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The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells.
Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2015; 35(33):11462-11481. DOI:10.1523/JNEUROSCI.3046-14.2015 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The thalamus is thought to be crucially involved in the anesthetic state. Here, we investigated the effect of the inhaled anesthetic xenon on stimulus-evoked thalamocortical network activity and on excitability of thalamocortical neurons. Because hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are key regulators of neuronal excitability in the thalamus, the effect of xenon on HCN channels was examined.
The effects of xenon on thalamocortical network activity were investigated in acutely prepared brain slices from adult wild-type and HCN2 knockout mice by means of voltage-sensitive dye imaging. The influence of xenon on single-cell excitability in brain slices was investigated using the whole-cell patch-clamp technique. Effects of xenon on HCN channels were verified in human embryonic kidney cells expressing HCN2 channels.
Xenon concentration-dependently diminished thalamocortical signal propagation. In neurons, xenon reduced HCN channel-mediated Ih current amplitude by 33.4 ± 12.2% (at -133 mV; n = 7; P = 0.041) and caused a left-shift in the voltage of half-maximum activation (V1/2) from -98.8 ± 1.6 to -108.0 ± 4.2 mV (n = 8; P = 0.035). Similar effects were seen in human embryonic kidney cells. The impairment of HCN channel function was negligible when intracellular cyclic adenosine monophosphate level was increased. Using HCN2 mice, we could demonstrate that xenon did neither attenuate in vitro thalamocortical signal propagation nor did it show sedating effects in vivo.
Here, we clearly showed that xenon impairs HCN2 channel function, and this impairment is dependent on intracellular cyclic adenosine monophosphate levels. We provide evidence that this effect reduces thalamocortical signal propagation and probably contributes to the hypnotic properties of xenon.
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.
[Show abstract][Hide abstract] ABSTRACT: In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-β (IFN-β) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-β ( IFN-α/β) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
[Show abstract][Hide abstract] ABSTRACT: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I(h), which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.
We investigated the effects of Kcne2 gene deletion on I(h) properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2(+/+) and Kcne2(-/-) mice. Kcne2 deletion shifted the voltage-dependence of I(h) activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I(h) density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2(-/-) neurons.
Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.
PLoS ONE 08/2012; 7(8):e42756. DOI:10.1371/journal.pone.0042756 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, I(h), which regulates neuronal excitability and oscillatory activity in the brain. Although all four HCN isoforms are expressed in the brain, the functional contribution of HCN3 is unknown. Using immunohistochemistry, confocal microscopy, and whole-cell patch-clamp recording techniques, we investigated HCN3 function in thalamic intergeniculate leaflet (IGL) neurons, as HCN3 is reportedly preferentially expressed in these cells. We observed that I(h) recorded from IGL, but not ventral geniculate nucleus, neurons in HCN2(+/+) mice and rats activated slowly and were cAMP insensitive, which are hallmarks of HCN3 channels. We also observed strong immunolabeling for HCN3, with no labeling for HCN1 and HCN4, and only very weak labeling for HCN2. Deletion of HCN2 did not alter I(h) characteristics in mouse IGL neurons. These data together indicate that the HCN3 channel isoform generated I(h) in IGL neurons. Intracellular phosphatidylinositol-4,5-bisphosphate (PIP(2)) shifted I(h) activation to more depolarized potentials and accelerated activation kinetics. Upregulation of HCN3 function by PIP(2) augmented low-threshold burst firing and spontaneous oscillations; conversely, depletion of PIP(2) or pharmacologic block of I(h) resulted in a profound inhibition of excitability. The results indicate that functional expression of HCN3 channels in IGL neurons is crucial for intrinsic excitability and rhythmic burst firing, and PIP(2) serves as a powerful modulator of I(h)-dependent properties via an effect on HCN3 channel gating. Since the IGL is a major input to the suprachiasmatic nucleus, regulation of pacemaking function by PIP(2) in the IGL may influence sleep and circadian rhythms.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2011; 31(28):10412-23. DOI:10.1523/JNEUROSCI.0021-11.2011 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GABAergic neurons in the reticular thalamic nucleus (RTN) synapse onto thalamocortical neurons in the ventrobasal (VB) thalamus, and this reticulo-thalamocortical pathway is considered an anatomic target for general anesthetic-induced unconsciousness. A mutant mouse was engineered to harbor two amino acid substitutions (S270H, L277A) in the GABA(A) receptor (GABA(A)-R) alpha1 subunit; this mutation abolished sensitivity to the volatile anesthetic isoflurane in recombinant GABA(A)-Rs, and reduced in vivo sensitivity to isoflurane in the loss-of-righting-reflex assay. We examined the effects of the double mutation on GABA(A)-R-mediated synaptic currents and isoflurane sensitivity by recording from thalamic neurons in brain slices. The double mutation accelerated the decay, and decreased the (1/2) width of, evoked inhibitory postsynaptic currents (eIPSCs) in VB neurons and attenuated isoflurane-induced prolongation of the eIPSC. The hypnotic zolpidem, a selective modulator of GABA(A)-Rs containing the alpha1 subunit, prolonged eIPSC duration regardless of genotype, indicating that mutant mice incorporate alpha1 subunit-containing GABA(A)-Rs into synapses. In RTN neurons, which lack the alpha1 subunit, eIPSC duration was longer than in VB, regardless of genotype. Isoflurane