GRID1/GluD1 homozygous variants linked to intellectual disability and spastic paraplegia impair mGlu1/5 receptor signaling and excitatory synapses.

The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.

Parvalbumin interneuron-derived tissue-type plasminogen activator shapes perineuronal net structure.

BACKGROUND: Perineuronal nets (PNNs) are specialized extracellular matrix structures mainly found around fast-spiking parvalbumin (FS-PV) interneurons. In the adult, their degradation alters FS-PV-driven functions, such as brain plasticity and memory, and altered PNN structures have been found in neurodevelopmental and central nervous system disorders such as Alzheimer's disease, leading to interest in identifying targets able to modify or participate in PNN metabolism. The serine protease tissue-type plasminogen activator (tPA) plays multifaceted roles in brain pathophysiology. However, its cellular expression profile in the brain remains unclear and a possible role in matrix plasticity through PNN remodeling has never been investigated. RESULT: By combining a GFP reporter approach, immunohistology, electrophysiology, and single-cell RT-PCR, we discovered that cortical FS-PV interneurons are a source of tPA in vivo. We found that mice specifically lacking tPA in FS-PV interneurons display denser PNNs in the somatosensory cortex, suggesting a role for tPA from FS-PV interneurons in PNN remodeling. In vitro analyses in primary cultures of mouse interneurons also showed that tPA converts plasminogen into active plasmin, which in turn, directly degrades aggrecan, a major structural chondroitin sulfate proteoglycan (CSPG) in PNNs. CONCLUSIONS: We demonstrate that tPA released from FS-PV interneurons in the central nervous system reduces PNN density through CSPG degradation. The discovery of this tPA-dependent PNN remodeling opens interesting insights into the control of brain plasticity.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Structural biology of ionotropic glutamate delta receptors and their crosstalk with metabotropic glutamate receptors.

Enigmatic orphan glutamate delta receptors (GluD) are one of the four classes of the ionotropic glutamate receptors (iGluRs) that play key roles in synaptic transmission and plasticity. While members of other iGluR families viz AMPA, NMDA, and kainate receptors are gated by glutamate, the GluD receptors neither bind glutamate nor evoke ligand-induced currents upon binding of glycine and D-serine. Thus, the GluD receptors were considered to function as structural proteins that facilitate the formation, maturation, and maintenance of synapses in the hippocampus and cerebellum. Recent work has revealed that GluD receptors have extensive crosstalk with metabotropic glutamate receptors (mGlus) and are also gated by their activation. The latest development of a novel optopharamcological tool and the cryoEM structures of GluD receptors would help define the molecular and chemical basis of the GluD receptor's role in synaptic physiology. This article is part of the special Issue on "Glutamate Receptors - Orphan iGluRs".

Regulation of perineuronal nets in the adult cortex by the activity of the cortical network.

Perineuronal net (PNN) accumulation around parvalbumin-expressing (PV) inhibitory interneurons marks the closure of critical periods of high plasticity, whereas PNN removal reinstates juvenile plasticity in the adult cortex. Using targeted chemogenetic in vivo approaches in the adult mouse visual cortex, we found that transient inhibition of PV interneurons, through metabotropic or ionotropic chemogenetic tools, induced PNN regression. Electroencephalographic recordings indicated that inhibition of PV interneurons did not elicit unbalanced network excitation. Likewise, inhibition of local excitatory neurons also induced PNN regression, whereas chemogenetic excitation of either PV or excitatory neurons did not reduce the PNN. We also observed that chemogenetically inhibited PV interneurons exhibited reduced PNN compared to their untransduced neighbors, and confirmed that single PV interneurons express multiple genes enabling individual regulation of their own PNN density. Our results indicate that PNN density is regulated in the adult cortex by local changes of network activity that can be triggered by modulation of PV interneurons. PNN regulation may provide adult cortical circuits with an activity-dependent mechanism to control their local remodeling.SIGNIFICANCE STATEMENTThe perineuronal net is an extracellular matrix, which accumulates around individual parvalbumin-expressing inhibitory neurons during postnatal development, and is seen as a barrier that prevents plasticity of neuronal circuits in the adult cerebral cortex. We found that transiently inhibiting parvalbumin-expressing or excitatory cortical neurons triggers a local decrease of perineuronal net density. Our results indicate that perineuronal nets are regulated in the adult cortex depending on the activity of local microcircuits. These findings uncover an activity-dependent mechanism by which adult cortical circuits may locally control their plasticity.

Bioluminescence Imaging of Neuronal Network Dynamics Using Aequorin-Based Calcium Sensors.

