Transcriptomic changes due to early, chronic intermittent alcohol exposure during forebrain development implicate WNT signaling, cell-type specification, and cortical regionalization as primary determinants of fetal alcohol syndrome.

BACKGROUND: Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS. METHODS: In this study, we utilized an in vitro human pluripotent stem cell-based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester-equivalent cortical neurons. RESULTS: We used RNA sequencing of developing hPSC-derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell-type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling-associated transcripts observed in alcohol-exposed cultures relative to alcohol-naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain-related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90-4), indicating that these patterning alterations are not cell line-specific. CONCLUSIONS: We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high-dose prenatal alcohol exposure, as well as patients with FAS.

Genetic inactivation of synaptosomal-associated protein 25 (SNAP-25) in adult hippocampal neural progenitors impairs pattern discrimination learning but not survival or structural maturation of newborn dentate granule cells.

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreERT2 :Snap25fl/fl : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreERT2 :Snap25fl/fl : tdTomato SNAP compared to tamoxifen-treated NestinCreERT2 :Snap25wt/wt : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

Effects of Ethanol on Cellular Composition and Network Excitability of Human Pluripotent Stem Cell-Derived Neurons.

BACKGROUND: Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus, E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD), but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here, we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause the aberrant production of glutamatergic and GABAergic neurons, resulting in E/I imbalance. METHODS: We applied 50 mM EtOH daily to differentiating hPSNs for 50 days to model chronic first-trimester exposure. We used quantitative polymerase chain reaction, immunocytochemical, and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. RESULTS: We found that EtOH did not alter neural induction nor general forebrain patterning and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast, our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g., GSX2, DLX1/2/5/6, NR2F2) as well as mature IN specification (e.g., SST, NPY). Interestingly, EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. CONCLUSIONS: Similar to in vivo rodent studies, EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs, but did not cause imbalances of synaptic excitation-inhibition. Thus, our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However, in contrast to rodent binge models, our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance.

Deficits in human trisomy 21 iPSCs and neurons.

Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.

Microglial derived tumor necrosis factor-α drives Alzheimer's disease-related neuronal cell cycle events.

Massive neuronal loss is a key pathological hallmark of Alzheimer's disease (AD). However, the mechanisms are still unclear. Here we demonstrate that neuroinflammation, cell autonomous to microglia, is capable of inducing neuronal cell cycle events (CCEs), which are toxic for terminally differentiated neurons. First, oligomeric amyloid-beta peptide (AßO)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-α (TNFα) and the c-Jun Kinase (JNK) signaling pathway. Second, adoptive transfer of CD11b+ microglia from AD transgenic mice (R1.40) induced neuronal cyclin D1 expression via TNFα signaling pathway. Third, genetic deficiency of TNFα in R1.40 mice (R1.40-Tnfα(-/-)) failed to induce neuronal CCEs. Finally, the mitotically active neurons spatially co-exist with F4/80+ activated microglia in the human AD brain and that a portion of these neurons are apoptotic. Together our data suggest a cell-autonomous role of microglia, and identify TNFα as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD.

Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network.

Whether hESC-derived neurons can fully integrate with and functionally regulate an existing neural network remains unknown. Here, we demonstrate that hESC-derived neurons receive unitary postsynaptic currents both in vitro and in vivo and adopt the rhythmic firing behavior of mouse cortical networks via synaptic integration. Optical stimulation of hESC-derived neurons expressing Channelrhodopsin-2 elicited both inhibitory and excitatory postsynaptic currents and triggered network bursting in mouse neurons. Furthermore, light stimulation of hESC-derived neurons transplanted to the hippocampus of adult mice triggered postsynaptic currents in host pyramidal neurons in acute slice preparations. Thus, hESC-derived neurons can participate in and modulate neural network activity through functional synaptic integration, suggesting they are capable of contributing to neural network information processing both in vitro and in vivo.

Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

Distribution and inflammatory cell response to intracranial delivery of radioluminescent Y2(SiO4)O:Ce particles.

