Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome.

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%

Btbd11 supports cell-type-specific synaptic function.

Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific specializations in the composition of glutamatergic synapses, identifying Btbd11 as an inhibitory interneuron-specific, synapse-enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins, including Psd-95. Intriguingly, we show that Btbd11 can undergo liquid-liquid phase separation when expressed with Psd-95, supporting the idea that the glutamatergic postsynaptic density in synapses in inhibitory interneurons exists in a phase-separated state. Knockout of Btbd11 decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons alters exploratory behavior, measures of anxiety, and sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific mechanism that supports glutamatergic synapse function in inhibitory interneurons-with implications for circuit function and animal behavior.

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina.

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

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.

An optimized CRISPR/Cas9 approach for precise genome editing in neurons.

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

Lack of redundancy between electrophysiological measures of long-range neuronal communication.

BACKGROUND: Communication between brain areas has been implicated in a wide range of cognitive and emotive functions and is impaired in numerous mental disorders. In rodent models, various metrics have been used to quantify inter-regional neuronal communication. However, in individual studies, typically, only very few measures of coupling are reported and, hence, redundancy across such indicators is implicitly assumed. RESULTS: In order to test this assumption, we here comparatively assessed a broad range of directional and non-directional metrics like coherence, Weighted Phase Lag Index (wPLI), phase-locking value (PLV), pairwise phase consistency (PPC), parametric and non-parametric Granger causality (GC), partial directed coherence (PDC), directed transfer function (DTF), spike-phase coupling (SPC), cross-regional phase-amplitude coupling, amplitude cross-correlations and others. We applied these analyses to simultaneous field recordings from the prefrontal cortex and the ventral and dorsal hippocampus in the schizophrenia-related Gria1-knockout mouse model which displays a robust novelty-induced hyperconnectivity phenotype. Using the detectability of coupling deficits in Gria1-/- mice and bivariate correlations within animals as criteria, we found that across such measures, there is a considerable lack of functional redundancy. Except for three pairwise correlations-PLV with PPC, PDC with DTF and parametric with non-parametric Granger causality-almost none of the analysed metrics consistently co-varied with any of the other measures across the three connections and two genotypes analysed. Notable exceptions to this were the correlation of coherence with PPC and PLV that was found in most cases, and partial correspondence between these three measures and Granger causality. Perhaps most surprisingly, partial directed coherence and Granger causality-sometimes regarded as equivalent measures of directed influence-diverged profoundly. Also, amplitude cross-correlation, spike-phase coupling and theta-gamma phase-amplitude coupling each yielded distinct results compared to all other metrics. CONCLUSIONS: Our analysis highlights the difficulty of quantifying real correlates of inter-regional information transfer, underscores the need to assess multiple coupling measures and provides some guidelines which metrics to choose for a comprehensive, yet non-redundant characterization of functional connectivity.

Hippocampal Hyperactivity as a Druggable Circuit-Level Origin of Aberrant Salience in Schizophrenia.

The development of current neuroleptics was largely aiming to decrease excessive dopaminergic signaling in the striatum. However, the notion that abnormal dopamine creates psychotic symptoms by causing an aberrant assignment of salience that drives maladaptive learning chronically during disease development suggests a therapeutic value of early interventions that correct salience-related neural processing. The mesolimbic dopaminergic output is modulated by several interconnected brain-wide circuits centrally involving the hippocampus and key relays like the ventral and associative striatum, ventral pallidum, amygdala, bed nucleus of the stria terminalis, nucleus reuniens, lateral and medial septum, prefrontal and cingulate cortex, among others. Unraveling the causal relationships between these circuits using modern neuroscience techniques holds promise for identifying novel cellular-and ultimately molecular-treatment targets for reducing transition to psychosis and symptoms of schizophrenia. Imaging studies in humans have implicated a hyperactivity of the hippocampus as a robust and early endophenotype in schizophrenia. Experiments in rodents, in turn, suggested that the activity of its output region-the ventral subiculum-may modulate dopamine release from ventral tegmental area (VTA) neurons in the ventral striatum. Even though these observations suggested a novel circuit-level target for anti-psychotic action, no therapy has yet been developed along this rationale. Recently evaluated treatment strategies-at least in part-target excess glutamatergic activity, e.g. N-acetyl-cysteine (NAC), levetiracetam, and mGluR2/3 modulators. We here review the evidence for the central implication of the hippocampus-VTA axis in schizophrenia-related pathology, discuss its symptom-related implications with a particular focus on aberrant assignment of salience, and evaluate some of its short-comings and prospects for drug discovery.

Tyrosine phosphorylation of the AMPA receptor subunit GluA2 gates homeostatic synaptic plasticity.