Optogenetic calcium sensors enable the imaging in real-time of the activities of single or multiple neurons in brain slices and in vivo. Bioluminescent probes engineered from the natural calcium sensor aequorin do not require illumination, are virtually devoid of background signal, and exhibit wide dynamic range and low cytotoxicity. These probes are thus well suited for long-duration, whole-field recordings of multiple neurons simultaneously. Here, we describe a protocol for monitoring and analyzing the dynamics of neuronal ensembles using whole-field bioluminescence imaging of an aequorin-based sensor in brain slice.

Combined Optogenetic Approaches Reveal Quantitative Dynamics of Endogenous Noradrenergic Transmission in the Brain.

Little is known about the real-time cellular dynamics triggered by endogenous catecholamine release despite their importance in brain functions. To address this issue, we expressed channelrhodopsin in locus coeruleus neurons and protein kinase-A activity biosensors in cortical pyramidal neurons and combined two-photon imaging of biosensors with photostimulation of locus coeruleus cortical axons, in acute slices and in vivo. Burst photostimulation of axons for 5-10 s elicited robust, minutes-lasting kinase-A activation in individual neurons, indicating that a single burst firing episode of synchronized locus coeruleus neurons has rapid and lasting effects on cortical network. Responses were mediated by ß1 adrenoceptors, dampened by co-activation of α2 adrenoceptors, and dramatically increased upon inhibition of noradrenaline reuptake transporter. Dopamine receptors were not involved, showing that kinase-A activation was due to noradrenaline release. Our study shows that noradrenergic transmission can be characterized with high spatiotemporal resolution in brain slices and in vivo using optogenetic tools.

Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with genetically-engineered photopharmacology.

Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet they don't bind glutamate and are considered orphan. Progress in defining the ion channel function of GluDs in neurons has been hindered by a lack of pharmacological tools. Here, we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable pore blocker. In the constitutively open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with high pharmacological specificity that metabotropic glutamate receptor signaling triggers opening of GluD2. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.

Neurotransmitters are chemicals released by the body that trigger activity in neurons. Receptors on the surface of neurons detect these neurotransmitters, providing a link between the inside and the outside of the cell. Glutamate is one of the major neurotransmitters and is involved in virtually all brain functions. Glutamate binds to two different types of receptors in neurons. Ionotropic receptors have pores known as ion channels, which open when glutamate binds. This is a fast-acting response that allows sodium ions to flow into the neuron, triggering an electrical signal. Metabotropic receptors, on the other hand, trigger a series of events inside the cell that lead to a response. Metabotropic receptors take more time than ionotropic receptors to elicit a response in the cell, but their effects last much longer. One type of receptor, known as the GluD family, is very similar to ionotropic glutamate receptors but does not directly respond to glutamate. Instead, the ion channel of GluD receptors opens after being activated by glutamate metabotropic receptors. GluD receptors are produced throughout the brain and play roles in synapse formation and activity, but the way they work remains unclear. An obstacle to understanding how GluD receptors work is the lack of molecules that can specifically block these receptors' ion channel activity. Lemoine et al. have developed a tool that enables control of the ion channel in GluD receptors using light. Human cells grown in the lab were genetically modified to produce a version of GluD2 (a member of the GluD family) with a light-sensitive molecule attached. In darkness or under green light, the light-sensitive molecule blocks the channel and prevents ions from passing through. Under violet light, the molecule twists, and ions can flow through the channel. With this control over the GluD2 ion channel activity, Lemoine et al. were able to validate previous research showing that the activation of metabotropic glutamate receptors can trigger GluD2 to open. The next step will be to test this approach in neurons. This will help researchers to understand what role GluD ion channels play in neuron to neuron communication.

RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities.

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.

Gene Expression Analysis by Multiplex Single-Cell RT-PCR.

Brain circuit assemblies comprise different cellular subpopulations that exhibit morphological, electrophysiological, and molecular diversity. Here we describe a protocol which, combined with whole-cell patch-clamp recording and morphological reconstruction, allows the transcriptomic analysis of the recorded cell. This protocol provides recipes on how to detect simultaneously the expression of 24 genes/markers at the single-cell level using polymerase chain reaction (PCR), how to design gene-specific probes, and how to validate them. This technique provides multiplexed expression data that cannot be easily obtained by other approaches such as immunological co-labeling.

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

Target Interneuron Preference in Thalamocortical Pathways Determines the Temporal Structure of Cortical Responses.

