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Neuronal sensitivity to light stimulation can be a significant confounding factor for assays that use light to study neuronal processes, such as optogenetics and fluorescent imaging. While continuous light stimulation has been shown to be responsible for a decrease in firing activity in several neuronal subtypes, discontinuous light stimulation commonly used in optogenetic experiments is supposed to have a negligible action. In the present report, we experimentally test this theoretical prediction by evaluating the effect produced by ten of the most commonly used patterns of discontinuous light stimulation under several electrophysiological parameters.

SUMMARYStriatal cholinergic interneurons (CINs) respond to salient or reward prediction-related stimuli after conditioning with brief pauses in their activity, implicating them in learning and action selection. This pause is lost in animal models of Parkinson’s disease. How this signal regulates the functioning of the striatum remains an open question. To address this issue, we examined the impact of CIN firing inhibition on glutamatergic transmission between the cortex and the medium-sized spiny projection neurons expressing dopamine D1 receptors (D1 MSNs). Brief interruption of CIN activity had no effect in control condition whereas it increased glutamatergic responses in D1 MSNs after nigrostriatal dopamine denervation. This potentiation was dependent upon M4 muscarinic receptor and protein kinase A. Decreasing CIN firing by opto/chemogenetic strategies in vivo rescued long-term potentiation in some MSNs and alleviated motor learning deficits in parkinsonian mice. Taken together, our findings demonstrate that the control exerted by CINs on corticostriatal transmission and striatal-dependent motor-skill learning depends on the integrity of dopaminergic inputs.

Striatal cholinergic interneurons (CINs) respond to salient or reward prediction-related stimuli after conditioning with brief pauses in their activity, implicating them in learning and action selection. This pause is lost in animal models of Parkinson's disease. How this signal regulates the striatal network remains an open question. Here, we examine the impact of CIN firing inhibition on glutamatergic transmission between the cortex and the medium spiny neurons expressing dopamine D1 receptor (D1 MSNs). Brief interruption of CIN activity has no effect in control conditions, whereas it increases glutamatergic responses in D1 MSNs after dopamine denervation. This potentiation depends upon M4 muscarinic receptor and protein kinase A. Decreasing CIN firing by optogenetics/chemogenetics in vivo partially rescues long-term potentiation in MSNs and motor learning deficits in parkinsonian mice. Our findings demonstrate that the control exerted by CINs on corticostriatal transmission and striatal-dependent motor-skill learning depends on the integrity of dopaminergic inputs.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD., SCOPUS: ar.j, info:eu-repo/semantics/published

We identified seven families associatingNEUROD2pathogenic mutations with ASD and intellectual disability. To get insight into the pathophysiological mechanisms, we analyzed cortical development inNeurod2KO 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 inNeurod2KO mice were enriched for voltage-gated ion channels, and the human orthologs of these genes were strongly associated with ASD. Furthermore, adultNeurod2KO mice exhibited core ASD-like behavioral abnormalities. Finally, by generatingNeurod2conditional mutant mice we demonstrate that forebrain excitatory neuron-specificNeurod2deletion recapitulates cellular and behavioral ASD phenotypes found in full KO mice. Our findings demonstrate crucial roles forNeurod2in cortical development and function, whose alterations likely account for ASD and related symptoms in the newly definedNEUROD2mutation syndrome.

International audience; Optogenetics is based on the selective expression of exogenous opsins by neurons allowing experimental control of their electrical activity using visible light. The interpretation of the results of optogenetic experiments is based on the assumption that light stimulation selectively acts on those neurons expressing the exogenous opsins without perturbing the activity of naive ones. Here, we report that light stimulation, of wavelengths and power in the range of those normally used in optogenetic experiments, consistently reduces the firing activity of naive Mitral Cells (MCs) and Tufted Neurons in the olfactory bulb as well as in Medium Spiny Neurons (MSNs) in the striatum. No such effect was observed for cerebellar Purkinje and hippocampal CA1 neurons. The effects on MC firing appear to be mainly due to a light-induced increase in tissue temperature, between 0.1 and 0.4°C, associated with the generation of a hyperpolarizing current and a modification of action potential (AP) shape. Therefore, light in the visible range can affect neuronal physiology in a cell-specific manner. Beside the implications for optogenetic studies, our results pave the way to investigating the use of visible light for therapeutic purposes in pathologies associated with neuronal hyperexcitability.

