Calcium-permeable AMPA receptors govern PV neuron feature selectivity.

The brain helps us survive by forming internal representations of the external world1,2. Excitatory cortical neurons are often precisely tuned to specific external stimuli3,4. However, inhibitory neurons, such as parvalbumin-positive (PV) interneurons, are generally less selective5. PV interneurons differ from excitatory neurons in their neurotransmitter receptor subtypes, including AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs)6,7. Excitatory neurons express calcium-impermeable AMPARs that contain the GluA2 subunit (encoded by GRIA2), whereas PV interneurons express receptors that lack the GluA2 subunit and are calcium-permeable (CP-AMPARs). Here we demonstrate a causal relationship between CP-AMPAR expression and the low feature selectivity of PV interneurons. We find low expression stoichiometry of GRIA2 mRNA relative to other subunits in PV interneurons that is conserved across ferrets, rodents, marmosets and humans, and causes abundant CP-AMPAR expression. Replacing CP-AMPARs in PV interneurons with calcium-impermeable AMPARs increased their orientation selectivity in the visual cortex. Manipulations to induce sparse CP-AMPAR expression demonstrated that this increase was cell-autonomous and could occur with changes beyond development. Notably, excitatory-PV interneuron connectivity rates and unitary synaptic strength were unaltered by CP-AMPAR removal, which suggested that the selectivity of PV interneurons can be altered without markedly changing connectivity. In Gria2-knockout mice, in which all AMPARs are calcium-permeable, excitatory neurons showed significantly degraded orientation selectivity, which suggested that CP-AMPARs are sufficient to drive lower selectivity regardless of cell type. Moreover, hippocampal PV interneurons, which usually exhibit low spatial tuning, became more spatially selective after removing CP-AMPARs, which indicated that CP-AMPARs suppress the feature selectivity of PV interneurons independent of modality. These results reveal a new role of CP-AMPARs in maintaining low-selectivity sensory representation in PV interneurons and implicate a conserved molecular mechanism that distinguishes this cell type in the neocortex.

Stimulus-dependent synaptic plasticity underlies neuronal circuitry refinement in the mouse primary visual cortex.

Perceptual learning improves our ability to interpret sensory stimuli present in our environment through experience. Despite its importance, the underlying mechanisms that enable perceptual learning in our sensory cortices are still not fully understood. In this study, we used in vivo two-photon imaging to investigate the functional and structural changes induced by visual stimulation in the mouse primary visual cortex (V1). Our results demonstrate that repeated stimulation leads to a refinement of V1 circuitry by decreasing the number of responsive neurons while potentiating their response. At the synaptic level, we observe a reduction in the number of dendritic spines and an overall increase in spine AMPA receptor levels in the same subset of neurons. In addition, visual stimulation induces synaptic potentiation in neighboring spines within individual dendrites. These findings provide insights into the mechanisms of synaptic plasticity underlying information processing in the neocortex.

SynGAP regulates synaptic plasticity and cognition independently of its catalytic activity.

SynGAP is an abundant synaptic GTPase-activating protein (GAP) critical for synaptic plasticity, learning, memory, and cognition. Mutations in SYNGAP1 in humans result in intellectual disability, autistic-like behaviors, and epilepsy. Heterozygous Syngap1-knockout mice display deficits in synaptic plasticity, learning, and memory and exhibit seizures. It is unclear whether SynGAP imparts structural properties at synapses independently of its GAP activity. Here, we report that inactivating mutations within the GAP domain do not inhibit synaptic plasticity or cause behavioral deficits. Instead, SynGAP modulates synaptic strength by physically competing with the AMPA-receptor-TARP excitatory receptor complex in the formation of molecular condensates with synaptic scaffolding proteins. These results have major implications for developing therapeutic treatments for SYNGAP1-related neurodevelopmental disorders.

Mouse models of SYNGAP1-related intellectual disability.

SYNGAP1 is a Ras-GTPase-activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDDs). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), an NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances. Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning, and memory and have seizures. However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A. While reduction in Syngap1 mRNA varies from 30 to 50% depending on the specific mutation, both models show ~50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder.