Sensory processing relies on fast detection of changes in environment, as well as integration of contextual cues over time. The mechanisms by which local circuits of the cerebral cortex simultaneously perform these opposite processes remain obscure. Thalamic "specific" nuclei relay sensory information, whereas "nonspecific" nuclei convey information on the environmental and behavioral contexts. We expressed channelrhodopsin in the ventrobasal specific (sensory) or the rhomboid nonspecific (contextual) thalamic nuclei. By selectively activating each thalamic pathway, we found that nonspecific inputs powerfully activate adapting (slow-responding) interneurons but weakly connect fast-spiking interneurons, whereas specific inputs exhibit opposite interneuron preference. Specific inputs thereby induce rapid feedforward inhibition that limits response duration, whereas, in the same cortical area, nonspecific inputs elicit delayed feedforward inhibition that enables lasting recurrent excitation. Using a mean field model, we confirm that cortical response dynamics depends on the type of interneuron targeted by thalamocortical inputs and show that efficient recruitment of adapting interneurons prolongs the cortical response and allows the summation of sensory and contextual inputs. Hence, target choice between slow- and fast-responding inhibitory neurons endows cortical networks with a simple computational solution to perform both sensory detection and integration.

A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats.

Tobacco addiction is a chronic and relapsing disorder with an important genetic component that represents a major public health issue. Meta-analysis of large-scale human genome-wide association studies (GWASs) identified a frequent non-synonymous SNP in the gene coding for the α5 subunit of nicotinic acetylcholine receptors (α5SNP), which significantly increases the risk for tobacco dependence and delays smoking cessation. To dissect the neuronal mechanisms underlying the vulnerability to nicotine addiction in carriers of the α5SNP, we created rats expressing this polymorphism using zinc finger nuclease technology and evaluated their behavior under the intravenous nicotine-self-administration paradigm. The electrophysiological responses of their neurons to nicotine were also evaluated. α5SNP rats self-administered more nicotine at high doses and exhibited higher nicotine-induced reinstatement of nicotine seeking than wild-type rats. Higher reinstatement was associated with altered neuronal activity in several discrete areas that are interconnected, including in the interpeduncular nucleus (IPN), a GABAergic structure that strongly expresses α5-containing nicotinic receptors. The altered reactivity of IPN neurons of α5SNP rats to nicotine was confirmed electrophysiologically. In conclusion, the α5SNP polymorphism is a major risk factor for nicotine intake at high doses and for relapse to nicotine seeking in rats, a dual effect that reflects the human condition. Our results also suggest an important role for the IPN in the higher relapse to nicotine seeking observed in α5SNP rats.

Tissue Plasminogen Activator Expression Is Restricted to Subsets of Excitatory Pyramidal Glutamatergic Neurons.

Although the extracellular serine protease tissue plasminogen activator (tPA) is involved in pathophysiological processes such as learning and memory, anxiety, epilepsy, stroke, and Alzheimer's disease, information about its regional, cellular, and subcellular distribution in vivo is lacking. In the present study, we observed, in healthy mice and rats, the presence of tPA in endothelial cells, oligodendrocytes, mastocytes, and ependymocytes, but not in pericytes, microglial cells, and astrocytes. Moreover, blockage of the axo-dendritic transport unmasked tPA expression in neurons of cortical and hippocampal areas. Interestingly, combined electrophysiological recordings, single-cell reverse transcription polymerase chain reaction (RT-PCR), and immunohistological analyses revealed that the presence of tPA is restricted to subsets of excitatory pyramidal glutamatergic neurons. We further evidenced that tPA is stored in synaptobrevin-2-positive glutamatergic synaptic vesicles. Based on all these data, we propose the existence of tPA-ergic neurons in the mature brain.

Nicotinic Transmission onto Layer 6 Cortical Neurons Relies on Synaptic Activation of Non-α7 Receptors.

Nicotinic excitation in neocortex is mediated by low-affinity α7 receptors and by high-affinity α4ß2 receptors. There is evidence that α7 receptors are synaptic, but it is unclear whether high-affinity receptors are activated by volume transmission or synaptic transmission. To address this issue, we characterized responses of excitatory layer 6 (L6) neurons to optogenetic release of acetylcholine (ACh) in cortical slices. L6 responses consisted in a slowly decaying α4ß2 current and were devoid of α7 component. Evidence that these responses were mediated by synapses was 4-fold. 1) Channelrhodopsin-positive cholinergic varicosities made close appositions onto responsive neurons. 2) Inhibition of ACh degradation failed to alter onset kinetics and amplitude of currents. 3) Quasi-saturation of α4ß2 receptors occurred upon ACh release. 4) Response kinetics were unchanged in low release probability conditions. Train stimulations increased amplitude and decay time of responses and these effects appeared to involve recruitment of extrasynaptic receptors. Finally, we found that the α5 subunit, known to be associated with α4ß2 in L6, regulates short-term plasticity at L6 synapses. Our results are consistent with previous anatomical observations of widespread cholinergic synapses and suggest that a significant proportion of these small synapses operate via high-affinity nicotinic receptors.

