Dopamine and glutamate regulate striatal acetylcholine in decision-making.

Striatal dopamine and acetylcholine are essential for the selection and reinforcement of motor actions and decision-making1. In vitro studies have revealed an intrastriatal circuit in which acetylcholine, released by cholinergic interneurons (CINs), drives the release of dopamine, and dopamine, in turn, inhibits the activity of CINs through dopamine D2 receptors (D2Rs). Whether and how this circuit contributes to striatal function in vivo is largely unknown. Here, to define the role of this circuit in a living system, we monitored acetylcholine and dopamine signals in the ventrolateral striatum of mice performing a reward-based decision-making task. We establish that dopamine and acetylcholine exhibit multiphasic and anticorrelated transients that are modulated by decision history and reward outcome. Dopamine dynamics and reward encoding do not require the release of acetylcholine by CINs. However, dopamine inhibits acetylcholine transients in a D2R-dependent manner, and loss of this regulation impairs decision-making. To determine how other striatal inputs shape acetylcholine signals, we assessed the contribution of cortical and thalamic projections, and found that glutamate release from both sources is required for acetylcholine release. Altogether, we uncover a dynamic relationship between dopamine and acetylcholine during decision-making, and reveal multiple modes of CIN regulation. These findings deepen our understanding of the neurochemical basis of decision-making and behaviour.

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Reversing a model of Parkinson's disease with in situ converted nigral neurons.

Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2. Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson's disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.

Dopamine Deficiency Reduces Striatal Cholinergic Interneuron Function in Models of Parkinson's Disease.

Motor and cognitive functions depend on the coordinated interactions between dopamine (DA) and acetylcholine (ACh) at striatal synapses. Increased ACh availability was assumed to accompany DA deficiency based on the outcome of pharmacological treatments and measurements in animals that were critically depleted of DA. Using Slc6a3DTR/+ diphtheria-toxin-sensitive mice, we demonstrate that a progressive and L-dopa-responsive DA deficiency reduces ACh availability and the transcription of hyperpolarization-activated cation (HCN) channels that encode the spike timing of ACh-releasing tonically active striatal interneurons (ChIs). Although the production and release of ACh and DA are reduced, the preponderance of ACh over DA contributes to the motor deficit. The increase in striatal ACh relative to DA is heightened via D1-type DA receptors that activate ChIs in response to DA release from residual axons. These results suggest that stabilizing the expression of HCN channels may improve ACh-DA reciprocity and motor function in Parkinson's disease (PD). VIDEO ABSTRACT.

Chemogenetic Targeting of Dorsomedial Direct-pathway Striatal Projection Neurons Selectively Elicits Rotational Behavior in Mice.

The striatum of the basal ganglia is pivotal for voluntary movements and is implicated in debilitating movement disorders such as Parkinsonism and dystonia. Striatum projects to downstream nuclei through direct (dSPN) and indirect (iSPN) pathway projection neurons thought to exert opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation induces ipsiversive rotational behavior. The dSPNs of the dorsal striatum are believed to engage distinct motor programs but underlying mechanisms remain unclear. Here, we show by employing chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] that unilateral inhibition of dorsomedial dSPNs is sufficient to selectively impair contraversive movement and elicit ipsiversive rotational behavior in mice. Adeno-associated virus (AAV) encoding Cre-dependent Gi-coupled DREADD was injected unilaterally into the dorsomedial striatum of Drd1-Cre mice, resulting in expression of the modified human M4 muscarinic receptor (hM4Di) in ∼20% of dorsostriatal dSPNs. Upon hM4Di activation, a striking positive linear correlation was found between turn ratio and viral expression, which corroborates a relationship between unilateral inhibition of dorsomedial dSPNs and rotational behavior. Bursts of ipsiversive rotations were interspersed with normal ambulation. However, partial unilateral inhibition of ∼20% of dorsostriatal dSPNs did not affect horizontal and vertical locomotion or forelimb use preference. Overall, our results substantiate a unique role of dSPNs in promoting response bias in rotational behavior and show this to be a highly sensitive measure of dSPN performance.