Mouse models of SYNGAP1 -related intellectual disability.

SYNGAP1 is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDD). These mutations are highly penetrant and cause SYNGAP1 -related intellectual disability (SRID), a NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances (1-5). Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function (6-11), and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning and memory, and have seizures (9, 12-14). However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A . While reduction in Syngap1 mRNA varies from 30-50% depending on the specific mutation, both models show ∼50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder. Significance Statement: SYNGAP1 is a protein enriched at excitatory synapses in the brain that is an important regulator of synapse structure and function. SYNGAP1 mutations cause SYNGAP1 -related intellectual disability (SRID), a neurodevelopmental disorder with cognitive impairment, social deficits, seizures, and sleep disturbances. To explore how SYNGAP1 mutations found in humans lead to disease, we generated the first knock-in mouse models with causal SRID variants: one with a frameshift mutation and a second with an intronic mutation that creates a cryptic splice acceptor site. Both models show decreased Syngap1 mRNA and Syngap1 protein and recapitulate key features of SRID including hyperactivity and impaired working memory. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies. Highlights: Two mouse models with SYNGAP1 -related intellectual disability (SRID) mutations found in humans were generated: one with a frameshift mutation that results in a premature stop codon and the other with an intronic mutation resulting in a cryptic splice acceptor site and premature stop codon. Both SRID mouse models show 35∼50% reduction in mRNA and ∼50% reduction in Syngap1 protein.Both SRID mouse models display deficits in synaptic plasticity and behavioral phenotypes found in people. RNA-seq confirmed cryptic splice acceptor activity in one SRID mouse model and revealed broad transcriptional changes also identified in Syngap1 +/- mice. Novel SRID mouse models generated here provide a resource and establish a framework for development of future therapeutic intervention.

Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors.

Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking their dynamic expression during behavior is crucial to understand cognition and disease, but current technological barriers preclude large-scale exploration of molecular dynamics in vivo. We have developed a suite of innovative methodologies that break through these barriers: a new knockin mouse line with fluorescently tagged endogenous AMPA receptors, two-photon imaging of hundreds of thousands of labeled synapses in behaving mice, and computer vision-based automatic synapse detection. Using these tools, we can longitudinally track how the strength of populations of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synapses undergoing changes in strength following sensory experience. More generally, these tools can be used as an optical probe capable of measuring functional synapse strength across entire brain areas during any behavioral paradigm, describing complex system-wide changes with molecular precision.

All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity.

Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected form of experience-dependent circuit plasticity. In the layer 2/3 of mouse visual cortex, monocular deprivation triggers a complete, "all-or-none," elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (Pyr→PV). This binary form of circuit plasticity is unique, as it is transient, local, and discrete. It lasts only 1 d, and it does not manifest as widespread changes in synaptic strength; rather, only about half of local connections are lost, and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of Pyr→PV is contingent on a reduction of the protein neuropentraxin2. Functionally, the loss of Pyr→PV is absolutely necessary for ocular dominance plasticity, a canonical model of deprivation-induced model of cortical remodeling. We surmise, therefore, that this all-or-none loss of local Pyr→PV circuitry gates experience-dependent cortical plasticity.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

GSK-3ß activation is required for ZIP-induced disruption of learned fear.

The myristoylated zeta inhibitory peptide (ZIP), which was originally developed as a protein kinase C/Mζ (PKCζ/PKMζ) inhibitor, is known to produce the loss of different forms of memories. However, ZIP induces memory loss even in the absence of PKMζ, and its mechanism of action, therefore, remains elusive. Here, through a kinome-wide screen, we found that glycogen synthase kinase 3 beta (GSK-3ß) was robustly activated by ZIP in vitro. ZIP induced depotentiation (a cellular substrate of memory erasure) of conditioning-induced potentiation at LA synapses, and the ZIP-induced depotentiation was prevented by a GSK-3ß inhibitor, 6-bromoindirubin-3-acetoxime (BIO-acetoxime). Consistently, GSK-3ß inhibition by BIO-acetoxime infusion or GSK-3ß knockdown by GSK-3ß shRNA in the LA attenuated ZIP-induced disruption of learned fear. Furthermore, conditioned fear was decreased by expression of a non-inhibitable form of GSK-3ß in the LA. Our findings suggest that GSK-3ß activation is a critical step for ZIP-induced disruption of memory.