Due to increasing advances in their manufacture and functionalization, nanoparticle-based systems have become a popular tool for in vivo drug delivery and biodetection. Recently, scintillating nanoparticles such as yttrium orthosilicate doped with cerium (Y2(SiO4)O:Ce) have come under study for their potential utility in optogenetic applications, as they emit photons upon low levels of stimulation from remote x-ray sources. The utility of such nanoparticles in vivo is hampered by rapid clearance from circulation by the mononuclear phagocytic system, which heavily restricts nanoparticle accumulation at target tissues. Local transcranial injection of nanoparticles may deliver scintillating nanoparticles to highly specific brain regions by circumventing the blood-brain barrier and avoiding phagocytic clearance. Few studies to date have examined the distribution and response to nanoparticles following localized delivery to cerebral cortex, a crucial step in understanding the therapeutic potential of nanoparticle-based biodetection in the brain. Following the synthesis and surface modification of these nanoparticles, two doses (1 and 3 mg/ml) were introduced into mouse secondary motor cortex (M2). This region was chosen as the site for RLP delivery, as it represents a common target for optogenetic manipulations of mouse behavior, and RLPs could eventually serve as an injectable x-ray inducible light delivery system. The spread of particles through the target tissue was assessed 24 hours, 72 hours, and 9 days post-injection. Y2(SiO4)O:Ce nanoparticles were found to be detectable in the brain for up to 9 days, initially diffusing through the tissue until 72 hours before achieving partial clearance by the final endpoint. Small transient increases in the presence of IBA-1+ microglia and GFAP+ astrocytic cell populations were detected near nanoparticle injection sites of both doses tested 24 hours after surgery. Taken together, these data provide evidence that Y2(SiO4)O:Ce nanoparticles coated with BSA can be injected directly into mouse cortex in vivo, where they persist for days and are broadly tolerated, such that they may be potentially utilized for remote x-ray activated stimulation and photon emission for optogenetic experiments in the near future.

Global loss of Neuron-specific gene 1 causes alterations in motor coordination, increased anxiety, and diurnal hyperactivity in male mice.

The Neuron-specific gene family (NSG1-3) consists of small endolysosomal proteins that are critical for trafficking multiple receptors and signaling molecules in neurons. NSG1 has been shown to play a critical role in AMPAR recycling from endosomes to plasma membrane during synaptic plasticity. However, to date nothing is known about whether NSG1 is required for normal behavior at an organismal level. Here we performed a battery of behavioral tests to determine whether loss of NSG1 would affect motor, cognitive, and/or affective behaviors, as well as circadian-related activity. Consistent with unique cerebellar expression of NSG1 among family members, we found that NSG1 was obligatory for motor coordination but not for gross motor function or learning. NSG1 knockout (KO) also altered performance across other behavioral modalities including anxiety-related and diurnal activity paradigms. Surprisingly, NSG1 KO did not cause significant impairments across all tasks within a given modality, but had specific effects within each modality. For instance, we found increases in anxiety-related behaviors in tasks with multiple stressors (e.g., elevation and exposure), but not those with a single main stressor (e.g., exposure). Interestingly, NSG1 KO animals displayed a significant increase in locomotor activity during subjective daytime, suggesting a possible impact on diurnal activity rhythms or vigilance. Surprisingly, loss of NSG1 had no effect on hippocampal-dependent learning despite previous studies showing deficits in CA1 long-term potentiation. Together, these findings do not support a role of NSG1 in hippocampal-dependent learning, but support a role in mediating proper neuronal function across amygdalar and cerebellar circuits.

Functional control of transplantable human ESC-derived neurons via optogenetic targeting.

Current methods to examine and regulate the functional integration and plasticity of human ESC (hESC)-derived neurons are cumbersome and technically challenging. Here, we engineered hESCs and their derivatives to express the light-gated channelrhodopsin-2 (ChR2) protein to overcome these deficiencies. Optogenetic targeting of hESC-derived neurons with ChR2 linked to the mCherry fluorophore allowed reliable cell tracking as well as light-induced spiking at physiological frequencies. Optically induced excitatory and inhibitory postsynaptic currents could be elicited in either ChR2(+) or ChR2(-) neurons in vitro and in acute brain slices taken from transplanted severe combined immunodeficient (SCID) mice. Furthermore, we created a clonal hESC line that expresses ChR2-mCherry under the control of the synapsin-1 promoter. On neuronal differentiation, ChR2-mCherry expression was restricted to neurons and was stably expressed for at least 6 months, providing more predictable light-induced currents than transient infections. This pluripotent cell line will allow both in vitro and in vivo analysis of functional development as well as the integration capacity of neuronal populations for cell-replacement strategies.