Differences in Nicotine Encoding Dopamine Release between the Striatum and Shell Portion of the Nucleus Accumbens.

The aim of this work was to determine the effect of nicotine desensitization on dopamine (DA) release in the dorsal striatum and shell of the nucleus accumbens (NAc) from brain slices. In vitro fast-scan cyclic voltammetry analysis was used to evaluate dopamine release in the dorsal striatum and the NAc shell of Sprague-Dawley rats after infusion of nicotine, a nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (Mec), and an α4ß2 cholinergic receptor antagonist (DHße). DA release related to nicotine desensitization in the striatum and NAc shell was compared. In both structures, tonic release was suppressed by inhibition of the nicotine receptor (via Mec) and the α4ß2 receptor (via DHße). Paired-pulse ratio (PPR) was facilitated in both structures after nicotine and Mec infusion, and this facilitation was suppressed by increasing the stimulation interval. After variable frequency stimulation (simulating phasic burst), nicotine infusion induced significant augmentation of DA release in the striatum that was not seen in the absence of nicotine. In contrast, nicotine reduced phasic DA release in NAc, although frequency augmentation was seen both with and without nicotine. Evaluation of DA release evoked by various trains (high-frequency stimulation (HFS) 100 Hz) of high-frequency stimulation revealed significant enhancement after a train of three or more pulses in the striatum and NAc. The concentration differences between tonic and phasic release related to nicotine desensitization were more pronounced in the NAc shell. Nicotine desensitization is associated with suppression of tonic release of DA in both the striatum and NAc shell that may occur via the α4ß2 subtype of nAChR, whereas phasic frequency-dependent augmentation and HFS-related gating release is more pronounced in the striatum than in the NAc shell. Differences between phasic and tonic release associated with nicotine desensitization may underlie processing of reward signals in the NAc shell, and this may have major implications for addictive behavior.

Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.

Exosomal miR-9 Released from HIV Tat Stimulated Astrocytes Mediates Microglial Migration.

Chronic neuroinflammation still remains a common underlying feature of HIV-infected patients on combined anti-retroviral therapy (cART). Previous studies have reported that despite near complete suppression of virus replication by cART, cytotoxic viral proteins such as HIV trans-activating regulatory protein (Tat) continue to persist in tissues such as the brain and the lymph nodes, thereby contributing, in part, to chronic glial activation observed in HIV-associated neurological disorders (HAND). Understanding how the glial cells cross talk to mediate neuropathology is thus of paramount importance. MicroRNAs (miR) also known as regulators of gene expression, have emerged as key paracrine signaling mediators that regulate disease pathogenesis and cellular crosstalk, through their transfer via the extracellular vesicles (EV). In the current study we have identified a novel function of miR-9, that of mediating microglial migration. We demonstrate that miR-9 released from Tat-stimulated astrocytes can be taken up by microglia resulting in their migratory phenotype. Exposure of human astrocytoma (A172) cells to HIV Tat resulted in induction and release of miR-9 in the EVs, which, was taken up by microglia, leading in turn, increased migration of the latter cells, a process that could be blocked by both an exosome inhibitor GW4869 or a specific target protector of miR-9. Furthermore, it was also demonstrated that EV miR-9 mediated inhibition of the expression of target PTEN, via its binding to the 3'UTR seed sequence of the PTEN mRNA, was critical for microglial migration. To validate the role of miR-9 in this process, microglial cells were treated with EVs loaded with miR-9, which resulted in significant downregulation of PTEN expression with a concomitant increase in microglial migration. These findings were corroborated by transfecting microglia with a specific target protector of PTEN, that blocked miR-9-mediated downregulation of PTEN as well as microglial migration. In vivo studies wherein the miR-9 precursor-transduced microglia were transplanted into the striatum of mice, followed by assessing their migration in response to a stimulus administered distally, further validated the role of miR-9 in mediating microglial migration. Collectively, our findings provide evidence that glial crosstalk via miRs released from EVs play a vital role in mediating disease pathogenesis and could provide new avenues for development of novel therapeutic strategies aimed at dampening neuropathogenesis.

