomegaly, enlarged subarachnoid spaces, molar tooth malformation. Ultrasound generally detected most of the associated intracranial anomalies, prompting the MR investigation; on the contrary in none of the cases, GE anomalies had been detected by ultrasound. CONCLUSIONS: Our observation expands the spectrum of human GE anomalies, demonstrating that these may take place also without associated severe micro-lissencephalies.

Increased pCREB expression and the spontaneous epileptiform activity in a BCNU-treated rat model of cortical dysplasia.

OBJECTIVE: Cortical dysplasias (CDs) represent a wide range of cortical abnormalities that closely correlate with intractable epilepsy. Rats prenatally exposed to 1-3-bis-chloroethyl-nitrosurea (BCNU) represent an injury-based model that reproduces many histopathologic features of human CD. Previous studies reported in vivo hyperexcitability in this model, but in vivo epileptogenicity has not been confirmed. METHODS: To determine whether cortical and hippocampal lesions lead to epileptiform discharges and/or seizures in the BCNU model, rats at three different ages (3, 5, and 9 months old) were implanted for long-term video electroencephalographic recording. At the end of the recording session, brain tissue was processed for histologic and immunohistochemical investigation including cAMP response element binding protein (CREB) phosphorylation, as a biomarker of epileptogenicity. RESULTS: BCNU-treated rats showed spontaneous epileptiform activity (67%) in the absence of a second seizure-provoking hit. Such activity originated mainly from one hippocampus and propagated to the ipsilateral neocortex. No epileptiform activity was found in age-matched control rats. The histopathologic investigation revealed that all BCNU rats with epileptiform activity showed neocortical and hippocampal abnormalities; the presence and the severity of these lesions did not correlate consistently with the propensity to generate epileptiform discharges. Epileptiform activity was found only in cortical areas of BCNU-treated rats in which a correlation between brain abnormalities and increased pCREB expression was observed. SIGNIFICANCE: This study demonstrates the in vivo occurrence of spontaneous epileptiform discharges in the BCNU model and shows that increased pCREB expression can be utilized as a reliable biomarker of epileptogenicity.

Epileptiform activity and cognitive deficits in SNAP-25(+/-) mice are normalized by antiepileptic drugs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.

7T MRI features in control human hippocampus and hippocampal sclerosis: an ex vivo study with histologic correlations.

OBJECTIVE: Hippocampal sclerosis (HS) is the major structural brain lesion in patients with temporal lobe epilepsy (TLE). However, its internal anatomic structure remains difficult to recognize at 1.5 or 3 Tesla (T) magnetic resonance imaging (MRI), which allows neither identification of specific pathology patterns nor their proposed value to predict postsurgical outcome, cognitive impairment, or underlying etiologies. We aimed to identify specific HS subtypes in resected surgical TLE samples on 7T MRI by juxtaposition with corresponding histologic sections. METHODS: Fifteen nonsclerotic and 18 sclerotic hippocampi were studied ex vivo using an experimental 7T MRI scanner. T2 -weighted images (T2wi) and diffusion tensor imaging (DTI) data were acquired and validated using a systematic histologic analysis of same specimens along the anterior-posterior axis of the hippocampus. RESULTS: In nonsclerotic hippocampi, differences in MR intensity could be assigned to seven clearly recognizable layers and anatomic boundaries as confirmed by histology. All hippocampal subfields could be visualized also in the hippocampal head with three-dimensional imaging and angulated coronal planes. Only four discernible layers were identified in specimens with histopathologically confirmed HS. All sclerotic hippocampi showed a significant atrophy and increased signal intensity along the pyramidal cell layer. Changes in DTI parameters such as an increased mean diffusivity, allowed to distinguish International League Against Epilepsy (ILAE) HS type 1 from type 2. Whereas the increase in T2wi signal intensities could not be attributed to a distinct specific histopathologic substrate, that is, decreased neuronal or increased glial cell densities, intrahippocampal projections and fiber tracts were distorted in HS specimens suggesting a complex disorganization of the cellular composition, fiber networks, as well as its extracellular matrix. SIGNIFICANCE: Our data further advocate high-resolution MRI as a helpful and promising diagnostic tool for the investigation of hippocampal pathology along the anterior-posterior extent in TLE, as well as in other neurologic and neurodegenerative disorders.

