Interhemispheric reactivity of the subthalamic nucleus sustains progressive dopamine neuron loss in asymmetrical parkinsonism.

Parkinson's disease (PD) is characterized by the progressive and asymmetrical degeneration of the nigrostriatal dopamine neurons and the unilateral presentation of the motor symptoms at onset, contralateral to the most impaired hemisphere. We previously developed a rat PD model that mimics these typical features, based on unilateral injection of a substrate inhibitor of excitatory amino acid transporters, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), in the substantia nigra (SN). Here, we used this progressive model in a multilevel study (behavioral testing, in vivo 1H-magnetic resonance spectroscopy, slice electrophysiology, immunocytochemistry and in situ hybridization) to characterize the functional changes occurring in the cortico-basal ganglia-cortical network in an evolving asymmetrical neurodegeneration context and their possible contribution to the cell death progression. We focused on the corticostriatal input and the subthalamic nucleus (STN), two glutamate components with major implications in PD pathophysiology. In the striatum, glutamate and glutamine levels increased from presymptomatic stages in the PDC-injected hemisphere only, which also showed enhanced glutamatergic transmission and loss of plasticity at corticostriatal synapses assessed at symptomatic stage. Surprisingly, the contralateral STN showed earlier and stronger reactivity than the ipsilateral side (increased intraneuronal cytochrome oxidase subunit I mRNA levels; enhanced glutamate and glutamine concentrations). Moreover, its lesion at early presymptomatic stage halted the ongoing neurodegeneration in the PDC-injected SN and prevented the expression of motor asymmetry. These findings reveal the existence of endogenous interhemispheric processes linking the primary injured SN and the contralateral STN that could sustain progressive dopamine neuron loss, opening new perspectives for disease-modifying treatment of PD.

Targeted Tshz3 deletion in corticostriatal circuit components segregates core autistic behaviors.

We previously linked TSHZ3 haploinsufficiency to autism spectrum disorder (ASD) and showed that embryonic or postnatal Tshz3 deletion in mice results in behavioral traits relevant to the two core domains of ASD, namely social interaction deficits and repetitive behaviors. Here, we provide evidence that cortical projection neurons (CPNs) and striatal cholinergic interneurons (SCINs) are two main and complementary players in the TSHZ3-linked ASD syndrome. In the cerebral cortex, TSHZ3 is expressed in CPNs and in a proportion of GABAergic interneurons, but not in cholinergic interneurons or glial cells. In the striatum, TSHZ3 is expressed in all SCINs, while its expression is absent or partial in the other main brain cholinergic systems. We then characterized two new conditional knockout (cKO) models generated by crossing Tshz3flox/flox with Emx1-Cre (Emx1-cKO) or Chat-Cre (Chat-cKO) mice to decipher the respective role of CPNs and SCINs. Emx1-cKO mice show altered excitatory synaptic transmission onto CPNs and impaired plasticity at corticostriatal synapses, with neither cortical neuron loss nor abnormal layer distribution. These animals present social interaction deficits but no repetitive patterns of behavior. Chat-cKO mice exhibit no loss of SCINs but changes in the electrophysiological properties of these interneurons, associated with repetitive patterns of behavior without social interaction deficits. Therefore, dysfunction in either CPNs or SCINs segregates with a distinct ASD behavioral trait. These findings provide novel insights onto the implication of the corticostriatal circuitry in ASD by revealing an unexpected neuronal dichotomy in the biological background of the two core behavioral domains of this disorder.

Postsynaptic autism spectrum disorder genes and synaptic dysfunction.

This review provides an overview of the synaptic dysfunction of neuronal circuits and the ensuing behavioral alterations caused by mutations in autism spectrum disorder (ASD)-linked genes directly or indirectly affecting the postsynaptic neuronal compartment. There are plenty of ASD risk genes, that may be broadly grouped into those involved in gene expression regulation (epigenetic regulation and transcription) and genes regulating synaptic activity (neural communication and neurotransmission). Notably, the effects mediated by ASD-associated genes can vary extensively depending on the developmental time and/or subcellular site of expression. Therefore, in order to gain a better understanding of the mechanisms of disruptions in postsynaptic function, an effort to better model ASD in experimental animals is required to improve standardization and increase reproducibility within and among studies. Such an effort holds promise to provide deeper insight into the development of these disorders and to improve the translational value of preclinical studies.

Camk2a-Cre and Tshz3 Expression in Mouse Striatal Cholinergic Interneurons: Implications for Autism Spectrum Disorder.