Pramipexole reduces soluble mutant huntingtin and protects striatal neurons through dopamine D3 receptors in a genetic model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.

Regulation of Striatal Neuron Activity by Cyclic Nucleotide Signaling and Phosphodiesterase Inhibition: Implications for the Treatment of Parkinson's Disease.

Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the hydrolysis and inactivation of cyclic nucleotides (cAMP/cGMP) in the brain. Several classes of PDE enzymes with distinct tissue distributions, cyclic nucleotide selectivity, and regulatory factors are highly expressed in brain regions subserving cognitive and motor processes known to be disrupted in neurodegenerative diseases such as Parkinson's disease (PD). Furthermore, small-molecule inhibitors of several different PDE family members alter cyclic nucleotide levels and favorably enhance motor performance and cognition in animal disease models. This chapter will explore the roles and therapeutic potential of non-selective and selective PDE inhibitors on neural processing in fronto-striatal circuits in normal animals and models of DOPA-induced dyskinesias (LIDs) associated with PD. The impact of selective PDE inhibitors and augmentation of cAMP and cGMP signaling on the membrane excitability of striatal medium-sized spiny projection neurons (MSNs) will be discussed. The effects of cyclic nucleotide signaling and PDE inhibitors on synaptic plasticity of striatonigral and striatopallidal MSNs will be also be reviewed. New data on the efficacy of PDE10A inhibitors for reversing behavioral and electrophysiological correlates of L-DOPA-induced dyskinesias in a rat model of PD will also be presented. Together, these data will highlight the potential of novel PDE inhibitors for treatment of movement disorders such as PD which are associated with abnormal corticostriatal transmission.

Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

Cholinergic Interneurons Use Orbitofrontal Input to Track Beliefs about Current State.

UNLABELLED: When conditions change, organisms need to learn about the changed conditions without interfering with what they already know. To do so, they can assign the new learning to a new "state" and the old learning to a previous state. This state assignment is fundamental to behavioral flexibility. Cholinergic interneurons (CINs) in the dorsomedial striatum (DMS) are necessary for associative information to be compartmentalized in this way, but the mechanism by which they do so is unknown. Here we addressed this question by recording putative CINs from the DMS in rats performing a task consisting of a series of trial blocks, or states, that required the recall and application of contradictory associative information. We found that individual CINs in the DMS represented the current state throughout each trial. These state correlates were not observed in dorsolateral striatal CINs recorded in the same rats. Notably, DMS CIN ensembles tracked rats' beliefs about the current state such that, when states were miscoded, rats tended to make suboptimal choices reflecting the miscoding. State information held by the DMS CINs also depended completely on the orbitofrontal cortex, an area that has been proposed to signal environmental states. These results suggest that CINs set the stage for recalling associative information relevant to the current environment by maintaining a real-time representation of the current state. Such a role has novel implications for understanding the neural basis of a variety of psychiatric diseases, such as addiction or anxiety disorders, in which patients generalize inappropriately (or fail to generalize) between different environments. SIGNIFICANCE STATEMENT: Striatal cholinergic interneurons (CINs) are thought to be identical to tonically active neurons. These neurons have long been thought to have an important influence on striatal processing during reward-related learning. Recently, a more specific function for striatal CINs has been suggested, which is that they are necessary for striatal learning to be compartmentalized into different states as the state of the environment changes. Here we report that putative CINs appear to track rats' beliefs about which environmental state is current. We further show that this property of CINs depends on orbitofrontal cortex input and is correlated with choices made by rats. These findings could provide new insight into neuropsychiatric diseases that involve improper generalization between different contexts.

Functional Indicators of Glutamate Transport in Single Striatal Astrocytes and the Influence of Kir4.1 in Normal and Huntington Mice.

