Corinne Beurrier

Subthalamic Nucleus Neurons Switch from Single-Spike Activity to Burst-Firing Mode

The modification of the discharge pattern of subthalamic nucleus (STN) neurons from single-spike activity to mixed burst-firing mode is one of the characteristics of parkinsonism in rat and primates. However, the mechanism of this process is not yet understood. Intrinsic firing patterns of STN neurons were examined in rat brain slices with intracellular and patch-clamp techniques. Almost half of the STN neurons that spontaneously discharged in the single-spike mode had the intrinsic property of switching to pure or mixed burst-firing mode when the membrane was hyperpolarized from −41.3 ± 1.0 mV (range, −35 to −50 mV; n = 15) to −51.0 ± 1.0 mV (range, −42 to −60 mV; n = 20). This switch was greatly facilitated by activation of metabotropic glutamate receptors with 1S,3R-ACPD. Recurrent membrane oscillations underlying burst-firing mode were endogenous and Ca2+-dependent because they were largely reduced by nifedipine (3 μm), Ni2+ (40 μm), and BAPTA-AM (10–50 μm) at any potential tested, whereas TTX (1 μm) had no effect. In contrast, simultaneous application of TEA (1 mm) and apamin (0.2 μm) prolonged burst duration. Moreover, in response to intracellular stimulation at hyperpolarized potentials, a plateau potential with a voltage and ionic basis similar to those of spontaneous bursts was recorded in 82% of the tested STN neurons, all of which displayed a low-threshold Ni2+-sensitive spike. We propose that recurrent membrane oscillations during bursts result from the sequential activation of T/R- and L-type Ca2+ currents, a Ca2+-activated inward current, and Ca2+-activated K+ currents.

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

Developing nondopaminergic palliative treatments for Parkinsons 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.—Beurrier, C., Lopez, S., Révy, D., Selvam, C., Goudet, C., Lhérondel, M., Gubellini, P., Kerkerian‐LeGoff, L., Acher, F., Pin, J.‐P., Amalric, M. Electrophysiological and behavioral evidence that modulation of metabotropic glutamate receptor 4 with a new agonist reverses experimental parkinsonism. FASEB J. 23, 3619–3628 (2009). www.fasebj.org

Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson’s disease treatment

Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson’s disease. For this reason, we investigated the role of group‐III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3S)‐3‐[(3‐amino‐3‐carboxypropyl(hydroxy)phosphinyl)‐hydroxymethyl]‐5‐nitrothiophene (LSP1‐3081) and l‐2‐amino‐4‐phosphonobutanoate (L‐AP4). Here, we show that both drugs dose‐dependently reduced glutamate‐ and GABA‐mediated post‐synaptic potentials, and increased the paired‐pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post‐synaptic currents. Their inhibitory effect was abolished by (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine and was lost in slices from mGlu4 knock‐out mice. Furthermore, (S)‐3,4‐dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1‐3081 or L‐AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.

Striatal Cholinergic Interneurons Control Motor Behavior and Basal Ganglia Function in Experimental Parkinsonism.

Despite evidence showing that anticholinergic drugs are of clinical relevance in Parkinsons disease (PD), the causal role of striatal cholinergic interneurons (CINs) in PD pathophysiology remains elusive. Here, we show that optogenetic inhibition of CINs alleviates motor deficits in PD mouse models, providing direct demonstration for their implication in parkinsonian motor dysfunctions. As neural correlates, CIN inhibition in parkinsonian mice differentially impacts the excitability of striatal D1 and D2 medium spiny neurons, normalizes pathological bursting activity in the main basal ganglia output structure, and increases the functional weight of the direct striatonigral pathway in cortical information processing. By contrast, CIN inhibition in non-lesioned mice does not affect locomotor activity, equally modulates medium spiny neuron excitability, and does not modify spontaneous or cortically driven activity in the basal ganglia output, suggesting that the role of these interneurons in motor function is highly dependent on dopamine tone.

Subthalamic stimulation elicits hemiballismus in normal monkey

HIGH frequency stimulation (HFS) of the subthalamic nucleus (STN) reduces parkinsonian symptoms in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkey and in human patients. The effects of stimulation on normal waking primates have never been evaluated. While low frequency stimulation has no effect, HFS induces dyskinesias contralateral to the stimulated STN resembling human hemiballismus and those obtained in primates after neurotoxic lesion or pharmacological blockade of the STN. In the normal monkey, HFS appears reversibly to incapacitate the STN and allow the emergence of involuntary proximal displacements, due to disinhibition of the thalamo-cortical pathway. In the MPTP-treated monkey HFS buffers STN overactivity and alleviates akinesia and rigidity by reducing inputs to the internal segment of the globus pallidus.

