Publisher Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Author Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

Operation and plasticity of hippocampal CA3 circuits: implications for memory encoding.

The CA3 region of the hippocampus is important for rapid encoding of memory. Computational theories have proposed specific roles in hippocampal function and memory for the sparse inputs from the dentate gyrus to CA3 and for the extended local recurrent connectivity that gives rise to the CA3 autoassociative network. Recently, we have gained considerable new insight into the operation and plasticity of CA3 circuits, including the identification of novel forms of synaptic plasticity and their underlying mechanisms, and structural plasticity in the GABAergic control of CA3 circuits. In addition, experimental links between synaptic plasticity of CA3 circuits and memory are starting to emerge.

Formin 2 links neuropsychiatric phenotypes at young age to an increased risk for dementia.

Age-associated memory decline is due to variable combinations of genetic and environmental risk factors. How these risk factors interact to drive disease onset is currently unknown. Here we begin to elucidate the mechanisms by which post-traumatic stress disorder (PTSD) at a young age contributes to an increased risk to develop dementia at old age. We show that the actin nucleator Formin 2 (Fmn2) is deregulated in PTSD and in Alzheimer's disease (AD) patients. Young mice lacking the Fmn2 gene exhibit PTSD-like phenotypes and corresponding impairments of synaptic plasticity, while the consolidation of new memories is unaffected. However, Fmn2 mutant mice develop accelerated age-associated memory decline that is further increased in the presence of additional risk factors and is mechanistically linked to a loss of transcriptional homeostasis. In conclusion, our data present a new approach to explore the connection between AD risk factors across life span and provide mechanistic insight to the processes by which neuropsychiatric diseases at a young age affect the risk for developing dementia.

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Activity-dependent control of NMDA receptor subunit composition at hippocampal mossy fibre synapses.

KEY POINTS: CA3 pyramidal cells display input-specific differences in the subunit composition of synaptic NMDA receptors (NMDARs). Although at low density, GluN2B contributes significantly to NMDAR-mediated EPSCs at mossy fibre synapses. Long-term potentiation (LTP) of NMDARs triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. GluN2B subunits are essential for the expression of LTP of NMDARs at mossy fibre synapses. ABSTRACT: Single neurons express NMDA receptors (NMDARs) with distinct subunit composition and biophysical properties that can be segregated in an input-specific manner. The dynamic control of the heterogeneous distribution of synaptic NMDARs is crucial to control input-dependent synaptic integration and plasticity. In hippocampal CA3 pyramidal cells from mice of both sexes, we found that mossy fibre (MF) synapses display a markedly lower proportion of GluN2B-containing NMDARs than associative/commissural synapses. The mechanism involved in such heterogeneous distribution of GluN2B subunits is not known. Here we show that long-term potentiation (LTP) of NMDARs, which is selectively expressed at MF-CA3 pyramidal cell synapses, triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. This activity-dependent recruitment of GluN2B at mature MF-CA3 pyramidal cell synapses contrasts with the removal of GluN2B subunits at other glutamatergic synapses during development and in response to activity. Furthermore, although expressed at low levels, GluN2B is necessary for the expression of LTP of NMDARs at MF-CA3 pyramidal cell synapses. Altogether, we reveal a previously unknown activity-dependent regulation and function of GluN2B subunits that may contribute to the heterogeneous plasticity induction rules in CA3 pyramidal cells.

Non-associative potentiation of perisomatic inhibition alters the temporal coding of neocortical layer 5 pyramidal neurons.

In the neocortex, the coexistence of temporally locked excitation and inhibition governs complex network activity underlying cognitive functions, and is believed to be altered in several brain diseases. Here we show that this equilibrium can be unlocked by increased activity of layer 5 pyramidal neurons of the mouse neocortex. Somatic depolarization or short bursts of action potentials of layer 5 pyramidal neurons induced a selective long-term potentiation of GABAergic synapses (LTPi) without affecting glutamatergic inputs. Remarkably, LTPi was selective for perisomatic inhibition from parvalbumin basket cells, leaving dendritic inhibition intact. It relied on retrograde signaling of nitric oxide, which persistently altered presynaptic GABA release and diffused to inhibitory synapses impinging on adjacent pyramidal neurons. LTPi reduced the time window of synaptic summation and increased the temporal precision of spike generation. Thus, increases in single cortical pyramidal neuron activity can induce an interneuron-selective GABAergic plasticity effectively altering the computation of temporally coded information.

Feedforward and disinhibitory circuits differentially control activity of cortical somatostatin interneurons during behavioral state transitions.