reduced the efficacy of GABAergic transmission from RTN to VB, independent of genotype, suggesting a presynaptic action in RTN neurons. Consistent with this observation, isoflurane inhibited both tonic action potential and rebound burst firing in the presence of GABA(A)-R blockade. The suppressed excitability in RTN neurons is likely mediated by isoflurane-enhanced Ba(2+)-sensitive, but 4-aminopyridine-insenstive, potassium conductances. We conclude that isoflurane enhances inhibition of thalamic neurons in VB via GABA(A)-R-dependent, but in RTN via GABA(A)-R-independent, mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Hyperpolarization activated cyclic nucleotide (HCN) gated channels conduct a current, I(h); how I(h) influences excitability and spike firing depends primarily on channel distribution in subcellular compartments. For example, dendritic expression of HCN1 normalizes somatic voltage responses and spike output in hippocampal and cortical neurons. We reported previously that HCN2 is predominantly expressed in dendritic spines in reticular thalamic nucleus (RTN) neurons, but the functional impact of such nonsomatic HCN2 expression remains unknown. We examined the role of HCN2 expression in regulating RTN excitability and GABAergic output from RTN to thalamocortical relay neurons using wild-type and HCN2 knock-out mice. Pharmacological blockade of I(h) significantly increased spike firing in RTN neurons and large spontaneous IPSC frequency in relay neurons; conversely, pharmacological enhancement of HCN channel function decreased spontaneous IPSC frequency. HCN2 deletion abolished I(h) in RTN neurons and significantly decreased sensitivity to 8-bromo-cAMP and lamotrigine. Recapitulating the effects of I(h) block, HCN2 deletion increased both temporal summation of EPSPs in RTN neurons as well as GABAergic output to postsynaptic relay neurons. The enhanced excitability of RTN neurons after I(h) block required activation of ionotropic glutamate receptors; consistent with this was the colocalization of HCN2 and glutamate receptor 4 subunit immunoreactivities in dendritic spines of RTN neurons. The results indicate that, in mouse RTN neurons, HCN2 is the primary functional isoform underlying I(h) and expression of HCN2 constrains excitatory synaptic integration.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2007; 27(32):8719-32. DOI:10.1523/JNEUROSCI.1630-07.2007 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve gamma-aminobutyric acid (GABA)(A) receptors, other mechanisms may be involved. The hyperpolarization-activated cation current (I(h)) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on I(h)-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23-35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of I(h). In whole-cell voltage-clamp recordings, propofol suppressed the I(h) conductance and slowed the kinetics of activation. The block of I(h) by propofol was associated with decreased regularity and frequency of delta-oscillations in VB neurons. The principal source of the I(h) current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased I(h) and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), I(h) was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of I(h) may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of I(h).
European Journal of Neuroscience 02/2006; 23(2):465-80. DOI:10.1111/j.1460-9568.2005.04587.x · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The GABAergic reticular thalamic nucleus (RTN) is a major source of inhibition for thalamocortical neurons in the ventrobasal complex (VB). Thalamic circuits are thought to be an important anatomic target for general anesthetics. We investigated presynaptic actions of the intravenous anesthetic propofol in RTN neurons, using RTN-retained and RTN-removed brain slices. In RTN-retained slices, focal and bath application of propofol increased intrinsic excitability, temporal summation, and spike firing rate in RTN neurons. Propofol-induced activation was associated with suppression of medium afterhyperpolarization potentials. This activation was mimicked and completely occluded by the small conductance calcium-activated potassium (SK) channel blocker apamin, indicating that propofol could enhance RTN excitability by blocking SK channels. Propofol increased GABAergic transmission at RTN-VB synapses, consistent with excitation of presynaptic RTN neurons. Stimulation of RTN resulted in synaptic inhibition in postsynaptic neurons in VB, and this inhibition was potentiated by propofol in a concentration-dependent manner. Removal of RTN resulted in a dramatic reduction of both spontaneous postsynaptic inhibitory current frequency and propofol-mediated inhibition of VB neurons. Thus the existence and activation of RTN input were essential for propofol to elicit thalamocortical suppression; such suppression resulted from shunting through the postsynaptic GABA(A) receptor-mediated chloride conductance. The results indicate that propofol enhancement of RTN-mediated inhibitory input via blockade of SK channels may play a critical role in "gating" spike firing in thalamocortical relay neurons.
Journal of Neurophysiology 05/2005; 93(4):1935-48. DOI:10.1152/jn.01058.2004 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 - 3 microM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.
[Show abstract][Hide abstract] ABSTRACT: GABA(A) receptors (GABA(A)-Rs) are pentameric structures consisting of two alpha, two beta, and one gamma subunit. The alpha subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the alpha1 subunit to native GABA(A)-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and alpha1 subunit knock-out (alpha1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from alpha1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the alpha1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from alpha1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the alpha1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the alpha1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.
Journal of Neurophysiology 01/2003; 88(6):3208-17. DOI:10.1152/jn.00885.2001 · 2.89 Impact Factor