International audience; 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 ChAT(cre/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 Parkinson's 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 Parkinson's disease.SIGNIFICANCE STATEMENT:The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinson's 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.

International audience; Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons.

High-affinity glutamate transporters (GTs) play a major role in controlling the extracellular level of this excitatory neurotransmitter in the CNS. Here we have characterized, by means of in vitro patch-clamp recordings from medium spiny neurons (MSNs), the role of GTs in regulating corticostriatal glutamatergic synaptic transmission in the adult rat. Charge transfer and decay-time, but not amplitude, of excitatory postsynaptic currents (EPSCs) were enhanced by dl -threo-β-benzyloxyaspartate (TBOA), a broad inhibitor of GTs. Moreover, TBOA also potentiated currents induced by high-frequency stimulation (HFS) protocols. Interestingly, the effect of TBOA on EPSCs was lost when MSNs were clamped at +40 mV, a condition in which neuronal GTs, that are voltage-dependent, are blocked. However, in this condition TBOA was still able to enhance HFS-induced currents, suggesting that glial GT's role is to regulate synaptic transmission when glutamate release is massive. These data suggest that neuronal GTs, rather than glial, shape EPSCs' kinetics and modulate glutamate transmission at corticostriatal synapse. Moreover, the control of glutamate concentration in the synaptic cleft by GTs may play a role in a number of degenerative disorders characterized by the hyperactivity of corticostriatal pathway, as well as in synaptic plasticity.

International audience; 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.

One-half of the subthalamic nucleus (STN) neurons switch from single-spike activity to burst-firing mode according to membrane potential. In an earlier study, the ionic mechanisms of the bursting mode were studied but the ionic currents underlying single-spike activity were not determined. The single-spike mode of activity of STN neurons recorded from acute slices in the current clamp mode is TTX-sensitive but is not abolished by antagonists of ionotropic glutamatergic and GABAergic receptors, blockers of calcium currents (2 mM cobalt or 40 μM nickel), or intracellular Ca2+ ions chelators. Tonic activity is characterized by a pacemaker depolarization that spontaneously brings the membrane from the peak of the afterspike hyperpolarization (AHP) to firing threshold (from −57.1 ± 0.5 mV to −42.2 ± 0.3 mV). Voltage-clamp recordings suggest that the Ni2+-sensitive, T-type Ca2+ current does not play a significant role in single-spike activity because it is totally inactivated at potentials more depolarized than −60 mV. In contrast, the TTX-sensitive, I NaP that activated at −54.4 ± 0.6 mV fulfills the conditions for underlying pacemaker depolarization because it is activated below spike threshold and is not fully inactivated in the pacemaker range. In some cases, the depolarization required to reach the threshold for I NaP activation is mediated by hyperpolarization-activated cation current ( I h). This was directly confirmed by the cesium-induced shift from single-spike to burst-firing mode which was observed in some STN neurons. Therefore, a fraction of I h which is tonically activated at rest, exerts a depolarizing influence and enables membrane potential to reach the threshold for I NaP activation, thus favoring the single-spike mode. The combined action of I NaP and I h is responsible for the dual mode of discharge of STN neurons.

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.