Cytoarchitectural, behavioural and neurophysiological dysfunctions in the BCNU-treated rat model of cortical dysplasia.

Cortical dysplasias (CDs) include a spectrum of cerebral lesions resulting from cortical development abnormalities during embryogenesis that lead to cognitive disabilities and epilepsy. The experimental model of CD obtained by means of in utero administration of BCNU (1-3-bis-chloroethyl-nitrosurea) to pregnant rats on embryonic day 15 mimics the histopathological abnormalities observed in many patients. The aim of this study was to investigate the behavioural, electrophysiological and anatomical profile of BCNU-treated rats in order to determine whether cortical and hippocampal lesions can directly lead to cognitive dysfunction. The BCNU-treated rats showed impaired short-term working memory but intact long-term aversive memory, whereas their spontaneous motor activity and anxiety-like response were normal. The histopathological and immunohistochemical analyses, made after behavioural tests, revealed the disrupted integrity of neuronal populations and connecting fibres in hippocampus and prefrontal and entorhinal cortices, which are involved in memory processes. An electrophysiological evaluation of the CA1 region of in vitro hippocampal slices indicated a decrease in the efficiency of excitatory synaptic transmission and impaired paired pulse facilitation, but enhanced long-term potentiation (LTP) associated with hyperexcitability in BCNU-treated rats compared with controls. The enhanced LTP, associated with hyperexcitability, may indicate a pathological distortion of long-term plasticity. These findings suggest that prenatal developmental insults at the time of peak cortical neurogenesis can induce anatomical abnormalities associated with severe impairment of spatial working memory in adult BCNU-treated rats and may help to clarify the pathophysiological mechanisms of cognitive dysfunction that is often associated with epilepsy in patients with CD.

Genesis of heterotopia in BCNU model of cortical dysplasia, detected by means of in utero electroporation.

Derangements of cortical development can cause a wide spectrum of malformations, generally termed 'cortical dysplasia' (CD), which are frequently associated with drug-resistant epilepsy and other neurological and mental disorders. 1,3-Bis-chloroethyl-nitrosurea (BCNU)-treated rats represent a model of CD due to the presence of histological alterations similar to those observed in human CD. BCNU is an alkylating agent that, administered at embryonic day 15 (E15), causes the loss of many cells destined to cortical layers; this results in cortical thinning but also in histological alterations imputable to migration defects, such as laminar disorganization and cortical and periventricular heterotopia. In the present study we investigated the genesis of heterotopia in BCNU-treated rats by labeling cortical ventricular zone (VZ) cells with a green fluorescent protein (GFP) expression vector by means of in utero electroporation. Here, we compared the migratory pattern and subsequent distribution of the GFP-labeled cells in the developing somatosensory cortex of control and BCNU-treated animals. To this aim, we investigated the expression of a panel of developmental marker genes which identified radial glia cells (Pax6), intermediate precursors cells (Tbr2), and postmitotic neurons destined to infragranular (Tbr1) or supragranular layers (Satb2). The VZ of BCNU-treated rats appeared disorganized since E18 and at E21 the embryos showed an altered migratory pattern: migration of superficial layers appeared delayed, with a number of migrating cells in the intermediate zone and some neurons destined to superficial layers arrested in the VZ, thus forming periventricular heterotopia. Moreover, neurons that reached their correct position did not extend their axons through the corpus callosum in the contralateral hemisphere as in the control, but toward the ipsilateral cingulated cortex. Our analysis sheds light on how a malformed cortex develops after a temporally discrete environmental insult.

TI-VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes.

BACKGROUND INFORMATION: ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. RESULTS: In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. CONCLUSIONS: Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions.

Dynamic expression of NR2F1 and SOX2 in developing and adult human cortex: comparison with cortical malformations.

