Specific Neuronal Subtypes Research Articles

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16–17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.

Specification of transplantable astroglial subtypes from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have been differentiated efficiently to neuronal cell types. However, directed differentiation of hPSCs to astrocytes and astroglial subtypes remains elusive. In this study, hPSCs were directed to nearly uniform populations of immature astrocytes (>90% S100β(+) and GFAP(+)) in large quantities. The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels after transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that express distinct homeodomain transcription factors and display phenotypic differences of different astroglial subtypes. These human astroglial progenitors and immature astrocytes will be useful for studying astrocytes in brain development and function, understanding the roles of astrocytes in disease processes and developing novel treatments for neurological disorders.

Cellular stress response pathways and ageing: intricate molecular relationships.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

A5-Positive Primary Sensory Neurons Are Nonpermissive for Productive Infection with Herpes Simplex Virus 1 In Vitro

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency and express the latency-associated transcript (LAT) preferentially in different murine sensory neuron populations, with most HSV-1 LAT expression in A5(+) neurons and most HSV-2 LAT expression in KH10(+) neurons. To study the mechanisms regulating the establishment of HSV latency in specific subtypes of neurons, cultured dissociated adult murine trigeminal ganglion (TG) neurons were assessed for relative permissiveness for productive infection. In contrast to that for neonatal TG, the relative distribution of A5(+) and KH10(+) neurons in cultured adult TG was similar to that seen in vivo. Productive infection with HSV was restricted, and only 45% of cultured neurons could be productively infected with either HSV-1 or HSV-2. A5(+) neurons supported productive infection with HSV-2 but were selectively nonpermissive for productive infection with HSV-1, a phenomenon that was not due to restricted viral entry or DNA uncoating, since HSV-1 expressing β-galactosidase under the control of the neurofilament promoter was detected in ∼90% of cultured neurons, with no preference for any neuronal subtype. Infection with HSV-1 reporter viruses expressing enhanced green fluorescent protein (EGFP) from immediate early (IE), early, and late gene promoters indicated that the block to productive infection occurred before IE gene expression. Trichostatin A treatment of quiescently infected neurons induced productive infection preferentially from non-A5(+) neurons, demonstrating that the nonpermissive neuronal subtype is also nonpermissive for reactivation. Thus, HSV-1 is capable of entering the majority of sensory neurons in vitro; productive infection occurs within a subset of these neurons; and this differential distribution of productive infection is determined at or before the expression of the viral IE genes.

Neuronal activity is required for the development of specific cortical interneuron subtypes

Electrical activity has been shown to regulate development in a variety of species and in various structures, including the retina, spinal cord and cortex. Within the mammalian cortex specifically, the development of dendrites and commissural axons in pyramidal cells is activity-dependent. However, little is known about the developmental role of activity in the other major cortical population of neurons, the GABA-producing interneurons. These neurons are morphologically and functionally heterogeneous and efforts over the past decade have focused on determining the mechanisms that contribute to this diversity. It was recently discovered that 30% of all cortical interneurons arise from a relatively novel source within the ventral telencephalon, the caudal ganglionic eminence (CGE). Owing to their late birth date, these interneurons populate the cortex only after the majority of other interneurons and pyramidal cells are already in place and have started to functionally integrate. Here we demonstrate in mice that for CGE-derived reelin (Re)-positive and calretinin (Cr)-positive (but not vasoactive intestinal peptide (VIP)-positive) interneurons, activity is essential before postnatal day 3 for correct migration, and that after postnatal day 3, glutamate-mediated activity controls the development of their axons and dendrites. Furthermore, we show that the engulfment and cell motility 1 gene (Elmo1), a target of the transcription factor distal-less homeobox 1 (Dlx1), is selectively expressed in Re(+) and Cr(+) interneurons and is both necessary and sufficient for activity-dependent interneuron migration. Our findings reveal a selective requirement for activity in shaping the cortical integration of specific neuronal subtypes.

Spatiotemporal fate map of neurogenin1 (Neurog1) lineages in the mouse central nervous system

Neurog1 (Ngn1, Neurod3, neurogenin1) is a basic helix-loop-helix (bHLH) transcription factor essential for neuronal differentiation and subtype specification during embryogenesis. Due to the transient expression of Neurog1 and extensive migration of neuronal precursors, it has been challenging to understand the full complement of Neurog1 lineage cells throughout the central nervous system (CNS). Here we labeled and followed Neurog1 lineages using inducible Cre-flox recombination systems with Neurog1-Cre and Neurog1-CreER(T2) BAC (bacterial artificial chromosome) transgenic mice. Neurog1 lineage cells are restricted to neuronal fates and contribute to diverse but discrete populations in each brain region. In the forebrain, Neurog1 lineages include mitral cells and glutamatergic interneurons in the olfactory bulb, pyramidal and granule neurons in the hippocampus, and pyramidal cells in the cortex. In addition, most of the thalamus, but not the hypothalamus, arises from Neurog1 progenitors. Although Neurog1 lineages are largely restricted to glutamatergic neurons, there are multiple exceptions including Purkinje cells and other GABAergic neurons in the cerebellum. This study provides the first overview of the spatiotemporal fate map of Neurog1 lineages in the CNS.