SynGAP isoforms differentially regulate synaptic plasticity and dendritic development.

SynGAP is a synaptic Ras GTPase-activating protein (GAP) with four C-terminal splice variants: α1, α2, ß, and γ. Although studies have implicated SYNGAP1 in several cognitive disorders, it is not clear which SynGAP isoforms contribute to disease. Here, we demonstrate that SynGAP isoforms exhibit unique spatiotemporal expression patterns and play distinct roles in neuronal and synaptic development in mouse neurons. SynGAP-α1, which undergoes liquid-liquid phase separation with PSD-95, is highly enriched in synapses and is required for LTP. In contrast, SynGAP-ß, which does not bind PSD-95 PDZ domains, is less synaptically targeted and promotes dendritic arborization. A mutation in SynGAP-α1 that disrupts phase separation and synaptic targeting abolishes its ability to regulate plasticity and instead causes it to drive dendritic development like SynGAP-ß. These results demonstrate that distinct intrinsic biochemical properties of SynGAP isoforms determine their function, and individual isoforms may differentially contribute to the pathogenesis of SYNGAP1-related cognitive disorders.

Cortical Synaptic AMPA Receptor Plasticity during Motor Learning.

Modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) is a fundamental mechanism underlying synaptic plasticity, learning, and memory. However, the dynamics of AMPAR trafficking in vivo and its correlation with learning have not been resolved. Here, we used in vivo two-photon microscopy to visualize surface AMPARs in mouse cortex during the acquisition of a forelimb reaching task. Daily training leads to an increase in AMPAR levels at a subset of spatially clustered dendritic spines in the motor cortex. Surprisingly, we also observed increases in spine AMPAR levels in the visual cortex. There, synaptic potentiation depends on the availability of visual input during motor training, and optogenetic inhibition of visual cortex activity impairs task performance. These results indicate that motor learning induces widespread cortical synaptic potentiation by increasing the net trafficking of AMPARs into spines, including in non-motor brain regions.

Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-Dependent Homeostatic Scaling in Cortical Neurons.

Regulation of AMPA-type glutamate receptor (AMPAR) number at synapses is a major mechanism for controlling synaptic strength during homeostatic scaling in response to global changes in neural activity. We show that the secreted guidance cue semaphorin 3F (Sema3F) and its neuropilin-2 (Npn-2)/plexinA3 (PlexA3) holoreceptor mediate homeostatic plasticity in cortical neurons. Sema3F-Npn-2/PlexA3 signaling is essential for cell surface AMPAR homeostatic downscaling in response to an increase in neuronal activity, Npn-2 associates with AMPARs, and Sema3F regulates this interaction. Therefore, Sema3F-Npn-2/PlexA3 signaling controls both synapse development and synaptic plasticity.

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy.

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases.

Sound tuning of amygdala plasticity in auditory fear conditioning.

Various auditory tones have been used as conditioned stimuli (CS) for fear conditioning, but researchers have largely neglected the effect that different types of auditory tones may have on fear memory processing. Here, we report that at lateral amygdala (LA) synapses (a storage site for fear memory), conditioning with different types of auditory CSs (2.8 kHz tone, white noise, FM tone) recruits distinct forms of long-term potentiation (LTP) and inserts calcium permeable AMPA receptor (CP-AMPAR) for variable periods. White noise or FM tone conditioning produced brief insertion (<6 hr after conditioning) of CP-AMPARs, whereas 2.8 kHz tone conditioning induced more persistent insertion (≥6 hr). Consistently, conditioned fear to 2.8 kHz tone but not to white noise or FM tones was erased by reconsolidation-update (which depends on the insertion of CP-AMPARs at LA synapses) when it was performed 6 hr after conditioning. Our data suggest that conditioning with different auditory CSs recruits distinct forms of LA synaptic plasticity, resulting in more malleable fear memory to some tones than to others.

