Shui-Wang Ying

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

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.

Propofol block of Ih contributes to the suppression of neuronal excitability and rhythmic burst firing in thalamocortical neurons

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve γ‐aminobutyric acid (GABA)A receptors, other mechanisms may be involved. The hyperpolarization‐activated cation current (Ih) 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 Ih‐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 Ih. In whole‐cell voltage‐clamp recordings, propofol suppressed the Ih conductance and slowed the kinetics of activation. The block of Ih by propofol was associated with decreased regularity and frequency of δ‐oscillations in VB neurons. The principal source of the Ih 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 Ih 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), Ih 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 Ih may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer‐term effects represent a novel form of propofol‐mediated regulation of Ih.

Dendritic HCN2 Channels Constrain Glutamate-Driven Excitability in Reticular Thalamic Neurons

Hyperpolarization activated cyclic nucleotide (HCN) gated channels conduct a current, Ih; how Ih 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 Ih 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 Ih in RTN neurons and significantly decreased sensitivity to 8-bromo-cAMP and lamotrigine. Recapitulating the effects of Ih 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 Ih 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 Ih and expression of HCN2 constrains excitatory synaptic integration.

Compartmental distribution of hyperpolarization-activated cyclic-nucleotide-gated channel 2 and hyperpolarization-activated cyclic-nucleotide-gated channel 4 in thalamic reticular and thalamocortical relay neurons

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels conduct a monovalent cationic current, I(h), which contributes to the electrophysiological properties of neurons and regulates thalamic oscillations in circuits containing the glutamatergic ventrobasal complex (VB) and GABAergic reticular thalamic nucleus (RTN). Four distinct HCN channel isoforms (HCN1-4) have been identified, and mRNAs and proteins for HCN channels have been detected in the RTN and VB, with HCN2 and HCN4 being the predominant isoforms. RTN and VB neurons have distinct electrophysiological properties, and those differences may reflect variable compartmental distribution of HCN channels. Whole cell patch clamp recordings from thalamic neurons in brain slices obtained from C57/Bl6 mice demonstrate that I(h) is much smaller in RTN than in VB neurons although the time constants for I(h) current activation are very similar. To study the compartmental distribution of the underlying channels, we performed qualitative and quantitative examination of HCN2 and HCN4 expression using fluorescent immunohistochemistry and confocal microscopy. HCN2-immunoreactivity (IR) on the somata of RTN neurons was approximately 10-fold less than that seen in VB neurons while HCN4-IR was detected on the somata of RTN and VB neurons to an equal degree. HCN2-IR in RTN and VB did not overlap with synaptophysin-IR, but strongly colocalized with cortactin-IR, indicating that HCN2 was not present in axon terminals but was present in dendritic spines. Although HCN2-IR in spines was more pronounced in VB than in RTN, the ratio of spinous to somatic expression in RTN was dramatically higher than that in VB, strongly suggesting that HCN2-IR in RTN is principally located in sites distal to the soma. In contrast, HCN4-IR did not colocalize with either synaptophysin or cortactin. The colocalization of HCN2-IR with HCN4-IR was greater in VB than in RTN. The results suggest that the distinct compartmental distribution of HCN2 channels in RTN and VB neurons contributes to the profound differences in the I(h)-dependent properties of these cells.

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABAA receptor chloride channels

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 μM) 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.

A gain-of-function mutation in the GABAA receptor produces synaptic and behavioral abnormalities in the mouse

In mammalian species, inhibition in the brain is mediated predominantly by the activation of GABAA receptors. We report here changes in inhibitory synaptic function and behavior in a mouse line harboring a gain‐of‐function mutation at Serine 270 (S270) in the GABAA receptor α1 subunit. In recombinant α1β2γ2 receptors, replacement of S270 by Histidine (H) results in an increase in sensitivity to γ‐aminobutyric acid (GABA), and slowing of deactivation following transient activation by saturating concentrations of GABA. Heterozygous mice expressing the S270H mutation are hyper‐responsive to human contact, exhibit intention tremor, smaller body size and reduced viability. These mice also displayed reduced motor coordination, were hypoactive in the home cage, but paradoxically were hyperactive in a novel open field environment. Heterozygous knockin mice of both sexes were fertile but females failed to care for offspring. This deficit in maternal behavior prevented production of homozygous animals. Recordings from brain slices prepared from these animals revealed a substantial prolongation of miniature inhibitory postsynaptic currents (IPSCs) and a loss of sensitivity to the anesthetic isoflurane, in neurons that express a substantial amount of the α1 subunit. The results suggest that the biophysical properties of GABAA receptors are important in determining the time‐course of inhibition in vivo, and suggest that the duration of synaptic inhibition is a critical determinant that influences a variety of behaviors in the mouse.

PIP2-Mediated HCN3 Channel Gating Is Crucial for Rhythmic Burst Firing in Thalamic Intergeniculate Leaflet Neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, Ih, 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 Ih 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 Ih characteristics in mouse IGL neurons. These data together indicate that the HCN3 channel isoform generated Ih in IGL neurons. Intracellular phosphatidylinositol-4,5-bisphosphate (PIP2) shifted Ih activation to more depolarized potentials and accelerated activation kinetics. Upregulation of HCN3 function by PIP2 augmented low-threshold burst firing and spontaneous oscillations; conversely, depletion of PIP2 or pharmacologic block of Ih 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 PIP2 serves as a powerful modulator of Ih-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 PIP2 in the IGL may influence sleep and circadian rhythms.

Targeted deletion of Kcne2 impairs HCN channel function in mouse thalamocortical circuits.

Background Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, Ih, 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. Methodology/Principal Findings We investigated the effects of Kcne2 gene deletion on Ih 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 Ih activation to more hyperpolarized potentials, slowed gating kinetics, and decreased Ih 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. Conclusions/Significance 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.

Isoflurane modulates excitability in the mouse thalamus via GABA-dependent and GABA-independent mechanisms

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.

Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

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. SIGNIFICANCE STATEMENT 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.