A concurrent excitation and inhibition of dopaminergic subpopulations in response to nicotine.

Midbrain dopamine (DA) neurons are key players in motivation and reward processing. Increased DA release is thought to be central in the initiation of drug addiction. Whereas dopamine neurons are generally considered to be activated by drugs such as nicotine, we report here that nicotine not only induces excitation of ventral tegmental area (VTA) DA cells but also induces inhibition of a subset of VTA DA neurons that are anatomically segregated in the medial part of the VTA. These opposite responses do not correlate with the inhibition and excitation induced by noxious stimuli. We show that this inhibition requires D2 receptor (D2-R) activation, suggesting that a dopaminergic release is involved in the mechanism. Our findings suggest a principle of concurrent excitation and inhibition of VTA DA cells in response to nicotine. It promotes unexplored roles for DA release in addiction contrasting with the classical views of reinforcement and motivation, and give rise to a new interpretation of the mode of operation of the reward system.

Orexin-dependent activation of layer VIb enhances cortical network activity and integration of non-specific thalamocortical inputs.

Neocortical layer VI is critically involved in thalamocortical activity changes during the sleep/wake cycle. It receives dense projections from thalamic nuclei sensitive to the wake-promoting neuropeptides orexins, and its deepest part, layer VIb, is the only cortical lamina reactive to orexins. This convergence of wake-promoting inputs prompted us to investigate how layer VIb can modulate cortical arousal, using patch-clamp recordings and optogenetics in rat brain slices. We found that the majority of layer VIb neurons were excited by nicotinic agonists and orexin through the activation of nicotinic receptors containing α4-α5-ß2 subunits and OX2 receptor, respectively. Specific effects of orexin on layer VIb neurons were potentiated by low nicotine concentrations and we used this paradigm to explore their intracortical projections. Co-application of nicotine and orexin increased the frequency of excitatory post-synaptic currents in the ipsilateral cortex, with maximal effect in infragranular layers and minimal effect in layer IV, as well as in the contralateral cortex. The ability of layer VIb to relay thalamocortical inputs was tested using photostimulation of channelrhodopsin-expressing fibers from the orexin-sensitive rhomboid nucleus in the parietal cortex. Photostimulation induced robust excitatory currents in layer VIa neurons that were not pre-synaptically modulated by orexin, but exhibited a delayed, orexin-dependent, component. Activation of layer VIb by orexin enhanced the reliability and spike-timing precision of layer VIa responses to rhomboid inputs. These results indicate that layer VIb acts as an orexin-gated excitatory feedforward loop that potentiates thalamocortical arousal.

Glutamate receptors of the delta family are widely expressed in the adult brain.

Recent reports point to critical roles of glutamate receptor subunit delta2 (GluD2) at excitatory synapses and link GluD1 gene alteration to schizophrenia but the expression patterns of these subunits in the brain remain almost uncharacterized. We examined the distribution of GluD1-2 mRNAs and proteins in the adult rodent brain, focusing mainly on GluD1. In situ hybridization revealed widespread neuronal expression of the GluD1 mRNA, with higher levels occurring in several forebrain regions and lower levels in cerebellum. Quantitative RT-PCR assessed differential GluD1 expression in cortex and cerebellum, and revealed GluD2 expression in cortex, albeit at markedly lower level than in cerebellum. Likewise, a high GluD1/GluD2 mRNA ratio was observed in cortex and a low ratio in cerebellum. GluD1 and GluD2 mRNAs were co-expressed in single cortical and hippocampal neurons, with a large predominance of GluD1. Western blots using GluD1- and GluD2-specific antibodies showed expression of both subunits in various brain structures, but not in non-nervous tissues examined. Both delta subunits were upregulated during postnatal development. Widespread neuronal expression of the GluD1 protein was confirmed using immunohistochemistry. Examination at the electron microscopic level in the hippocampus revealed that GluD1 was mainly localized at postsynaptic density of excitatory synapses on pyramidal cells. Control experiments performed using mice carrying deletion of the GluD1- or the GluD2-encoding gene confirmed the specificity of the present mRNA and protein analyses. Our results support a role for the delta family of glutamate receptors at excitatory synapses in neuronal networks throughout the adult brain.