Feasibility of cerium-doped LSO particles as a scintillator for x-ray induced optogenetics.

Objective.Non-invasive light delivery into the brain is needed forin vivooptogenetics to avoid physical damage. An innovative strategy could employ x-ray activation of radioluminescent particles (RLPs) to emit localized light. However, modulation of neuronal or synaptic function by x-ray induced radioluminescence from RLPs has not yet been demonstrated.Approach.Molecular and electrophysiological approaches were used to determine if x-ray dependent radioluminescence emitted from RLPs can activate light sensitive proteins. RLPs composed of cerium doped lutetium oxyorthosilicate (LSO:Ce), an inorganic scintillator that emits blue light, were used as they are biocompatible with neuronal function and synaptic transmission.Main results.We show that 30 min of x-ray exposure at a rate of 0.042 Gy s-1caused no change in the strength of basal glutamatergic transmission during extracellular field recordings in mouse hippocampal slices. Additionally, long-term potentiation, a robust measure of synaptic integrity, was induced after x-ray exposure and expressed at a magnitude not different from control conditions (absence of x-rays). We found that x-ray stimulation of RLPs elevated cAMP levels in HEK293T cells expressing OptoXR, a chimeric opsin receptor that combines the extracellular light-sensitive domain of rhodopsin with an intracellular second messenger signaling cascade. This demonstrates that x-ray radioluminescence from LSO:Ce particles can activate OptoXR. Next, we tested whether x-ray activation of the RLPs can enhance synaptic activity in whole-cell recordings from hippocampal neurons expressing channelrhodopsin-2, both in cell culture and acute hippocampal slices. Importantly, x-ray radioluminescence caused an increase in the frequency of spontaneous excitatory postsynaptic currents in both systems, indicating activation of channelrhodopsin-2 and excitation of neurons.Significance.Together, our results show that x-ray activation of LSO:Ce particles can heighten cellular and synaptic function. The combination of LSO:Ce inorganic scintillators and x-rays is therefore a viable method for optogenetics as an alternative to more invasive light delivery methods.

Proteopathic tau primes and activates interleukin-1ß via myeloid-cell-specific MyD88- and NLRP3-ASC-inflammasome pathway.

Pathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1ß (IL-1ß), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1ß. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1ß signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1ß activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1ß expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1ß activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1ß and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1ß via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.

Developmental regulation of human embryonic stem cell-derived neurons by calcium entry via transient receptor potential channels.

Spontaneous calcium (Ca(2+)) transients in the developing nervous system can affect proliferation, migration, neuronal subtype specification, and neurite outgrowth. Here, we show that telencephalic human neuroepithelia (hNE) and postmitotic neurons (PMNs) generated from embryonic stem cells display robust Ca(2+) transients. Unlike previous reports in animal models, transients occurred by a Gd(3+)/La(3+)-sensitive, but thapsigargin- and Cd(2+)-insensitive, mechanism, strongly suggestive of a role for transient receptor potential (Trp) channels. Furthermore, Ca(2+) transients in PMNs exhibited an additional sensitivity to the canonical Trp (TrpC) antagonist SKF96365 and shRNA-mediated knockdown of the TrpC1 subunit. Functionally, inhibition of Ca(2+) transients in dividing hNE cells led to a significant reduction in proliferation, whereas either pharmacological inhibition or shRNA-mediated knockdown of the TrpC1 and TrpC4 subunits significantly reduced neurite extension in PMNs. Primary neurons cultured from fetal human cortex displayed nearly identical Ca(2+) transients and pharmacological sensitivities to Trp channel antagonists. Together these data suggest that Trp channels present a novel mechanism for controlling Ca(2+) transients in human neurons and may offer a target for regulating proliferation and neurite outgrowth when engineering cells for therapeutic transplantation.