UNLABELLED: This study evaluates single-cell indicators of glutamate transport in sulforhodamine 101-positive astrocytes of Q175 mice, a knock-in model of Huntington's disease (HD). Transport-related fluorescent ratio signals obtained with sodium-binding benzofuran isophtalate (SBFI) AM from unperturbed or voltage-clamped astrocytes and respective glutamate transporter currents (GTCs) were induced by photolytic or synaptic glutamate release and isolated pharmacologically. The HD-induced deficit ranged from -27% (GTC maximum at -100 mV in Ba(2+)) to -41% (sodium transients in astrocytes after loading SBFI-AM). Our specific aim was to clarify the mechanism(s) by which Kir4.1 channels can influence glutamate transport, as determined by either Na(+) imaging or transport-associated electrical signals. A decrease of Kir4.1 conductance was mimicked with Ba(2+) (200 µm), and an increase of Kir4.1 expression was obtained by intravenous administration of AAV9-gfaABC1D-Kir4.1-EGFP. The decrease of Kir4.1 conductance reduced the sodium transients but increased the amplitudes of somatic GTCs. Accordingly, after genetic upregulation of Kir4.1, somatic GTCs were found to be decreased. In individual cells, there was a negative correlation between Kir4.1 currents and GTCs. The relative effect of the Kir4.1 conductance was higher in the astrocyte periphery. These and other results suggest that the Kir4.1 conductance affects glutamate transporter activity in a dual manner: (1) by providing the driving force (voltage dependency of the transport itself) and (2) by limiting the lateral charge transfer (thereby reducing the interference with other electrogenic transporter functions). This leads to the testable prediction that restoring the high conductance state of passive astrocytes will not only normalize glutamate uptake but also restore other astrocytic transporter activities afflicted with HD. SIGNIFICANCE STATEMENT: Insufficiency of astrocytic glutamate uptake is a major element in the pathophysiology of neurodegenerative diseases. Considering the heterogeneity of astrocytes and their differential susceptibility to therapeutic interventions, it becomes necessary to evaluate the determinants of transport activity in individual astroglial cells. We have examined intracellular Na(+) transients and glutamate transporter currents as the most telling indicators of glutamate clearance after synaptic or photolytic release of glutamate in striatal slices. The results show that, in Huntington's disease, glutamate uptake activity critically depends on Kir4.1. These channels enable the high conductance state of the astrocytic plasma membrane, which ensures the driving force for glutamate transport and dumps the transport-associated depolarization along the astrocyte processes. This has significant implications for developing therapeutic targets.

Enhanced cocaine-induced locomotor sensitization and intrinsic excitability of NAc medium spiny neurons in adult but not in adolescent rats susceptible to diet-induced obesity.

RATIONALE: Basal and diet-induced differences in mesolimbic function, particularly within the nucleus accumbens (NAc), may contribute to human obesity; these differences may be more pronounced in susceptible populations. OBJECTIVES: We examined differences in cocaine-induced behavioral plasticity in rats that are susceptible vs. resistant to diet-induced obesity and basal differences in striatal neuron function in adult and in adolescent obesity-prone and obesity-resistant rats. METHODS: Susceptible and resistant outbred rats were identified based on "junk-food" diet-induced obesity. Then, the induction and expression of cocaine-induced locomotor sensitization, which is mediated by enhanced striatal function and is associated with increased motivation for rewards and reward-paired cues, were evaluated. Basal differences in mesolimbic function were examined in selectively bred obesity-prone and obesity-resistant rats (P70-80 and P30-40) using both cocaine-induced locomotion and whole-cell patch clamping approaches in NAc core medium spiny neurons (MSNs). RESULTS: In rats that became obese after eating junk-food, the expression of locomotor sensitization was enhanced compared to non-obese rats, with similarly strong responses to 7.5 and 15 mg/kg cocaine. Without diet manipulation, obesity-prone rats were hyper-responsive to the acute locomotor-activating effects of cocaine, and the intrinsic excitability of NAc core MSNs was enhanced by ∼60 % at positive and negative potentials. These differences were present in adult, but not adolescent rats. Post-synaptic glutamatergic transmission was similar between groups. CONCLUSIONS: Mesolimbic systems, particularly NAc MSNs, are hyper-responsive in obesity-prone individuals, and interactions between predisposition and experience influence neurobehavioral plasticity in ways that may promote weight gain and hamper weight loss in susceptible rats.

The neostriatum: two entities, one structure?