Interneurons (INs), specifically those in disinhibitory circuits like somatostatin (SST) and vasoactive intestinal peptide (VIP)-INs, are strongly modulated by the behavioral context. Yet, the mechanisms by which these INs are recruited during active states and whether their activity is consistent across sensory cortices remain unclear. We now report that in mice, locomotor activity strongly recruits SST-INs in the primary somatosensory (S1) but not the visual (V1) cortex. This diverse engagement of SST-INs cannot be explained by differences in VIP-IN function but is absent in the presence of visual input, suggesting the involvement of feedforward sensory pathways. Accordingly, inactivating the somatosensory thalamus, but not decreasing VIP-IN activity, significantly reduces the modulation of SST-INs by locomotion. Model simulations suggest that the differences in SST-INs across behavioral states can be explained by varying ratios of VIP- and thalamus-driven activity. By integrating feedforward activity with neuromodulation, SST-INs are anticipated to be crucial for adapting sensory processing to behavioral states.

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD.

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.

Aberrant survival of hippocampal Cajal-Retzius cells leads to memory deficits, gamma rhythmopathies and susceptibility to seizures in adult mice.

Cajal-Retzius cells (CRs) are transient neurons, disappearing almost completely in the postnatal neocortex by programmed cell death (PCD), with a percentage surviving up to adulthood in the hippocampus. Here, we evaluate CR's role in the establishment of adult neuronal and cognitive function using a mouse model preventing Bax-dependent PCD. CRs abnormal survival resulted in impairment of hippocampus-dependent memory, associated in vivo with attenuated theta oscillations and enhanced gamma activity in the dorsal CA1. At the cellular level, we observed transient changes in the number of NPY+ cells and altered CA1 pyramidal cell spine density. At the synaptic level, these changes translated into enhanced inhibitory currents in hippocampal pyramidal cells. Finally, adult mutants displayed an increased susceptibility to lethal tonic-clonic seizures in a kainate model of epilepsy. Our data reveal that aberrant survival of a small proportion of postnatal hippocampal CRs results in cognitive deficits and epilepsy-prone phenotypes in adulthood.

Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses.

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Activity-dependent modulation of NMDA receptors by endogenous zinc shapes dendritic function in cortical neurons.

NMDA receptors (NMDARs) have been proposed to control single-neuron computations in vivo. However, whether specific mechanisms regulate the function of such receptors and modulate input-output transformations performed by cortical neurons under in vivo-like conditions is understudied. Here, we report that in layer 2/3 pyramidal neurons (L2/3 PNs), repeated synaptic stimulation results in an activity-dependent decrease in NMDAR function by vesicular zinc. Such a mechanism shifts the threshold for dendritic non-linearities and strongly reduces LTP. Modulation of NMDARs is cell and pathway specific, being present selectively in L2/3-L2/3 connections but absent in inputs originating from L4 neurons. Numerical simulations highlight that activity-dependent modulation of NMDARs influences dendritic computations, endowing L2/3 PN dendrites with the ability to sustain non-linear integrations constant across different regimes of synaptic activity like those found in vivo. Our results unveil vesicular zinc as an important endogenous modulator of dendritic function in cortical PNs.

GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits.

GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.

Visual-area-specific tonic modulation of GABA release by endocannabinoids sets the activity and coordination of neocortical principal neurons.

Perisomatic inhibition of pyramidal neurons (PNs) coordinates cortical network activity during sensory processing, and this role is mainly attributed to parvalbumin-expressing basket cells (BCs). However, cannabinoid receptor type 1 (CB1)-expressing interneurons are also BCs, but the connectivity and function of these elusive but prominent neocortical inhibitory neurons are unclear. We find that their connectivity pattern is visual area specific. Persistently active CB1 signaling suppresses GABA release from CB1 BCs in the medial secondary visual cortex (V2M), but not in the primary visual cortex (V1). Accordingly, in vivo, tonic CB1 signaling is responsible for higher but less coordinated PN activity in the V2M than in the V1. These differential firing dynamics in the V1 and V2M can be captured by a computational network model that incorporates visual-area-specific properties. Our results indicate a differential CB1-mediated mechanism controlling PN activity, suggesting an alternative connectivity scheme of a specific GABAergic circuit in different cortical areas.

Adenosine A2A receptors control neuroinflammation and consequent hippocampal neuronal dysfunction.