Regulation of neural specification from human embryonic stem cells by BMP and FGF.

Inhibition of bone morphogenetic protein (BMP) signaling is required for vertebrate neural induction, and fibroblast growth factors (FGFs) may affect neural induction through phosphorylation at the linker region of Smad1, thus regulating BMP signaling. Here we show that human embryonic stem cells efficiently convert to neuroepithelial cells in the absence of BMP antagonists, or even when exposed to high concentrations of exogenous BMP4. Molecular and functional analyses revealed multiple levels of endogenous BMP signaling inhibition that may account for the efficient neural differentiation. Blocking FGF signaling inhibited neural induction, but did not alter the phosphorylation of the linker region of Smad1, suggesting that FGF enhances human neural specification independently of BMP signaling.

Optical induction of autophagy via Transcription factor EB (TFEB) reduces pathological tau in neurons.

Pathological accumulation of microtubule associated protein tau in neurons is a major neuropathological hallmark of Alzheimer's disease (AD) and related tauopathies. Several attempts have been made to promote clearance of pathological tau (p-Tau) from neurons. Transcription factor EB (TFEB) has shown to clear p-Tau from neurons via autophagy. However, sustained TFEB activation and autophagy can create burden on cellular bioenergetics and can be deleterious. Here, we modified previously described two-plasmid systems of Light Activated Protein (LAP) from bacterial transcription factor-EL222 and Light Responsive Element (LRE) to encode TFEB. Upon blue-light (465 nm) illumination, the conformation changes in LAP induced LRE-driven expression of TFEB, its nuclear entry, TFEB-mediated expression of autophagy-lysosomal genes and clearance of p-Tau from neuronal cells and AD patient-derived human iPSC-neurons. Turning the blue-light off reversed the expression of TFEB-target genes and attenuated p-Tau clearance. Together, these results suggest that optically regulated TFEB expression unlocks the potential of opto-therapeutics to treat AD and other dementias.

Exosomal secretion of a psychosis-altered miRNA that regulates glutamate receptor expression is affected by antipsychotics.

The ability of small secretory microvesicles known as exosomes to influence neuronal and glial function via their microRNA (miRNA) cargo has positioned them as a novel and effective method of cell-to-cell communication. However, little is known about the role of exosome-secreted miRNAs in the regulation of glutamate receptor gene expression and their relevance for schizophrenia (SCZ) and bipolar disorder (BD). Using mature miRNA profiling and quantitative real-time PCR (qRT-PCR) in the orbitofrontal cortex (OFC) of SCZ (N = 29; 20 male and 9 female), BD (N = 26; 12 male and 14 female), and unaffected control (N = 25; 21 male and 4 female) subjects, we uncovered that miR-223, an exosome-secreted miRNA that targets glutamate receptors, was increased at the mature miRNA level in the OFC of SCZ and BD patients with positive history of psychosis at the time of death and was inversely associated with deficits in the expression of its targets glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B) and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2). Furthermore, changes in miR-223 levels in the OFC were positively and negatively correlated with inflammatory and GABAergic gene expression, respectively. Moreover, miR-223 was found to be enriched in astrocytes and secreted via exosomes, and antipsychotics were shown to control its cellular and exosomal localization in a cell-specific manner. Furthermore, addition of astrocytic exosomes in neuronal cultures resulted in a significant increase in miR-223 expression and a notable reduction in Grin2b and Gria2 mRNA levels, which was strongly inversely associated with miR-223 expression. Lastly, inhibition of astrocytic miR-223 abrogated the exosomal-mediated reduction in neuronal Grin2b expression. Taken together, our results demonstrate that the exosomal secretion of a psychosis-altered and glial-enriched miRNA that controls neuronal gene expression is regulated by antipsychotics.

Neuron-Specific Gene 2 (NSG2) Encodes an AMPA Receptor Interacting Protein That Modulates Excitatory Neurotransmission.