Functional Coupling with Cardiac Muscle Promotes Maturation of hPSC-Derived Sympathetic Neurons.

Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.

The nutrient sensor OGT in PVN neurons regulates feeding.

Maintaining energy homeostasis is crucial for the survival and health of organisms. The brain regulates feeding by responding to dietary factors and metabolic signals from peripheral organs. It is unclear how the brain interprets these signals. O-GlcNAc transferase (OGT) catalyzes the posttranslational modification of proteins by O-GlcNAc and is regulated by nutrient access. Here, we show that acute deletion of OGT from αCaMKII-positive neurons in adult mice caused obesity from overeating. The hyperphagia derived from the paraventricular nucleus (PVN) of the hypothalamus, where loss of OGT was associated with impaired satiety. These results identify O-GlcNAcylation in αCaMKII neurons of the PVN as an important molecular mechanism that regulates feeding behavior.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

mGluR2/3 in the Lateral Amygdala is Required for Fear Extinction: Cortical Input Synapses onto the Lateral Amygdala as a Target Site of the mGluR2/3 Action.

Various subtypes of metabotropic glutamate receptors (mGluRs) have been implicated in fear extinction, but mGluR2/3 subtype has not been tested. Here, we found that microinjection of an mGluR2/3 antagonist, LY341495, into the lateral amygdala (LA), but not into the adjacent central amygdala (CeA), impaired extinction retention without affecting within-session extinction. In contrast, we failed to detect any significant changes in motility and anxiety during a period when extinction training or retention was performed after LY341495 injection, suggesting that the effect of LY341495 is specific to conditioned responses. Subsequently, on the basis of a previous finding that a long-term potentiation of presynaptic efficacy at cortical input synapses onto the lateral amygdala (C-LA synapses) supports conditioned fear, we tested the hypothesis that activation of mGluR2/3 leads to fear extinction via a long-term weakening of presynaptic functions at C-LA synapses. Fear extinction produced a decrease in C-LA synaptic efficacy, whereas LY341495 infusion into the LA blocked this extinction-induced C-LA efficacy decrease without altering synaptic efficacy at other LA synapses. Furthermore, extinction enhanced paired pulse ratio (PPR) of EPSCs, which inversely correlates with presynaptic release probability, whereas LY341495 infusion into the LA attenuated the extinction-induced increase in PPR, suggesting the presence of mGluR2/3-dependent presynaptic changes after extinction. Consistently, extinction occluded a presynaptic form of depression at C-LA synapses, whereas the LY341495 infusion into the LA rescued this occlusion. Together, our findings suggest that mGluR2/3 is required for extinction retention and that the mGluR2/3 action is mediated by the long-term weakening of release probability at C-LA synapses.

Group I mGluR-dependent depotentiation in the lateral amygdala does not require the removal of calcium-permeable AMPA receptors.

There is conflicting evidence regarding whether calcium-permeable receptors are removed during group I mGluR-mediated synaptic depression. In support of this hypothesis, AMPAR rectification, a correlative index of the synaptic expression of GluA2-lacking calcium-permeable AMPARs (CP-AMPARs), is known to decrease after the induction of several types of group I mGluR-mediated long-term depression (LTD), suggesting that a significant proportion of synaptic CP-AMPARs is removed during synaptic depression. We have previously demonstrated that fear conditioning-induced synaptic potentiation in the lateral amygdala is reversed by group 1 mGluR-mediated depotentiation. Here, we examined whether CP-AMPARs are removed by mGluR1-mediated depotentiation of fear conditioning-induced synaptic potentiation. The synaptic expression of CP-AMPARs was negligible before, increased significantly 12 h after, and returned to baseline 48 h after fear conditioning, as evidenced by the changes in the sensitivity of lateral amygdala synaptic responses to NASPM. Importantly, the sensitivity to NASPM was not altered after induction of depotentiation. Our findings, together with previous results, suggest that the removal of CP-AMPARs is not required for the depotentiation of fear conditioning-induced synaptic potentiation at lateral amygdala synapses.

Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment.

Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer's disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins. We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly-Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift. We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.