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 ∼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 (∼40% vs. ∼7%), confirming an enhanced resistance to excitotoxicity. The GT inhibitor dl-threo-β-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 (γ-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.

Involvement of Striatal Cholinergic Interneurons and M1 and M4 Muscarinic Receptors in Motor Symptoms of Parkinson's Disease

Over the last decade, striatal cholinergic interneurons (ChIs) have reemerged as key actors in the pathophysiology of basal-ganglia-related movement disorders. However, the mechanisms involved are still unclear. In this study, we address the role of ChI activity in the expression of parkinsonian-like motor deficits in a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion model using optogenetic and pharmacological approaches. Dorsal striatal photoinhibition of ChIs in lesioned ChATcre/cre mice expressing halorhodopsin in ChIs reduces akinesia, bradykinesia, and sensorimotor neglect. Muscarinic acetylcholine receptor (mAChR) blockade by scopolamine produces similar anti-parkinsonian effects. To decipher which of the mAChR subtypes provides these beneficial effects, systemic and intrastriatal administration of the selective M1 and M4 mAChR antagonists telenzepine and tropicamide, respectively, were tested in the same model of Parkinsons disease. The two compounds alleviate 6-OHDA lesion-induced motor deficits. Telenzepine produces its beneficial effects by blocking postsynaptic M1 mAChRs expressed on medium spiny neurons (MSNs) at the origin of the indirect striatopallidal and direct striatonigral pathways. The anti-parkinsonian effects of tropicamide were almost completely abolished in mutant lesioned mice that lack M4 mAChRs specifically in dopamine D1-receptor-expressing neurons, suggesting that postsynaptic M4 mAChRs expressed on direct MSNs mediate the antiakinetic action of tropicamide. The present results show that altered cholinergic transmission via M1 and M4 mAChRs of the dorsal striatum plays a pivotal role in the occurrence of motor symptoms in Parkinsons disease. SIGNIFICANCE STATEMENT The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinsons disease. Here, using optogenetic and pharmacological approaches, we investigated the involvement of striatal cholinergic interneurons (ChIs) and muscarinic receptor subtypes (mAChRs) in the occurrence of a wide range of motor deficits such as akinesia, bradykinesia, motor coordination, and sensorimotor neglect after unilateral nigrostriatal 6-hydroxydopamine lesion in mice. Our results show that photoinhibition of ChIs in the dorsal striatum and pharmacological blockade of muscarinic receptors, specifically postsynaptic M1 and M4 mAChRs, alleviate lesion-induced motor deficits. The present study points to these receptor subtypes as potential targets for the symptomatic treatment of parkinsonian-like motor symptoms.

Cellular and Behavioral Outcomes of Dorsal Striatonigral Neuron Ablation: New Insights into Striatal Functions

The striatum is the input structure of the basal ganglia network that contains heterogeneous neuronal populations, including two populations of projecting neurons called the medium spiny neurons (MSNs), and different types of interneurons. We developed a transgenic mouse model enabling inducible ablation of the striatonigral MSNs constituting the direct pathway by expressing the human diphtheria toxin (DT) receptor under the control of the Slc35d3 gene promoter, a gene enriched in striatonigral MSNs. DT injection into the striatum triggered selective elimination of the majority of striatonigral MSNs. DT-mediated ablation of striatonigral MSNs caused selective loss of cholinergic interneurons in the dorsal striatum but not in the ventral striatum (nucleus accumbens), suggesting a region-specific critical role of the direct pathway in striatal cholinergic neuron homeostasis. Mice with DT injection into the dorsal striatum showed altered basal and cocaine-induced locomotion and dramatic reduction of L-DOPA-induced dyskinesia in the parkinsonian condition. In addition, these mice exhibited reduced anxiety, revealing a role of the dorsal striatum in the modulation of behaviors involving an emotional component, behaviors generally associated with limbic structures. Altogether, these results highlight the implication of the direct striatonigral pathway in the regulation of heterogeneous functions from cell survival to regulation of motor and emotion-associated behaviors.

Morphofunctional deficits in the cerebral cortex of NeuroD2 mutant mice are associated with autism/schizophrenia-like behaviors