Hebbian plasticity, comprised of long-term potentiation (LTP) and depression (LTD), allows neurons to encode and respond to specific stimuli; while homeostatic synaptic scaling is a counterbalancing mechanism that enables the maintenance of stable neural circuits. Both types of synaptic plasticity involve the control of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) abundance, which is modulated by AMPAR phosphorylation. To address the necessity of GluA2 phospho-Y876 in synaptic plasticity, we generated phospho-deficient GluA2 Y876F knock-in mice. We show that, while GluA2 phospho-Y876 is not necessary for Hebbian plasticity, it is essential for both in vivo and in vitro homeostatic upscaling. Bidirectional changes in GluA2 phospho-Y876 were observed during homeostatic scaling, with a decrease during downscaling and an increase during upscaling. GluA2 phospho-Y876 is necessary for synaptic accumulation of glutamate receptor interacting protein 1 (GRIP1), a crucial scaffold protein that delivers AMPARs to synapses, during upscaling. Furthermore, increased phosphorylation at GluA2 Y876 increases GluA2 binding to GRIP1. These results demonstrate that AMPAR trafficking during homeostatic upscaling can be gated by a single phosphorylation site on the GluA2 subunit.

Can N-Methyl-D-Aspartate Receptor Hypofunction in Schizophrenia Be Localized to an Individual Cell Type?

Hypofunction of N-methyl-D-aspartate glutamate receptors (NMDARs), whether caused by endogenous factors like auto-antibodies or mutations, or by pharmacological or genetic manipulations, produces a wide variety of deficits which overlap with-but do not precisely match-the symptom spectrum of schizophrenia. In order to understand how NMDAR hypofunction leads to different components of the syndrome, it is necessary to take into account which neuronal subtypes are particularly affected by it in terms of detrimental functional alterations. We provide a comprehensive overview detailing findings in rodent models with cell type-specific knockout of NMDARs. Regarding inhibitory cortical cells, an emerging model suggests that NMDAR hypofunction in parvalbumin (PV) positive interneurons is a potential risk factor for this disease. PV interneurons display a selective vulnerability resulting from a combination of genetic, cellular, and environmental factors that produce pathological multi-level positive feedback loops. Central to this are two antioxidant mechanisms-NMDAR activity and perineuronal nets-which are themselves impaired by oxidative stress, amplifying disinhibition. However, NMDAR hypofunction in excitatory pyramidal cells also produces a range of schizophrenia-related deficits, in particular maladaptive learning and memory recall. Furthermore, NMDAR blockade in the thalamus disturbs thalamocortical communication, and NMDAR ablation in dopaminergic neurons may provoke over-generalization in associative learning, which could relate to the positive symptom domain. Therefore, NMDAR hypofunction can produce schizophrenia-related effects through an action on various different circuits and cell types.

Hippocampal-prefrontal coherence mediates working memory and selective attention at distinct frequency bands and provides a causal link between schizophrenia and its risk gene GRIA1.

Increased fronto-temporal theta coherence and failure of its stimulus-specific modulation have been reported in schizophrenia, but the psychological correlates and underlying neural mechanisms remain elusive. Mice lacking the putative schizophrenia risk gene GRIA1 (Gria1-/-), which encodes GLUA1, show strongly impaired spatial working memory and elevated selective attention owing to a deficit in stimulus-specific short-term habituation. A failure of short-term habituation has been suggested to cause an aberrant assignment of salience and thereby psychosis in schizophrenia. We recorded hippocampal-prefrontal coherence while assessing spatial working memory and short-term habituation in these animals, wildtype (WT) controls, and Gria1-/- mice in which GLUA1 expression was restored in hippocampal subfields CA2 and CA3. We found that beta (20-30 Hz) and low-gamma (30-48 Hz) frequency coherence could predict working memory performance, whereas-surprisingly-theta (6-12 Hz) coherence was unrelated to performance and largely unaffected by genotype in this task. In contrast, in novel environments, theta coherence specifically tracked exploration-related attention in WT mice, but was strongly elevated and unmodulated in Gria1-knockouts, thereby correlating with impaired short-term habituation. Strikingly, reintroduction of GLUA1 selectively into CA2/CA3 restored abnormal short-term habituation, theta coherence, and hippocampal and prefrontal theta oscillations. Although local oscillations and coherence in other frequency bands (beta, gamma), and theta-gamma cross-frequency coupling also showed dependence on GLUA1, none of them correlated with short-term habituation. Therefore, sustained elevation of hippocampal-prefrontal theta coherence may underlie a failure in regulating novelty-related selective attention leading to aberrant salience, and thereby represents a mechanistic link between GRIA1 and schizophrenia.

Optogenetic induction of the schizophrenia-related endophenotype of ventral hippocampal hyperactivity causes rodent correlates of positive and cognitive symptoms.