Camk2a-Cre mice have been widely used to study the postnatal function of several genes in forebrain projection neurons, including cortical projection neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). We linked heterozygous deletion of TSHZ3/Tshz3 gene to autism spectrum disorder (ASD) and used Camk2a-Cre mice to investigate the postnatal function of Tshz3, which is expressed by CPNs but not MSNs. Recently, single-cell transcriptomics of the adult mouse striatum revealed the expression of Camk2a in interneurons and showed Tshz3 expression in striatal cholinergic interneurons (SCINs), which are attracting increasing interest in the field of ASD. These data and the phenotypic similarity between the mice with Tshz3 haploinsufficiency and Camk2a-Cre-dependent conditional deletion of Tshz3 (Camk2a-cKO) prompted us to better characterize the expression of Tshz3 and the activity of Camk2a-Cre transgene in the striatum. Here, we show that the great majority of Tshz3-expressing cells are SCINs and that all SCINs express Tshz3. Using lineage tracing, we demonstrate that the Camk2a-Cre transgene is expressed in the SCIN lineage where it can efficiently elicit the deletion of the Tshz3-floxed allele. Moreover, transcriptomic and bioinformatic analysis in Camk2a-cKO mice showed dysregulated striatal expression of a number of genes, including genes whose human orthologues are associated with ASD and synaptic signaling. These findings identifying the expression of the Camk2a-Cre transgene in SCINs lineage lead to a reappraisal of the interpretation of experiments using Camk2a-Cre-dependent gene manipulations. They are also useful to decipher the cellular and molecular substrates of the ASD-related behavioral abnormalities observed in Tshz3 mouse models.

Construct Validity and Cross Validity of a Test Battery Modeling Autism Spectrum Disorder (ASD) in Mice.

Modeling in other organism species is one of the crucial stages in ascertaining the association between gene and psychiatric disorder. Testing Autism Spectrum Disorder (ASD) in mice is very popular but construct validity of the batteries is not available. We presented here the first factor analysis of a behavioral model of ASD-like in mice coupled with empirical validation. We defined fourteen measures aligning mouse-behavior measures with the criteria defined by DSM-5 for the diagnostic of ASD. Sixty-five mice belonging to a heterogeneous pool of genotypes were tested. Reliability coefficients vary from .68 to .81. The factor analysis resulted in a three- factor solution in line with DSM criteria: social behavior, stereotypy and narrowness of the field of interest. The empirical validation with mice sharing a haplo-insufficiency of the zinc-finger transcription factor TSHZ3/Tshz3 associated with ASD shows the discriminant power of the highly loaded items.

Postnatal Tshz3 Deletion Drives Altered Corticostriatal Function and Autism Spectrum Disorder-like Behavior.

BACKGROUND: Heterozygous deletion of the TSHZ3 gene, encoding for the teashirt zinc-finger homeobox family member 3 (TSHZ3) transcription factor that is highly expressed in cortical projection neurons (CPNs), has been linked to an autism spectrum disorder (ASD) syndrome. Similarly, mice with Tshz3 haploinsufficiency show ASD-like behavior, paralleled by molecular changes in CPNs and corticostriatal synaptic dysfunctions. Here, we aimed at gaining more insight into "when" and "where" TSHZ3 is required for the proper development of the brain, and its deficiency crucial for developing this ASD syndrome. METHODS: We generated and characterized a novel mouse model of conditional Tshz3 deletion, obtained by crossing Tshz3flox/flox with CaMKIIalpha-Cre mice, in which Tshz3 is deleted in CPNs from postnatal day 2 to 3 onward. We characterized these mice by a multilevel approach combining genetics, cell biology, electrophysiology, behavioral testing, and bioinformatics. RESULTS: These conditional Tshz3 knockout mice exhibit altered cortical expression of more than 1000 genes, ∼50% of which have their human orthologue involved in ASD, in particular genes encoding for glutamatergic synapse components. Consistently, we detected electrophysiological and synaptic changes in CPNs and impaired corticostriatal transmission and plasticity. Furthermore, these mice showed strong ASD-like behavioral deficits. CONCLUSIONS: Our study reveals a crucial postnatal role of TSHZ3 in the development and functioning of the corticostriatal circuitry and provides evidence that dysfunction in these circuits might be determinant for ASD pathogenesis. Our conditional Tshz3 knockout mouse constitutes a novel ASD model, opening the possibility for an early postnatal therapeutic window for the syndrome linked to TSHZ3 haploinsufficiency.

TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons.