How does the genetic assassin select its neuronal target?

Through many different routes of analysis, including human familial studies and animal models, we are identifying an increasing number of genes that are causative for human neurodegenerative disease and are now in a position for many such disorders to dissect the molecular pathology that gives rise to neuronal death. Yet a paradox remains: The majority of the genes identified cause neurodegeneration in specific neuronal subtypes, but the genes themselves are ubiquitously expressed. Furthermore, the different mutations in the same gene may cause quite different types of neurodegeneration. Something in our understanding of neurodegenerative disease is clearly missing, and we refer to this as the phenomenon of "neuronal targeting." Here we discuss possible explanations for neuronal targeting, why specific neuronal subtypes are vulnerable to specific mutations in ubiquitously expressed genes.

Restrictions in time and space – new insights into generation of specific neuronal subtypes in the adult mammalian brain

Key questions in regard to neuronal repair strategies are which cells are best suited to regenerate specific neuronal subtypes and how much of a neuronal circuit needs to persist in order to allow its functional repair. Here we discuss recent findings in the field of adult neurogenesis, which shed new light on these questions. Neural stem cells in the adult brain generate very distinct types of neurons depending on their regional and temporal specification. Moreover, distinct brain regions differ in the mode of neuron addition in adult neurogenesis, suggesting that different brain circuits may be able to cope differently with the incorporation of new neurons. These new insights are then considered in regard to the choice of cells with the appropriate region-specific identity for repair strategies.

Neuronal subtype specification in the spinal cord of a protovertebrate

The visceral ganglion (VG) comprises the basic motor pool of the swimming ascidian tadpole and has been proposed to be homologous to the spinal cord of vertebrates. Here, we use cis-regulatory modules, or enhancers, from transcription factor genes expressed in single VG neuronal precursors to label and identify morphologically distinct moto- and interneuron subtypes in the Ciona intestinalis tadpole larva. We also show that the transcription factor complement present in each differentiating neuron correlates with its unique morphology. Forced expression of putative interneuron markers Dmbx and Vsx results in ectopic interneuron-like cells at the expense of motoneurons. Furthermore, by perturbing upstream signaling events, we can change the transcription factor expression profile and subsequent identity of the different precursors. Perturbation of FGF signaling transforms the entire VG into Vsx+/Pitx+ putative cholinergic interneurons, while perturbation of Notch signaling results in duplication of Dmbx+ decussating interneurons. These experiments demonstrate the connection between transcriptional regulation and the neuronal subtype diversity underlying swimming behavior in a simple chordate.

Homeobox genes Gsx1 and Gsx2 differentially regulate telencephalic progenitor maturation

Homeobox genes Gsx1 and Gsx2 (formerly Gsh1 and Gsh2) are among the earliest transcription factors expressed in neuronal progenitors of the lateral ganglionic eminence (LGE) in the ventral telencephalon. Gsx2 is required for the early specification of LGE progenitor cells and recently has been shown to specify different LGE neuronal subtypes at distinct time points. In Gsx2 mutants, Gsx1 compensates, at least in part, for the loss of Gsx2 in the specification of LGE neuronal subtypes. Because no specific phenotype has been described in Gsx1 mutants, it is unclear what role this factor plays in the development of the ventral telencephalon. Here, we used a gain-of-function approach to express either Gsx1 or Gsx2 throughout the telencephalon and found that Gsx1 functions similarly to Gsx2 in the specification of LGE identity. However, our results show that Gsx1 and Gsx2 differentially regulate the maturation of LGE progenitors. Specifically, Gsx2 maintains LGE progenitors in an undifferentiated state, whereas Gsx1 promotes progenitor maturation and the acquisition of neuronal phenotypes, at least in part, through the down-regulation of Gsx2. These unique results indicate that the two closely related Gsx genes similarly regulate LGE patterning but oppositely control the balance between proliferation and differentiation in the neuronal progenitor pool.

Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion

Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis. Here, the cellular interaction network of secretagogin has been expanded with nine proteins: SNAP-23, DOC2alpha, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase and myeloid leukemia factor 2, based on screening of a high content protein array and validation and quantification of binding with surface plasmon resonance and GST pulldown assays. All targets have association rate constants in the range 10(4)-10(6) M(-1) s(-1), dissociation rate constants in the range 10(-3)-10(-5) s(-1) and equilibrium dissociation constants in the 100 pM to 10 nM range. The novel target SNAP23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion. Complementary roles in vesicle trafficking are known for ARFGAP2 and DOC2alpha in regulating fusion of vesicles to membranes, kinesin 5B and tubulin for transport of vesicles in the cell, while rootletin builds up the rootlet believed to function as a scaffold for vesicles. The identification of a discrete network of interacting proteins that mediate secretion and vesicle trafficking suggests a regulatory role for secretagogin in these processes.

Phylogeny and Ontogeny of the Habenular Structure

Habenula is an epithalamic nucleus connecting the forebrain with the ventral midbrain and hindbrain that plays a pivotal role in decision making by regulating dopaminergic and serotonergic activities. Intriguingly, habenula has also attracted interest as a model for brain asymmetry, since many vertebrates show left–right differences in habenula size and neural circuitry. Despite the functional significance of this nucleus, few studies have addressed the molecular mechanisms underlying habenular development. Mammalian habenula consists of the medial and lateral habenulae, which have distinct neural connectivity. The habenula shows phylogenetic conservation from fish to human, and studies using genetically accessible model animals have provided molecular insights into the developmental mechanisms of the habenula. The results suggest that development of the habenular asymmetry is mediated by differential regulation of the neurogenetic period for generating specific neuronal subtypes. Since the orientation and size ratio of the medial and lateral habenulae differ across species, the evolution of those subregions within the habenula may also reflect changes in neurogenesis duration for each habenular subdivision according to the evolutionary process.

Obesity Drug Update: The Lost Decade?

The growing worldwide obesity epidemic and obesity-related disorders present a huge unmet medical need for safe and effective anti-obesity medications. The discovery of leptin in 1994 was rapidly succeeded by a wave of related discoveries leading to the elaboration of a hypothalamic melanocortinergic neuronal circuit regulated by leptin and other central and peripheral signaling molecules to control energy homeostasis. The identification of specific neuronal subtypes along with their unique connections and expression products generated a rich target menu for anti-obesity drug discovery programs. Over the course of the last decade, several new chemical entities aimed at these targets have reached various stages or successfully completed the drug discovery/regulatory process only to be dropped or taken off the market. There are now in fact fewer options for anti-obesity drug therapies in late 2010 than were available in 2000. The challenge to discover safe and effective anti-obesity drugs is alive and well.

Differential requirements for neurogenin 3 in the development of POMC and NPY neurons in the hypothalamus

The neuroendocrine hypothalamus regulates a spectrum of essential biological processes and underlies a range of diseases from growth failure to obesity. While the exploration of hypothalamic function has progressed well, knowledge of hypothalamic development is poor. In particular, very little is known about the processes underlying the genesis and specification of the neurons in the arcuate and ventromedial nuclei. Recent studies demonstrate that the proneural basic helix-loop-helix transcription factor Mash1 is required for neurogenesis and neuronal subtype specification in the ventral hypothalamus. We demonstrate here that Ngn3, another basic helix-loop-helix transcription factor, is expressed in mitotic progenitors in the arcuate and ventromedial hypothalamic regions of mouse embryos from embryonic days 9.5–17.5. Genetic fate mapping and loss of function studies in mice demonstrate that Ngn3+ progenitors contribute to subsets of POMC, NPY, TH and SF1 neurons and is required for the specification of these neuronal subtypes in the ventral hypothalamus. Interestingly, while Ngn3 promotes the development of arcuate POMC and ventromedial SF1 neurons, it inhibits the development of NPY and TH neurons in the arcuate nuclei. Given the opposing roles of POMC and NPY neurons in regulating food intake, these results indicate that Ngn3 plays a central role in the generation of neuronal populations controlling energy homeostasis in mice.

From progenitor to unique neuron: Neuronal sub-type specification by the integration of positional and temporal cues

From progenitor to unique neuron: Neuronal sub-type specification by the integration of positional and temporal cues

The Transcription Factor Pax6 Regulates Survival of Dopaminergic Olfactory Bulb Neurons via Crystallin αA

Most neurons in the adult mammalian brain survive for the entire life of an individual. However, it is not known which transcriptional pathways regulate this survival in a healthy brain. Here, we identify a pathway regulating neuronal survival in a highly subtype-specific manner. We show that the transcription factor Pax6 expressed in dopaminergic neurons of the olfactory bulb regulates the survival of these neurons by directly controlling the expression of crystallin αA (CryαA), which blocks apoptosis by inhibition of procaspase-3 activation. Re-expression of CryαA fully rescues survival of Pax6-deficient dopaminergic interneurons invivo and knockdown of CryαA by shRNA in wild-type mice reduces the number of dopaminergic OB interneurons. Strikingly, Pax6 utilizes different DNA-binding domains for its well-known role in fate specification and this role of regulating the survival of specific neuronal subtypes in the mature, healthy brain.