The neocortex, the most recently evolved brain region in mammals, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the presence of graded expression of transcription factors and molecular determinants defining neuronal identity. However, little is known about the expression of key master genes orchestrating human cortical development. In this study, we explored the expression dynamics of NR2F1 and SOX2, key cortical genes whose mutations in human patients cause severe neurodevelopmental syndromes. We focused on physiological conditions, spanning from mid-late gestational ages to adulthood in unaffected specimens, but also investigated gene expression in a pathological context, a developmental cortical malformation termed focal cortical dysplasia (FCD). We found that NR2F1 follows an antero-dorsallow to postero-ventralhigh gradient as in the murine cortex, suggesting high evolutionary conservation. While SOX2 is mainly expressed in neural progenitors next to the ventricular surface, NR2F1 is found in both mitotic progenitors and post-mitotic neurons at GW18. Interestingly, both proteins are highly co-expressed in basal radial glia progenitors of the outer sub-ventricular zone (OSVZ), a proliferative region known to contribute to cortical expansion and complexity in humans. Later on, SOX2 becomes largely restricted to astrocytes and oligodendrocytes although it is also detected in scattered mature interneurons. Differently, NR2F1 maintains its distinct neuronal expression during the whole process of cortical development. Notably, we report here high levels of NR2F1 in dysmorphic neurons and NR2F1 and SOX2 in balloon cells of surgical samples from patients with FCD, suggesting their potential use in the histopathological characterization of this dysplasia.

Kir4.1 RNA Interference by In Utero Electroporation Fails to Affect Ictogenesis and Reveals a Possible role of Kir4.1 in Corticogenesis.

Astrocyte dysfunction, and in particular impaired extracellular potassium spatial buffering, has been postulated to have a potential role in seizure susceptibility and ictogenesis. Inwardly rectifying potassium (Kir) channels, and specifically KIR4.1, have a predominant role in K+ homeostasis and their involvement in neuronal excitability control have been hypothesized. To avoid the severe side effects observed in Kir4.1 cKO, we studied the effects of Kir4.1 down-regulation in cortical astrocytes by using Kir4.1 RNA interference (RNAi) technique combined with in utero electroporation (IUE) at E16 and a piggyBac transposon system. Kir4.1 down-regulation was confirmed by immunohistochemistry and field fraction analysis. To investigate if Kir4.1 silencing affects 4AP-induced seizure threshold and extracellular potassium homeostasis, simultaneous in vitro field potential and extracellular K+ recordings were performed on somatosensory cortex slices obtained from rats electroporated with a piggyBac-Kir4.1-shRNA (Kir4.1-) and scrambled shRNA (Kir4.1Sc). Electrophysiological data revealed no significant differences in terms of seizure onset and seizure-induced extracellular K+ changes between Kir4.1- and Kir4.1Sc rats. Intriguingly, immunohistochemical analysis performed on slices studied with electrophysiology revealed a reduced number of neurons generated from radial glial cells in Kir4.1- rats. We conclude that focal down-regulation of Kir4.1 channel in cortical astrocytes by Kir4.1 RNAi technique combined with IUE is not effective in altering potassium homeostasis and seizure susceptibility. This technique revealed a possible role of Kir4.1 during corticogenesis.

Sox2 Acts in Thalamic Neurons to Control the Development of Retina-Thalamus-Cortex Connectivity.

Visual system development involves the formation of neuronal projections connecting the retina to the thalamic dorso-lateral geniculate nucleus (dLGN) and the thalamus to the visual cerebral cortex. Patients carrying mutations in the SOX2 transcription factor gene present severe visual defects, thought to be linked to SOX2 functions in the retina. We show that Sox2 is strongly expressed in mouse postmitotic thalamic projection neurons. Cre-mediated deletion of Sox2 in these neurons causes reduction of the dLGN, abnormal distribution of retino-thalamic and thalamo-cortical projections, and secondary defects in cortical patterning. Reduced expression, in mutants, of Sox2 target genes encoding ephrin-A5 and the serotonin transport molecules SERT and vMAT2 (important for establishment of thalamic connectivity) likely provides a molecular contribution to these defects. These findings unveil thalamic SOX2 function as a novel regulator of visual system development and a plausible additional cause of brain-linked genetic blindness in humans.

SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization.

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.