TSHZ3, which encodes a zinc-finger transcription factor, was recently positioned as a hub gene in a module of the genes with the highest expression in the developing human neocortex, but its functions remained unknown. Here we identify TSHZ3 as the critical region for a syndrome associated with heterozygous deletions at 19q12-q13.11, which includes autism spectrum disorder (ASD). In Tshz3-null mice, differentially expressed genes include layer-specific markers of cerebral cortical projection neurons (CPNs), and the human orthologs of these genes are strongly associated with ASD. Furthermore, mice heterozygous for Tshz3 show functional changes at synapses established by CPNs and exhibit core ASD-like behavioral abnormalities. These findings highlight essential roles for Tshz3 in CPN development and function, whose alterations can account for ASD in the newly defined TSHZ3 deletion syndrome.

Metabolic, synaptic and behavioral impact of 5-week chronic deep brain stimulation in hemiparkinsonian rats.

The long-term effects and action mechanisms of subthalamic nucleus (STN) high-frequency stimulation (HFS) for Parkinson's disease still remain poorly characterized, mainly due to the lack of experimental models relevant to clinical application. To address this issue, we performed a multilevel study in freely moving hemiparkinsonian rats undergoing 5-week chronic STN HFS, using a portable constant-current microstimulator. In vivo metabolic neuroimaging by (1) H-magnetic resonance spectroscopy (11.7 T) showed that STN HFS normalized the tissue levels of the neurotransmission-related metabolites glutamate, glutamine and GABA in both the striatum and substantia nigra reticulata (SNr), which were significantly increased in hemiparkinsonian rats, but further decreased nigral GABA levels below control values; taurine levels, which were not affected in hemiparkinsonian rats, were significantly reduced. Slice electrophysiological recordings revealed that STN HFS was, uniquely among antiparkinsonian treatments, able to restore both forms of corticostriatal synaptic plasticity, i.e. long-term depression and potentiation, which were impaired in hemiparkinsonian rats. Behavior analysis (staircase test) showed a progressive recovery of motor skill during the stimulation period. Altogether, these data show that chronic STN HFS efficiently counteracts metabolic and synaptic defects due to dopaminergic lesion in both the striatum and SNr. Comparison of chronic STN HFS with acute and subchronic treatment further suggests that the long-term benefits of this treatment rely both on the maintenance of acute effects and on delayed actions on the basal ganglia network. We studied the effects of chronic (5 weeks) continuous subthalamic nucleus (STN) high-frequency stimulation (HFS) in hemiparkinsonian rats. The levels of glutamate and GABA in the striatum () and substantia nigra reticulata (SNr) (), measured by in vivo proton magnetic resonance spectroscopy ((1) H-MRS), were increased by 6-hydroxydopamine (6-OHDA) lesion, which also disrupted corticostriatal synaptic plasticity () and impaired forepaw skill () in the staircase test. Five-week STN HFS normalized glutamate and GABA levels and restored both synaptic plasticity and motor function. A partial behavioral recovery was observed at 2-week STN HFS.

Synaptic scaling up in medium spiny neurons of aged BACHD mice: A slow-progression model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disease that develops in midlife (~ 40 years-old at onset) and then progresses slowly. It is still unclear how striatal medium spiny neurons (MSNs), the most vulnerable neurons in HD, maintain their function for decades despite the chronic expression of mutant huntingtin (mHTT). In this study, we used aged BACHD mice, a HD model expressing the full-length human mHTT gene, to investigate the molecular, morphological and functional properties of striatal MSNs. We report that the density of dendritic spines in MSNs is substantially lower in aged BACHD mice than in wild-type (WT) mice, in the absence of major dendritic changes and neuronal loss. This spine loss is accompanied by changes in transcription, resulting in a low expression of the striatum-specific G protein-coupled receptor 88 (Gpr88) as well as a reorganization of the composition of AMPAR subunits (high Gria1/Gria2 mRNA ratio). We also detected functional changes in BACHD MSNs. Notably, BACHD MSNs were hyperexcitable and the amplitude of AMPAR-mediated synaptic currents was higher than in WT MSNs. Altogether, these data show that both the intrinsic properties and the strength of the remaining synapses are modified in MSNs with low dendritic spine density in aged BACHD mice. These homeostatic mechanisms may compensate for the substantial loss of synaptic inputs and thus alleviate the deleterious effects of mHTT expression on the activity of MSNs and also possibly on the motor phenotype in aged BACHD.

Distinct effects of mGlu4 receptor positive allosteric modulators at corticostriatal vs. striatopallidal synapses may differentially contribute to their antiparkinsonian action.