The role of the primary visual cortex in perceptual suppression of salient visual stimuli.

The role of primary visual cortex (area V1) in subjective perception has intrigued students of vision for decades. Specifically, the extent to which the activity of different types of cells (monocular versus binocular) and electrophysiological signals (i.e., local field potentials versus spiking activity) reflect perception is still debated. To address these questions we recorded from area V1 of the macaque using tetrodes during the paradigm of binocular flash suppression, where incongruent images presented dichoptically compete for perceptual dominance. We found that the activity of a minority (20%) of neurons reflect the perceived visual stimulus and these cells exhibited perceptual modulations substantially weaker compared with their sensory modulation induced by congruent stimuli. Importantly, perceptual modulations were found equally often for monocular and binocular cells, demonstrating that perceptual competition in V1 involves mechanisms across both types of neurons. The power of the local field potential (LFP) also showed moderate perceptual modulations with similar percentages of sites showing significant effects across frequency bands (18-22%). The possibility remains that perception may be strongly reflected in more elaborate aspects of activity in V1 circuits (e.g., specific neuronal subtypes) or perceptual states might have a modulatory role on more intricate aspects of V1 firing patterns (e.g., synchronization), not necessarily altering the firing rates of single cells or the LFP power dramatically.

Functional Diversity of ESC-Derived Motor Neuron Subtypes Revealed through Intraspinal Transplantation

Cultured ESCs can form different classes of neurons, but whether these neurons can acquire specialized subtype features typical of neurons in vivo remains unclear. We show here that mouse ESCs can be directed to form highly specific motor neuron subtypes in the absence of added factors, through a differentiation program that relies on endogenous Wnts, FGFs, and Hh-mimicking the normal program of motor neuron subtype differentiation. Molecular markers that characterize motor neuron subtypes anticipate the functional properties of these neurons in vivo: ESC-derived motor neurons grafted isochronically into chick spinal cord settle in appropriate columnar domains and select axonal trajectories with a fidelity that matches that of their in vivo generated counterparts. ESC-derived motor neurons can therefore be programmed in a predictive manner to acquire molecular and functional properties that characterize one of the many dozens of specialized motor neuron subtypes that exist in vivo.

Imaging-based chemical screens using normal and glioma-derived neural stem cells

The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.

Autonomous firing of low-threshold spike interneurons in the striatum

Specific striatal neuronal subtypes are classified as either phasically or tonically active. Of the four major neuronal subtypes of the striatum, the firing patterns of three have been studied in detail. Spiny projection neurons and fast-spiking interneurons fire phasically in response to synaptic input and are silent when input is absent. Cholinergic (ACh) interneurons are tonically active even in the absence of synaptic input. These ACh interneurons were the only known tonically active neurons in the striatum until recently. We have found that the low-threshold spike (LTS) interneurons also belong to this classification of striatal tonically active neurons, presenting a new aspect to the striatal oscillatory network. Striatal LTS interneurons are GABAergic interneurons that also contain neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and somatostatin. We used transgenic mice expressing green fluorescent protein (GFP) under control of the nNOS or NPY promoters to help identify LTS interneurons. GFP positive neurons were examined using whole-cell and cell-attached recording techniques to investigate firing patterns and possible mechanisms of such activity. GFP positive neurons displayed the morphological properties characteristic of LTS interneurons, and fired strong rebound bursts at the offset of hyperpolarizing current pulses. LTS interneurons were spontaneously active and this activity persisted in the presence of synaptic blockers. When compared with ACh interneurons, LTS interneurons fired at higher rates and with less regularity. Similar to ACh interneurons, LTS interneurons were capable of firing in a variety of firing patterns, including regular single spiking, irregular single spiking, and bursting combined with periods of regular and irregular single spiking. LTS interneurons were observed spontaneously shifting between these firing patterns. Whole-cell voltage clamp recordings revealed a tetrodotoxin-sensitive region of negative slope conductance in the subthreshold range consistent with a persistent sodium current. An absence of a zero current crossing in the I-V curve combined with a linear F-I curve suggest these interneurons possess a Hodgkin’s class 1 oscillation mechanism. Tonically active neurons in the striatum have been shown to become strongly synchronized with the beta oscillation seen in Parkinson’s disease. These strongly synchronized neurons may include LTS interneurons. In addition, both ACh and LTS interneurons are capable of endogenous oscillations in the beta frequency range. Our findings place LTS interneurons in the subtype of tonically active neurons in the striatum and in turn change our understanding of how synchronization may occur in the striatum.