Neurons have evolved a number of unique protein-coding genes that regulate trafficking of protein complexes within small organelles throughout dendrites and axons. Neuron-specific gene 2 (NSG2) encodes for one of the most abundant proteins in the nervous system during perinatal development. NSG2 belongs to a family of small neuronal endosomal proteins but its function has remained uncharacterized to date. Here, we show that NSG2 is found in discrete punctae restricted to the somatodendritic arbors of developing mouse and human neurons, and a significant proportion of NSG2 punctae colocalize with postsynaptic HOMER1 and surface-expressed AMPA-type glutamate receptors (AMPARs) at excitatory synapses. Immunoprecipitation revealed that NSG2 physically interacts with both the GluA1 and GluA2 AMPAR subunits in mouse brain. Knock-out of NSG2 in mouse hippocampal neurons selectively impaired the frequency of miniature EPSCs (mEPSCs) and caused alterations in PSD95 expression at postsynaptic densities (PSDs). In contrast, NSG2 overexpression caused a significant increase in the amplitude of mEPSCs as well as GluA2 surface expression. Thus, NSG2 functions as an AMPAR-binding protein that is required for normal synapse formation and/or maintenance, and has unique functions compared with other NSG family members.

Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture.

How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na+ and K+ currents, and fire mature action potentials by 7 weeks of differentiation. This is similar to the maturation pattern observed in animals, albeit on a greatly expanded time scale. An additional 3 weeks of differentiation resulted in neurons that could fire repetitive trains of action potentials in response to depolarizing current pulses. The onset of spontaneous synaptic activity also occurred after 7 weeks of differentiation, in association with the differentiation of astrocytes within the culture. Cocultures of hES cell-derived neuroepithelial cells with exogenous astrocytes significantly accelerated the onset of synaptic currents but did not alter action potential generation. These findings suggest that the development of membrane characteristics and action potentials depend on the intrinsic maturation of Na+ and K+ currents, whereas synaptic transmission is enhanced by astrocytes, which may be achieved independently of the maturation of action potentials. Furthermore, we found that although astrocyte-conditioned medium accelerated synaptic protein localization, it did not increase synaptic activity, suggesting a contact-dependent mechanism by which astrocytes augment synaptic activity. These results lay the foundation for future studies examining the functional development of human neurons and provide support for the potential application of human cells in restorative neuronal therapies.

D1 dopamine receptor activation of NFAT-mediated striatal gene expression.

Exposure to drugs of abuse activates gene expression and protein synthesis that result in long-lasting adaptations in striatal signaling. Therefore, identification of the transcription factors that couple drug exposure to gene expression is of particular importance. Members of the nuclear factor of activated T-cells (NFATc) family of transcription factors have recently been implicated in shaping neuronal function throughout the rodent nervous system. Here we demonstrate that regulation of NFAT-mediated gene expression may also be a factor in drug-induced changes to striatal functioning. In cultured rat striatal neurons, stimulation of D1 dopamine receptors induces NFAT-dependent transcription through activation of L-type calcium channels. Additionally, the genes encoding inositol-1,4,5-trisphosphate receptor type 1 and glutamate receptor subunit 2 are regulated by striatal NFATc4 activity. Consistent with these in-vitro data, repeated exposure to cocaine triggers striatal NFATc4 nuclear translocation and the up-regulation of inositol-1,4,5-trisphosphate receptor type 1 and glutamate receptor subunit 2 gene expression in vivo, suggesting that cocaine-induced increases in gene expression may be partially mediated through activation of NFAT-dependent transcription. Collectively, these findings reveal a novel molecular pathway that may contribute to the enduring modifications in striatal functioning that occur following the administration of drugs of abuse.

Default Patterning Produces Pan-cortical Glutamatergic and CGE/LGE-like GABAergic Neurons from Human Pluripotent Stem Cells.

Default differentiation of human pluripotent stem cells has been promoted as a model of cortical development. In this study, a developmental transcriptome analysis of default-differentiated hPSNs revealed a gene expression program resembling in vivo CGE/LGE subpallial domains and GABAergic signaling. A combination of bioinformatic, functional, and immunocytochemical analysis further revealed that hPSNs consist of both cortical glutamatergic and CGE-like GABAergic neurons. This study provides a comprehensive characterization of the heterogeneous group of neurons produced by default differentiation and insight into future directed differentiation strategies.