Metabotropic glutamate 4 (mGlu4) receptor is a promising target for the treatment of motor deficits in Parkinson's disease (PD). This is due in part to its localization at key basal ganglia (BG) synapses that become hyperactive in this pathology, particularly striatopallidal synapses. In this context, mGlu4 receptor activation using either orthosteric agonists or positive allosteric modulators (PAMs) improves motor symptoms in rodent PD models in certain conditions. However, literature data show that mGlu4 receptor PAMs have no effect at striatopallidal GABAergic synapses (unless combined with an orthosteric agonist) and on the firing activity of pallidal neurons, and fail to provide significant motor improvement in relevant PD models. This questions the mechanistic hypothesis that mGlu4 receptor PAMs should act at striatopallidal synapses to alleviate PD motor symptoms. To shed light on this issue, we performed brain slice electrophysiology experiments. We show that Lu AF21934, an mGlu4 PAM small-molecule probe-compound, was ineffective at striatopallidal synapses at all concentrations tested, while it significantly inhibited corticostriatal synaptic transmission. Similarly, Lu AF21934 did not affect electrophysiology readouts at striatopallidal synapses in the presence of haloperidol or in 6-hydroxydopamine-lesioned rats. Interestingly, co-application of Lu AF21934 with a glutamate transporter inhibitor revealed a significant inhibitory action at striatopallidal synapses. Possibly, this effect could rely on increased level/permanence of glutamate in the synaptic cleft. Such differential efficacy of mGlu4 receptor PAMs at corticostriatal vs. striatopallidal synapses raises several issues regarding the synaptic target(s) of these drugs in the BG, and challenges the mechanisms by which they alleviate motor deficits in experimental PD models.

Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of pathogenic events. Here we explored the hypothesis that dysfunction of excitatory amino acid transporters (EAATs) might be involved. Acutely-induced dysfunction of EAATs in the rat SN, by single unilateral injection of their substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a neurodegenerative process mimicking several PD features. Dopamine neurons are selectively affected, consistent with their sustained excitation by PDC measured by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA receptor antagonists ifenprodil and memantine provide neuroprotection. Besides oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione depletion and neuroinflammation characterize the primary insult. Most interestingly, the degeneration progresses overtime with unilateral to bilateral and caudo-rostral evolution. Transient adaptive changes in dopamine function markers in SN and striatum accompany cell loss and axonal dystrophy, respectively. Motor deficits appear when neuron loss exceeds 50% in the most affected SN and striatal dopamine tone is dramatically reduced. These findings outline a functional link between EAAT dysfunction and several PD pathogenic mechanisms/pathological hallmarks, and provide a novel acutely-triggered model of progressive Parkinsonism.

Synergy between L-DOPA and a novel positive allosteric modulator of metabotropic glutamate receptor 4: implications for Parkinson's disease treatment and dyskinesia.

Group III metabotropic glutamate (mGlu) receptors are localized in presynaptic terminals within basal ganglia (BG) circuitry that become hyperactive due to dopamine depletion in Parkinson's disease (PD). For this reason, group III mGlu receptors, in particular mGlu4, have been considered as key strategic targets for non-dopaminergic pharmacological treatments aimed at modulating these synapses, without producing the well known side-effects of l-DOPA, in particular the highly disabling l-DOPA-induced dyskinesia (LID). Herein we add physiological and functional support to this hypothesis using Lu AF21934, a novel selective and brain-penetrant mGlu4 receptor positive allosteric modulator (PAM) tool compound. By in vitro electrophysiological recordings we demonstrate that Lu AF21934 inhibits corticostriatal synaptic transmission and enhances the effect of the orthosteric mGlu4 receptor-preferred agonist LSP1-2111. In naïve rats, Lu AF21934 dose-dependently (10 and 30 mg/kg) alleviated haloperidol-induced catalepsy. In hemiparkinsonian rats (unilateral 6-hydroxydopamine lesion of the substantia nigra pars compacta), Lu AF21934 alone did not affect akinesia at the doses tested (10 and 30 mg/kg). However, when Lu AF21934 was combined with sub-threshold doses of l-DOPA (1 and 5 mg/kg), it acted synergistically in alleviating akinesia in a dose-dependent manner and, notably, also reduced the incidence of LID but not its severity. Interestingly, these effects occurred at Lu AF21934 brain free concentrations that showed functional activity in in vitro screens (calcium flux and electrophysiology assays). These results support the potential for antiparkinsonian clinical use of a combined treatment consisting in l-DOPA and a mGlu4 receptor PAM to reduce efficacious l-DOPA doses (generally known as l-DOPA sparing), while maintaining the same benefit on PD motor troubles, and at the same time minimizing the development of LID. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Effects of GPi and STN inactivation on physiological, motor, cognitive and motivational processes in animal models of Parkinson's disease.