Heterogeneous expression of SNAP-25 in rat and human brain.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a SNARE component of the exocytotic apparatus involved in the release of neurotransmitter. We used multiple-labeling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to examine the expression of SNAP-25 in excitatory and inhibitory terminals from different rat and human brain areas. Glutamatergic and GABAergic terminals were identified by staining for the vesicular glutamate transporter (vGLUT1), glutamic acid decarboxylase (GAD67), or the vesicular GABA transporter (vGAT). In all examined areas GABAergic terminals did not display detectable levels of SNAP-25, whereas glutamatergic terminals expressed the protein to a variable extent. Codistribution analysis revealed a high colocalization between pixels detecting SNAP-25 labeling and pixels detecting vGLUT1 immunoreactivity. On the contrary, a low degree of pixel colocalization, comparable to that between two unrelated antigens, was detected between SNAP-25 and vGAT, thus suggesting a random overlap of immunofluorescence signals. Our immunofluorescence evidence was supported by ultrastructural data, which clearly confirmed that SNAP-25 was undetectable in GABAergic terminals identified by both their typical morphology and specific staining for GABA. Interestingly, our ultrastructural results confirmed that a subset of glutamatergic synapses do not contain detectable levels of SNAP-25. The present study extends our previous findings obtained in rodent hippocampus and provides evidence that SNAP-25 expression is highly variable between different axon terminals both in rat and human brain. The heterogeneous distribution of SNAP-25 may have important implications not only in relation to the function of the protein as a SNARE but also in the control of network excitability.

Proliferative cells in the rat developing neocortical grey matter: new insights into gliogenesis.

The postnatal brain development is characterized by a substantial gain in weight and size, ascribed to increasing neuronal size and branching, and to massive addition of glial cells. This occurs concomitantly to the shrinkage of VZ and SVZ, considered to be the main germinal zones, thus suggesting the existence of other germinative niches. The aim of this study is to characterize the cortical grey matter proliferating cells during postnatal development, providing their stereological quantification and identifying the nature of their cell lineage. We performed double immunolabeling for the proliferation marker Ki67 and three proteins which identify either astrocytes (S100ß) or oligodendrocytes (Olig2 and NG2), in addition to a wider panel of markers apt to validate the former markers or to investigate other cell lineages. We found that proliferating cells increase in number during the first postnatal week until P10 and subsequently decreased until P21. Cell lineage characterization revealed that grey matter proliferating cells are prevalently oligodendrocytes and astrocytes along with endothelial and microglial cells, while no neurons have been detected. Our data showed that astrogliogenesis occurs prevalently during the first 10 days of postnatal development, whereas contrary to the expected peak of oligodendrogenesis at the second postnatal week, we found a permanent pool of proliferating oligodendrocytes enduring from birth until P21. These data support the relevance of glial proliferation within the grey matter and could be a point of departure for further investigations of this complex process.

Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood brain barrier.

Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases and tumors, would benefit from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To assess the mechanistic aspect of penetration of nanoparticles (NPs) into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood-brain barrier and the interactions between vascular and neuronal compartments are morphologically and functionally preserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS:OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by TEM analysis of the electron-dense core of the MUS:OT Au/Fe NPs was conducted to understand NPs' brain penetration through the BBB after in vitro arterial perfusion and their distribution in the parenchyma. Our data suggest that MUS:OT Au/Fe NPs enter the brain utilizing a physiological route and therefore can be exploited as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities.

PSA-NCAM in the developing and mature thalamus.

The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is involved in several morphogenetic processes of the central nervous system. In the present study the expression of PSA-NCAM has been investigated in the rat thalamus during embryonic and postnatal development using light and electron microscopic immunocytochemical techniques. At all the examined ages, PSA-NCAM staining in the thalamus was mainly observed along neuronal plasmatic membranes and absent in astrocytes identified by labelling with cytoskeletal (vimentin and glial fibrillary acidic protein) and membrane (GABA transporter-3) markers. At embryonic day 14 the immunoreactivity was restricted to the dorsal thalamic mantle and to the region of reticular thalamic migration and subsequently it extended throughout the whole thalamic primordium. PSA-NCAM labelling remained intense and homogeneously distributed along perinatal period, but from P4 it began to decrease selectively, persisting throughout adulthood only in the reticular nucleus, ventral lateral geniculate nucleus and midline and intralaminar nuclei. The expression of this adhesion molecule differed in areas characterized by the presence of neurons containing distinct calcium binding proteins, as PSA-NCAM labelling was intense around calretinin-positive neurons, whereas it decreased in some calbindin-immunoreactive regions. These findings show evidence of a selective neuronal expression of PSA-NCAM in developing thalamus, supporting its suggested role in cell migration and synaptogenesis as it occurs in the cerebral cortex. In adulthood PSA-NCAM could instead be a marker of thalamic nuclei that retain a potential for synaptic plasticity.