The striosome (or patch) was first identified with anatomical techniques as neurons organized in a three-dimensional labyrinth inserted in and interdigitating the rest of neostriatum: the matrix. Striosome and matrix rapidly became known as two neuronal compartments expressing different biochemical markers, embryonic development and afferent and efferent connectivity. In spite of extensive intrinsic neuronal axonal and dendritic extensions supposed to exchange information between matrix and striosomes, evidence suggested the presence of independent areas. Here, we report that indeed these two areas do not exchange synaptic information. We used genetic expression of channel rhodopsin 2 carried by adeno-associated virus serotype 10 (AAVrh10) that only expresses in neurons of the matrix compartment. Whole-cell patch-clamp recordings of matrix neurons activated by light pulses consistently produced inhibitory postsynaptic currents (IPSCs), but the same manipulation did not evoke IPSCs in striosome neurons. The matrix contains both direct and indirect striatal output pathways. By targeting striatal matrix expression of designer receptors exclusively activated by a designer drug (DREADD) hM3di carried by AAVrh10, we were able to inhibit the matrix neuronal compartment of the dorsolateral striatum during performance of a learned single-pellet reach-to-grasp task. As expected, inhibition of matrix neurons by systemic administration of DREADD agonist clozapine-n-oxide interfered with performance of the learned task.

DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons.

Environmental enrichment has multiple effects on behaviour, including modification of responses to psychostimulant drugs mediated by striatal neurons. However, the underlying molecular and cellular mechanisms are not known. Here we show that DARPP-32, a hub signalling protein in striatal neurons, interacts with adducins, which are cytoskeletal proteins that cap actin filaments' fast-growing ends and regulate synaptic stability. DARPP-32 binds to adducin MARCKS domain and this interaction is modulated by DARPP-32 Ser97 phosphorylation. Phospho-Thr75-DARPP-32 facilitates ß-adducin Ser713 phosphorylation through inhibition of a cAMP-dependent protein kinase/phosphatase-2A cascade. Caffeine or 24-h exposure to a novel enriched environment increases adducin phosphorylation in WT, but not T75A mutant mice. This cascade is implicated in the effects of brief exposure to novel enriched environment on dendritic spines in nucleus accumbens and cocaine locomotor response. Our results suggest a molecular pathway by which environmental changes may rapidly alter responsiveness of striatal neurons involved in the reward system.

KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons.

Striatal projection neurons (SPNs) process motor and cognitive information. Their activity is affected by Parkinson's disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit.

Activin A directs striatal projection neuron differentiation of human pluripotent stem cells.

The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells.

Striatal Transplantation of Human Dopaminergic Neurons Differentiated From Induced Pluripotent Stem Cells Derived From Umbilical Cord Blood Using Lentiviral Reprogramming.

Human induced pluripotent stem cells (hiPSCs) are promising sources for regenerative therapies like the replacement of dopaminergic neurons in Parkinson's disease. They offer an unlimited cell source that can be standardized and optimized to produce applicable cell populations to gain maximal functional recovery. In the present study, human cord blood-derived iPSCs (hCBiPSCs) were differentiated into dopaminergic neurons utilizing two different in vitro protocols for neural induction: (protocol I) by fibroblast growth factor (FGF-2) signaling, (protocol II) by bone morphogenetic protein (BMP)/transforming growth factor (TGF-ß) inhibition. After maturation, in vitro increased numbers of tyrosine hydroxylase (TH)-positive neurons (7.4% of total cells) were observed by protocol II compared to 3.5% in protocol I. Furthermore, 3 weeks after transplantation in hemiparkinsonian rats in vivo, a reduced number of undifferentiated proliferating cells was achieved with protocol II. In contrast, proliferation still occurred in protocol I-derived grafts, resulting in tumor-like growth in two out of four animals 3 weeks after transplantation. Protocol II, however, did not increase the number of TH(+) cells in the striatal grafts of hemiparkinsonian rats. In conclusion, BMP/TGF-ß inhibition was more effective than FGF-2 signaling with regard to dopaminergic induction of hCBiPSCs in vitro and prevented graft overgrowth in vivo.