Correction: GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

[This corrects the article DOI: 10.1371/journal.pbio.3002483.].

GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at subthreshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical, and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. In perforated patch recordings, activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in both juvenile and adult SPNs. Transcriptomic analysis and pharmacological work suggested that this relatively positive GABAAR reversal potential was not attributable to NKCC1 expression, but rather to HCO3- permeability. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to dendritic iGluR signaling and promoted dendritic spikes. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation for the role of intrastriatal GABAergic circuits.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔµH+) across the inner membrane. Although several drugs can modulate ΔµH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic ß-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Epileptogenic Q555X SYN1 mutant triggers imbalances in release dynamics and short-term plasticity.

Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.

Ca2+-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.

Ca 2+ -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

A signaling loop of REST, TSC2 and ß-catenin governs proliferation and function of PC12 neural cells.

The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-ß-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active ß-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

'Rejuvenation' protects neurons in mouse models of Parkinson's disease.

Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Ca(v)1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Ca(v)1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

State-dependent GABAergic regulation of striatal spiny projection neuron excitability.

Synaptic transmission mediated by GABA A receptors (GABA A Rs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABA A Rs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABA A Rs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell- type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser- scanning microscopy of genetically encoded sensors for bioenergetic and redox status. Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. Conclusions: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

An efficient rAAV vector for protein expression in cortical parvalbumin expressing interneurons.

Recombinant adeno-associated viruses (rAAV) are extensively used in both research and clinical applications. Despite significant advances, there is a lack of short promoters able to drive the expression of virus delivered genes in specific classes of neurons. We designed an efficient rAAV vector suitable for the rAAV-mediated gene expression in cortical interneurons, mainly in the parvalbumin expressing cells. The vector includes a short parvalbumin promoter and a specialized poly(A) sequence. The degree of conservation of the parvalbumin gene adjoining non-coding regions was used in both the promoter design and the selection of the poly(A) sequence. The specificity was established by co-localizing the fluorescence of the virus delivered eGFP and the antibody for a neuronal marker. rAAV particles were injected in the visual cortex area V1/V2 of adult rats (2-4 months old). Neurons expressing the virus delivered eGFP were mainly positive for interneuronal markers: 66.5 ± 2.8% for parvalbumin, 14.6 ± 2.4% for somatostatin, 7.1 ± 1.2% for vasoactive intestinal peptide, 2.8 ± 0.6% for cholecystokinin. Meanwhile, only 2.1 ± 0.5% were positive for CaMKII, a marker for principal cells in the cortex. The efficiency of the construct was verified by optogenetic experiments: the expression of the virus delivered ChR2 channels was sufficient to evoke by blue light laser high frequency bursts of action potentials in putative fast spiking neurons. We conclude that our promoter allows highly specific expression of the rAAV delivered cDNAs in cortical interneurons with a strong preference for the parvalbumin positive cells.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Selective participation of somatodendritic HCN channels in inhibitory but not excitatory synaptic integration in neurons of the subthalamic nucleus.

The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca(2+) imaging, and computational modeling revealed that HCN channels are activated by GABA(A) receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca(2+) channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABA(A) receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing.

Cholinergic modulation of Kir2 channels selectively elevates dendritic excitability in striatopallidal neurons.

Dopamine-depleting lesions of the striatum that mimic Parkinson's disease induce a profound pruning of spines and glutamatergic synapses in striatopallidal medium spiny neurons, leaving striatonigral medium spiny neurons intact. The mechanisms that underlie this cell type-specific loss of connectivity are poorly understood. The Kir2 K(+) channel is an important determinant of dendritic excitability in these cells. Here we show that opening of these channels is potently reduced by signaling through M1 muscarinic receptors in striatopallidal neurons, but not in striatonigral neurons. This asymmetry could be attributed to differences in the subunit composition of Kir2 channels. Dopamine depletion alters the subunit composition further, rendering Kir2 channels in striatopallidal neurons even more susceptible to modulation. Reduced opening of Kir2 channels enhances dendritic excitability and synaptic integration. This cell type-specific enhancement of dendritic excitability is an essential trigger for synaptic pruning after dopamine depletion, as pruning was prevented by genetic deletion of M1 muscarinic receptors.

Dopaminergic control of corticostriatal long-term synaptic depression in medium spiny neurons is mediated by cholinergic interneurons.

Long-term depression (LTD) of the synapse formed between cortical pyramidal neurons and striatal medium spiny neurons is central to many theories of motor plasticity and associative learning. The induction of LTD at this synapse is thought to depend upon D(2) dopamine receptors localized in the postsynaptic membrane. If this were true, LTD should be inducible in neurons from only one of the two projection systems of the striatum. Using transgenic mice in which neurons that contribute to these two systems are labeled, we show that this is not the case. Rather, in both cell types, the D(2) receptor dependence of LTD induction reflects the need to lower M(1) muscarinic receptor activity-a goal accomplished by D(2) receptors on cholinergic interneurons. In addition to reconciling discordant tracts of the striatal literature, these findings point to cholinergic interneurons as key mediators of dopamine-dependent striatal plasticity and learning.

RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion.

Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.

Neurite-Enriched MicroRNA-218 Stimulates Translation of the GluA2 Subunit and Increases Excitatory Synaptic Strength.

Local control of protein translation is a fundamental process for the regulation of synaptic plasticity. It has been demonstrated that the local protein synthesis occurring in axons and dendrites can be shaped by numerous mechanisms, including miRNA-mediated regulation. However, several aspects underlying this regulatory process have not been elucidated yet. Here, we analyze the differential miRNA profile in cell bodies and neurites of primary hippocampal neurons and find an enrichment of the precursor and mature forms of miR-218 in the neuritic projections. We show that miR-218 abundance is regulated during hippocampal development and by chronic silencing or activation of neuronal network. Overexpression and knockdown of miR-218 demonstrated that miR-218 targets the mRNA encoding the GluA2 subunit of AMPA receptors and modulates its expression. At the functional level, miR-218 overexpression increases glutamatergic synaptic transmission at both single neuron and network levels. Our data demonstrate that miR-218 may play a key role in the regulation of AMPA-mediated excitatory transmission and in the homeostatic regulation of synaptic plasticity.