Pathological over-activity of the CA1 subfield of the human anterior hippocampus has been identified as a potential predictive marker for transition from a prodromal state to overt schizophrenia. Psychosis, in turn, is associated with elevated activity in the anterior subiculum, the hippocampal output stage directly activated by CA1. Over-activity in these subfields may represent a useful endophenotype to guide translationally predictive preclinical models. To recreate this endophenotype and study its causal relation to deficits in the positive and cognitive symptom domains, we optogenetically activated excitatory neurons of the ventral hippocampus (vHPC; analogous to the human anterior hippocampus), targeting the ventral subiculum. Consistent with previous studies, we found that vHPC over-activity evokes hyperlocomotion, a rodent correlate of positive symptoms. vHPC activation also impaired performance on the spatial novelty preference (SNP) test of short-term memory, regardless of whether stimulation was applied during the encoding or retrieval stage of the task. Increasing dopamine transmission with amphetamine produced hyperlocomotion, but was not associated with SNP impairments. This suggests that short-term memory impairments resulting from hippocampal over-activity likely arise independently of a hyperdopaminergic state, a finding that is consistent with the pharmaco-resistance of cognitive symptoms in patients.

Gene-Environment Interaction in a Conditional NMDAR-Knockout Model of Schizophrenia.

Interactions between genetic and environmental risk factors take center stage in the pathology of schizophrenia. We assessed if the stressor of reduced environmental enrichment applied in adulthood provokes deficits in the positive, negative or cognitive symptom domains of schizophrenia in a mouse line modeling NMDA-receptor (NMDAR) hypofunction in forebrain inhibitory interneurons (Grin1 ΔPpp1r2 ). We find that Grin1 ΔPpp1r2 mice, when group-housed in highly enriched cages, appear largely normal across a wide range of schizophrenia-related behavioral tests. However, they display various short-term memory deficits when exposed to minimal enrichment. This demonstrates that the interaction between risk genes causing NMDA-receptor hypofunction and environmental risk factors may negatively impact cognition later in life.

PICK1 regulates AMPA receptor endocytosis via direct interactions with AP2 α-appendage and dynamin.

Clathrin-mediated endocytosis (CME) is used to internalize a diverse range of cargo proteins from the cell surface, often in response to specific signals. In neurons, the rapid endocytosis of GluA2-containing AMPA receptors (AMPARs) in response to NMDA receptor (NMDAR) stimulation causes a reduction in synaptic strength and is the central mechanism for long-term depression, which underlies certain forms of learning. The mechanisms that link NMDAR activation to CME of AMPARs remain elusive. PICK1 is a BAR domain protein required for NMDAR-dependent reductions in surface GluA2; however, the molecular mechanisms involved are unclear. In this study, we show that PICK1 makes direct, NMDAR-dependent interactions with the core endocytic proteins AP2 and dynamin. PICK1-AP2 interactions are required for clustering AMPARs at endocytic zones in dendrites in response to NMDAR stimulation and for consequent AMPAR internalization. We further show that PICK1 stimulates dynamin polymerization. We propose that PICK1 is a cargo-specific endocytic accessory protein required for efficient, activity-dependent AMPAR endocytosis.

The group II metabotropic glutamate receptor agonist LY354740 and the D2 receptor antagonist haloperidol reduce locomotor hyperactivity but fail to rescue spatial working memory in GluA1 knockout mice.

Group II metabotropic glutamate receptor agonists have been suggested as potential anti-psychotics, at least in part, based on the observation that the agonist LY354740 appeared to rescue the cognitive deficits caused by non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonists, including spatial working memory deficits in rodents. Here, we tested the ability of LY354740 to rescue spatial working memory performance in mice that lack the GluA1 subunit of the AMPA glutamate receptor, encoded by Gria1, a gene recently implicated in schizophrenia by genome-wide association studies. We found that LY354740 failed to rescue the spatial working memory deficit in Gria1-/- mice during rewarded alternation performance in the T-maze. In contrast, LY354740 did reduce the locomotor hyperactivity in these animals to a level that was similar to controls. A similar pattern was found with the dopamine receptor antagonist haloperidol, with no amelioration of the spatial working memory deficit in Gria1-/- mice, even though the same dose of haloperidol reduced their locomotor hyperactivity. These results with LY354740 contrast with the rescue of spatial working memory in models of glutamatergic hypofunction using non-competitive NMDAR antagonists. Future studies should determine whether group II mGluR agonists can rescue spatial working memory deficits with other NMDAR manipulations, including genetic models and other pharmacological manipulations of NMDAR function.

Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination.

Sparse coding may be a general strategy of neural systems for augmenting memory capacity. In Drosophila melanogaster, sparse odor coding by the Kenyon cells of the mushroom body is thought to generate a large number of precisely addressable locations for the storage of odor-specific memories. However, it remains untested how sparse coding relates to behavioral performance. Here we demonstrate that sparseness is controlled by a negative feedback circuit between Kenyon cells and the GABAergic anterior paired lateral (APL) neuron. Systematic activation and blockade of each leg of this feedback circuit showed that Kenyon cells activated APL and APL inhibited Kenyon cells. Disrupting the Kenyon cell-APL feedback loop decreased the sparseness of Kenyon cell odor responses, increased inter-odor correlations and prevented flies from learning to discriminate similar, but not dissimilar, odors. These results suggest that feedback inhibition suppresses Kenyon cell activity to maintain sparse, decorrelated odor coding and thus the odor specificity of memories.