Loss of the dopaminergic input to the striatum, characterizing Parkinson's disease, leads to the hyper-activity of two key nuclei of the basal ganglia (BG): the subthalamic nucleus (STN) and the internal segment of the globus pallidus (GPi). The anatomo-physiological organization of the BG and their output suggested that interfering with such hyper-activity could restore motor function and improve parkinsonism. Several animal models in rodents and primates, as well as clinical studies and neurosurgical treatments, have confirmed such hypothesis. This chapter will review the physiological and behavioural data obtained by inactivating the GPi or the STN by means of lesions, pharmacological approaches and deep brain stimulation. The consequences of these treatments will be examined at levels ranging from cellular to complex behavioural changes. Some of this experimental evidence suggested new and effective clinical treatments for PD, which are now routinely used worldwide. However, further studies are necessary to better understand the consequences of GPi and STN manipulation especially at the cognitive level in order to improve functional neurosurgical treatments for Parkinson's disease by minimizing risks of side-effects.

Ciliary neurotrophic factor protects striatal neurons against excitotoxicity by enhancing glial glutamate uptake.

Ciliary neurotrophic factor (CNTF) is a potent neuroprotective cytokine in different animal models of glutamate-induced excitotoxicity, although its action mechanisms are still poorly characterized. We tested the hypothesis that an increased function of glial glutamate transporters (GTs) could underlie CNTF-mediated neuroprotection. We show that neuronal loss induced by in vivo striatal injection of the excitotoxin quinolinic acid (QA) was significantly reduced (by approximately 75%) in CNTF-treated animals. In striatal slices, acute QA application dramatically inhibited corticostriatal field potentials (FPs), whose recovery was significantly higher in CNTF rats compared to controls (approximately 40% vs. approximately 7%), confirming an enhanced resistance to excitotoxicity. The GT inhibitor DL-threo-beta-benzyloxyaspartate greatly reduced FP recovery in CNTF rats, supporting the role of GT in CNTF-mediated neuroprotection. Whole-cell patch-clamp recordings from striatal medium spiny neurons showed no alteration of basic properties of striatal glutamatergic transmission in CNTF animals, but the increased effect of a low-affinity competitive glutamate receptor antagonist (gamma-D-glutamylglycine) also suggested an enhanced GT function. These data strongly support our hypothesis that CNTF is neuroprotective via an increased function of glial GTs, and further confirms the therapeutic potential of CNTF for the clinical treatment of progressive neurodegenerative diseases involving glutamate overflow.

Downstream mechanisms triggered by mitochondrial dysfunction in the basal ganglia: from experimental models to neurodegenerative diseases.

Mitochondrial dysfunctions have been implicated in the cellular processes underlying several neurodegenerative disorders affecting the basal ganglia. These include Huntington's chorea and Parkinson's disease, two highly debilitating motor disorders for which recent research has also involved gene mutation linked to mitochondrial deficits. Experimental models of basal ganglia diseases have been developed by using toxins able to disrupt mitochondrial function: these molecules act by selectively inhibiting mitochondrial respiratory complexes, uncoupling cellular respiration. This in turn leads to oxidative stress and energy deficit that trigger critical downstream mechanisms, ultimately resulting in neuronal vulnerability and loss. Here we review the molecular and cellular downstream effects triggered by mitochondrial dysfunction, and the different experimental models that are obtained by the administration of selective mitochondrial toxins or by the expression of mutant genes.

Electrophysiological and behavioral evidence that modulation of metabotropic glutamate receptor 4 with a new agonist reverses experimental parkinsonism.

Developing nondopaminergic palliative treatments for Parkinson's disease represents a major challenge to avoid the debilitating side effects produced by L-DOPA therapy. Increasing interest is addressed to the selective targeting of group III metabotropic glutamate (mGlu) receptors that inhibit transmitter release at presumably overactive synapses in the basal ganglia. Here we characterize the functional action of a new orthosteric group III mGlu agonist, LSP1-2111, with a preferential affinity for mGlu4 receptor. In mouse brain slices, LSP1-2111 inhibits striatopallidal GABAergic transmission by selectively activating the mGlu4 receptor but has no effect at a synapse modulated solely by the mGlu7 and mGlu8 receptors. Intrapallidal LSP1-2111 infusion reverses the akinesia produced by nigrostriatal dopamine depletion in a reaction time task, whereas an mGlu8-receptor agonist has no effect. Finally, systemic administration of LSP1-2111 counteracts haloperidol-induced catalepsy, opening promising perspectives for the development of antiparkinsonian therapeutic strategies focused on orthosteric mGlu4-receptor agonists.