The transcription factor NeuroD2 is a recent candidate for neuropsychiatric disorders but its impact in cortical networks and associated behaviors remains unknown. Here we show that in the mouse neocortex, NeuroD2 is restricted to pyramidal neurons, from development to adulthood. In NeuroD2 deficient mice, layer 5 pyramidal neurons of motor area displayed reduced dendritic complexity and reduced spine density. In contrast, production, radial migration, laminar organization and axonal target specificity of pyramidal neurons were normal, revealing a synaptopathy phenotype. Electrophysiologically, intrinsic excitability and inhibitory inputs onto pyramidal neurons were increased. Behaviorally, NeuroD2 homozygous and heterozygous mice exhibited normal interest and memory for objects but altered sociability and social memory, stereotypies, spontaneous epilepsy and hyperactivity. RNA sequencing from microdissected neocortex revealed that NeuroD2 target genes are highly associated with in cell intrinsic excitability, synaptic regulation, autism and schizophrenia. These results strongly reinforce the potential implication of NeuroD2 mutations in human neuropsychiatric disorders.We identified seven families associating NEUROD2 pathogenic mutations with ASD and intellectual disability. To get insight into the pathophysiological mechanisms, we analyzed cortical development in Neurod2 KO mice. Cortical projection neurons (CPNs) over-migrated during embryogenesis, inducing abnormal thickness and laminar positioning of cortical layers. At juvenile ages, dendritic spine turnover and intrinsic excitability were increased in L5 CPNs. Differentially expressed genes in Neurod2 KO mice were enriched for voltage-gated ion channels, and the human orthologs of these genes were strongly associated with ASD. Furthermore, adult Neurod2 KO mice exhibited core ASD-like behavioral abnormalities. Finally, by generating Neurod2 conditional mutant mice we demonstrate that forebrain excitatory neuron-specific Neurod2 deletion recapitulates cellular and behavioral ASD phenotypes found in full KO mice. Our findings demonstrate crucial roles for Neurod2 in cortical development and function, whose alterations likely account for ASD and related symptoms in the newly defined NEUROD2 mutation syndrome.Abstract The neuronal transcription factor NeuroD2 has recently been associated with early encephalopathic epilepsy 1 and genome-wide association studies (GWAS) have suggested that it might be a candidate for neuropsychiatric disorders 2. We set out to understand the function of NeuroD2 in cortex development and behavior and found that deleting this factor in mice results in altered migration, laminar positioning, structural synaptic maturation and physiology of cortical projection neurons (CPNs), as well as in differential expression of genes associated with neuronal excitability, synaptic transmission and neurodevelopmental disorders. These cellular and molecular defects were correlated with behavioral defects, namely locomotor hyperactivity, altered social interest and social memory, stereotypic behaviors and spontaneous seizures. Informed by these neurobehavioral features in mouse mutants, we identified individuals with de novo and familial heterozygous missense mutations in NEUROD2 or copy number variations involving NEUROD2, who shared clinical features such as intellectual disability (ID) and autism spectrum disorder (ASD), with sometimes hyperactivity and epilepsy. In vitro functional analyses showed that NEUROD2 missense mutations identified in a non-consanguineous family and a sporadic case were both pathogenic. Our study demonstrates that loss-of-function mutations in NEUROD2 cause a spectrum of neurobehavioral phenotypes including ID and ASD.

NS.4.3 - OPTOGENETIC AND PHARMACOLOGICAL CONTROL OF DOPAMINE/CHOLINERGIC BALANCE IN EXPERIMENTAL PARKINSONISM

The loss of striatal dopamine (DA) in Parkinson’s disease (PD) models triggers increased level of striatal cholinergic (ACh) activity. How this ACh hyperactivity contributes to motor dysfunction in PD models is still not clear. To address this question, the effects of optogenetic manipulation of dorsal striatal ACh interneurons were evaluated in normal and pathophysiological conditions. To specifically express the opsins in striatal ACh interneurons, we stereotaxically injected into the striatum a Cre-inducible adeno-associated virus (AAV) vector carrying the gene encoding channelrhodopsin (ChR2) or halorhodopsin (eNpHR) in transgenic mice expressing Cre-recombinase under the choline acetyltransferase promoter. Electrophysiological recordings of ACh interneurons in vitro in striatal slices and in vivo confirmed that under laser illumination both opsins were functional: ChR2 drove spike activity while eNpHR silenced firing. We then investigated the contribution of ACh interneurons to motor control in two experimental models of PD. In the haloperidolinduced catalepsy, optogenetic inhibition of ACh interneurons reduced the akinetic symptoms, while their activation had no effect. This antiparkinsonian benefit was also confirmed in the unilateral 6-OHDA nigrostriatal lesion model of PD. Inhibition of ACh interneurons led to a reduction of postural asymmetry and turning bias in the cylinder test and cross maze. Selective muscarinic receptor antagonists (telenzepine and tropicamide, M1 and M4 receptor antagonist, respectively), systemically administered in the same rodent model of PD, decreased the postural asymmetry in the same tests and reduced amphetamine-induced circling behavior. These optogenetic and pharmacological results emphasize the critical involvement of striatal ACh interneurons activity, mediated in part by muscarinic M1 and M4 receptors, in the motor symptoms of PD. This work is supported by ANR, France Parkinson, CNRS and AMU.