Publisher Summary This chapter provides an overview of the functional properties of the subthalamic nucleus (STN) and describes recent insights in its role in normal and patho­logical brain processes, with emphasis on Parkinson's disease (PD) and epileptic disorders. Due to its strategic position in the organization of the basal gangli, and its specific electrophysiological and synaptic properties, the STN has a cardinal and leading situation in basal ganglia functions. The chapter reviews the recent findings concerning the various types of normal spontaneous activities of STN neurons in vivo, and their relationships to cortical synchronization and vigilance states. The role of the dopaminergic system, and of its alteration, in the modulation of activity in the STN and related networks will be addressed, with a special emphasis on the abnormal oscillatory neuronal pattern associated with Parkinson's disease (PD). Finally, the functional implication of the STN in non-convulsive generalized epilepsy is also highlighted, based on the recent in vivo electrophysiological investigations performed in a genetic model of absence seizures.

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.

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)-alpha-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.

Neural adaptations in the nucleus accumbens (NAc), a key component of the mesolimbic dopamine (DA) system, are thought to mediate several of the long-term behavioral sequelas of chronicin vivoexposure to drugs of abuse. Here, we examine whether the modulation of excitatory synaptic transmission by DA in the NAc shell is modified after chronic cocaine exposure that induced behavioral sensitization. The DA-induced inhibition of AMPA receptor-mediated synaptic responses was enhanced in cocaine-treated mice, an effect that was caused by activation of D1-like receptors. DA did not enhance NMDA receptor-mediated synaptic responses in saline- and cocaine-treated mice or in the dorsal striatum of control mice. We hypothesize that the enhanced inhibitory effects of DA on synaptic transmission in the NAc are one of a number of adaptations that contribute to a decrease in excitatory drive to NAc after exposure to drugs of abuse.

A compelling model of experience-dependent plasticity is the long-lasting sensitization to the locomotor stimulatory effects of drugs of abuse. Adaptations in the nucleus accumbens (NAc), a component of the mesolimbic dopamine system, are thought to contribute to this behavioral change. Here we examine excitatory synaptic transmission in NAc slices prepared from animals displaying sensitization 10-14 days after repeated in vivo cocaine exposure. The ratio of AMPA (alpha-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid) receptor- to NMDA (N-methyl-d-aspartate) receptor-mediated excitatory postsynaptic currents (EPSCs) was decreased at synapses made by prefrontal cortical afferents onto medium spiny neurons in the shell of the NAc. The amplitude of miniature EPSCs at these synapses also was decreased, as was the magnitude of long-term depression. These data suggest that chronic in vivo administration of cocaine elicits a long-lasting depression of excitatory synaptic transmission in the NAc, a change that may contribute to behavioral sensitization and addiction.

The effect of high-frequency stimulation (HFS) of the subthalamic nucleus (STN) was analyzed with patch-clamp techniques (whole cell configuration, current- and voltage-clamp modes) in rat STN slices in vitro. A brief tetanus, consisting of 100-μs bipolar stimuli at a frequency of 100–250 Hz during 1 min, produced a full blockade of ongoing STN activity whether it was in the tonic or bursting mode. This HFS-induced silence lasted around 6 min after the end of stimulation, was frequency dependent, could be repeated without alteration, and was not synaptically induced as it was still observed in the presence of blockers of ionotropic GABA and glutamate receptors or in the presence of cobalt at a concentration (2 mM) that blocks voltage-gated Ca2+ channels and synaptic transmission. During HFS-induced silence, the following alterations were observed: the persistent Na+ current ( I NaP) was totally blocked (by 99%), the Ca2+-mediated responses were strongly reduced including the posthyperpolarization rebound (−62% in amplitude) and the plateau potential (−76% in duration), suggesting that T- and L-type Ca2+ currents are transiently depressed by HFS, whereas the Cs+-sensitive, hyperpolarization-activated cationic current ( I h) was little affected. Thus a high-frequency tetanus produces a blockade of the spontaneous activities of STN neurons as a result of a strong depression of intrinsic voltage-gated currents underlying single-spike and bursting modes of discharge. These effects of HFS, which are completely independent of synaptic transmission, provide a mechanism for interrupting ongoing activities of STN neurons.

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.