The blockade of adenosine A(2A) receptors (A2AR) affords a robust neuroprotection in different noxious brain conditions. However, the mechanisms underlying this general neuroprotection are unknown. One possible mechanism could be the control of neuroinflammation that is associated with brain damage, especially because A2AR efficiently control peripheral inflammation. Thus, we tested if the intracerebroventricular injection of a selective A2AR antagonist (SCH58261) would attenuate the changes in the hippocampus triggered by intraperitoneal administration of lipopolysaccharide (LPS) that induces neuroinflammation through microglia activation. LPS administration triggers an increase in inflammatory mediators like interleukin-1ß that causes biochemical changes (p38 and c-jun N-terminal kinase phosphorylation and caspase 3 activation) contributing to neuronal dysfunction typified by decreased long-term potentiation, a form of synaptic plasticity. Long-term potentiation, measured 30 min after the tetanus, was significantly lower in LPS-treated rats compared with control-treated rats, while SCH58261 attenuated the LPS-induced change. The LPS-induced increases in phosphorylation of c-jun N-terminal kinase and p38 and activation of caspase 3 were also prevented by SCH58261. Significantly, SCH58261 also prevented the LPS-induced recruitment of activated microglial cells and the increase in interleukin-1ß concentration in the hippocampus, indicating that A2AR activation is a pivotal step in mediating the neuroinflammation triggered by LPS. These results indicate that A2AR antagonists prevent neuroinflammation and support the hypothesis that this mechanism might contribute for the ability of A2AR antagonists to control different neurodegenerative diseases known to involve neuroinflammation.

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo.

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Activity-dependent synaptic plasticity of NMDA receptors.

Activity-dependent, bidirectional control of synaptic efficacy is thought to contribute to many forms of experience-dependent plasticity, including learning and memory. Although most excitatory synapses contain both AMPA and N-methyl-d-aspartate receptors (AMPARs and NMDARs), most studies have focused on the plasticity of synaptic AMPARs, and on the pivotal role of NMDA receptors for its induction. Here we review evidence that synaptic NMDARs themselves are subject to long-term activity-dependent changes by mechanisms that may differ from that of synaptic AMPARs. The bidirectional modulation of NMDAR-mediated synaptic responses is likely to have important functional implications for NMDAR-dependent forms of synaptic plasticity.

A critical role of the adenosine A2A receptor in extrastriatal neurons in modulating psychomotor activity as revealed by opposite phenotypes of striatum and forebrain A2A receptor knock-outs.

The function of striatal adenosine A(2A) receptors (A(2A)Rs) is well recognized because of their high expression levels and the documented antagonistic interaction between A(2A)Rs and dopamine D(2) receptors in the striatum. However, the role of extrastriatal A(2A)Rs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A(2A)Rs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A(2A)Rs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A(2A)R knock-out (st-A(2A)R KO) mice in comparison with forebrain-specific A(2A)R KO (fb-A(2A)R KO) mice. In contrast to fb-A(2A)R KO (deleting A(2A)Rs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A(2A)R KO mice exhibited Cre-mediated selective deletion of the A(2A)R gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A(2A)R KO but attenuated in fb-A(2A)R KO mice. Furthermore, selective inactivation of the A(2A)Rs in extrastriatal cells by administering the A(2A)R antagonist KW6002 into st-A(2A)R KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A(2A)Rs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A(2A)Rs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A(2A)Rs.

Adenosine A2A receptor antagonists exert motor and neuroprotective effects by distinct cellular mechanisms.

OBJECTIVE: To investigate whether the motor and neuroprotective effects of adenosine A(2A) receptor (A(2A)R) antagonists are mediated by distinct cell types in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. METHODS: We used the forebrain A(2A)R knock-out mice coupled with flow cytometric analyses and intracerebroventricular injection to determine the contribution of A(2A)Rs in forebrain neurons and glial cells to A(2A)R antagonist-mediated motor and neuroprotective effects. RESULTS: The selective deletion of A(2A)Rs in forebrain neurons abolished the motor stimulant effects of the A(2A)R antagonist KW-6002 but did not affect acute MPTP neurotoxicity. Intracerebroventricular administration of KW-6002 into forebrain A(2A)R knock-out mice reinstated protection against acute MPTP-induced dopaminergic neurotoxicity and attenuated MPTP-induced striatal microglial and astroglial activation. INTERPRETATION: A(2A)R activity in forebrain neurons is critical to the control of motor activity, whereas brain cells other than forebrain neurons (likely glial cells) are important components for protection against acute MPTP toxicity.

Distinct Nanoscale Calcium Channel and Synaptic Vesicle Topographies Contribute to the Diversity of Synaptic Function.

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.