AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons.

The metabolic prohormone pro-opiomelanocortin (POMC) is generally translocated into the endoplasmic reticulum (ER) for entry into the secretory pathway. Patients with mutations within the signal peptide (SP) of POMC or its adjoining segment develop metabolic disorders. However, the existence, metabolic fate, and functional outcomes of cytosol-retained POMC remain unclear. Here, we show that SP-uncleaved POMC is produced in the cytosol of POMC neuronal cells, thus inducing ER stress and ferroptotic cell death. Mechanistically, the cytosol-retained POMC sequesters the chaperone Hspa5 and subsequently accelerates degradation of the glutathione peroxidase Gpx4, a core regulator of ferroptosis, via the chaperone-mediated autophagy. We also show that the Marchf6 E3 ubiquitin ligase mediates the degradation of cytosol-retained POMC, thereby preventing ER stress and ferroptosis. Furthermore, POMC-Cre-mediated Marchf6-deficient mice exhibit hyperphagia, reduced energy expenditure, and weight gain. These findings suggest that Marchf6 is a critical regulator of ER stress, ferroptosis, and metabolic homeostasis in POMC neurons.

Npas4-mediated dopaminergic regulation of safety memory consolidation.

Amygdala circuitry encodes associations between conditioned stimuli and aversive unconditioned stimuli and also controls fear expression. However, whether and how non-threatening information for unpaired conditioned stimuli (CS-) is discretely processed remains unknown. The fear expression toward CS- is robust immediately after fear conditioning but then becomes negligible after memory consolidation. The synaptic plasticity of the neural pathway from the lateral to the anterior basal amygdala gates the fear expression of CS-, depending upon neuronal PAS domain protein 4 (Npas4)-mediated dopamine receptor D4 (Drd4) synthesis, which is precluded by stress exposure or corticosterone injection. Herein, we show cellular and molecular mechanisms that regulate the non-threatening (safety) memory consolidation, supporting the fear discrimination.

Rewiring of Prelimbic Inputs to the Nucleus Accumbens Core Underlies Cocaine-Induced Behavioral Sensitization.

BACKGROUND: Unbalanced activity of medium spiny neurons (MSNs) of the direct and indirect pathways mediates reward-related behaviors induced by addictive drugs. Prelimbic (PL) input to MSNs in the nucleus accumbens core (NAcC) plays a key role in cocaine-induced early locomotor sensitization (LS). However, the adaptive plastic changes at PL-to-NAcC synapses underlying early LS remain unclear. METHODS: Using transgenic mice and retrograde tracing, we identified NAcC-projecting pyramidal neurons (PNs) in the PL cortex based on the expression of dopamine receptor types (D1R or D2R). To examine cocaine-induced alterations in PL-to-NAcC synapses, we measured excitatory postsynaptic current amplitudes evoked by optostimulation of PL afferents to MSNs. Riluzole was chosen to test the effects of PL excitability on cocaine-induced changes of PL-to-NAcC synapses. RESULTS: NAcC-projecting PNs were segregated into D1R- and D2R-expressing PNs (D1- and D2-PNs, respectively), and their excitability was opposingly regulated by respective dopamine agonists. Both D1- and D2-PNs exhibited balanced innervation of direct MSNs and indirect MSNs in naïve animals. Repeated cocaine injections resulted in biased synaptic strength toward direct MSNs through presynaptic mechanisms in both D1- and D2-PNs, although D2R activation reduced the D2-PN excitability. Under group 1 metabotropic glutamate receptors coactivation, however, D2R activation enhanced the D2-PN excitability. The cocaine-induced rewiring accompanied LS, and both rewiring and LS were precluded by PL infusion of riluzole, which reduced the intrinsic excitability of PL neurons. CONCLUSIONS: These findings indicate that cocaine-induced rewiring of PL-to-NAcC synapses correlates well with early behavioral sensitization and that rewiring and LS can be prevented by riluzole-induced reduction of excitability of PL neurons.

Reversibility and developmental neuropathology of linear nevus sebaceous syndrome caused by dysregulation of the RAS pathway.

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous disorder caused by somatic gain-of-function mutations in KRAS or HRAS. LNSS brains have neurodevelopmental defects, including cerebral defects and epilepsy; however, its pathological mechanism and potentials for treatment are largely unclear. We show that introduction of KRASG12V in the developing mouse cortex results in subcortical nodular heterotopia and enhanced excitability, recapitulating major pathological manifestations of LNSS. Moreover, we show that decreased firing frequency of inhibitory neurons without KRASG12V expression leads to disrupted excitation and inhibition balance. Transcriptional profiling after destabilization domain-mediated clearance of KRASG12V in human neural progenitors and differentiating neurons identifies reversible functional networks underlying LNSS. Neurons expressing KRASG12V show molecular changes associated with delayed neuronal maturation, most of which are restored by KRASG12V clearance. These findings provide insights into the molecular networks underlying the reversibility of some of the neuropathologies observed in LNSS caused by dysregulation of the RAS pathway.

Deep brain stimulation by blood-brain-barrier-crossing piezoelectric nanoparticles generating current and nitric oxide under focused ultrasound.

Deep brain stimulation via implanted electrodes can alleviate neuronal disorders. However, its applicability is constrained by side effects resulting from the insertion of electrodes into the brain. Here, we show that systemically administered piezoelectric nanoparticles producing nitric oxide and generating direct current under high-intensity focused ultrasound can be used to stimulate deep tissue in the brain. The release of nitric oxide temporarily disrupted tight junctions in the blood-brain barrier, allowing for the accumulation of the nanoparticles into brain parenchyma, and the piezoelectrically induced output current stimulated the release of dopamine by dopaminergic neuron-like cells. In a mouse model of Parkinson's disease, the ultrasound-responsive nanoparticles alleviated the symptoms of the disease without causing overt toxicity. The strategy may inspire the development of other minimally invasive therapies for neurodegenerative diseases.

Synaptic ensembles between raphe and D1R-containing accumbens shell neurons underlie postisolation sociability in males.

Social animals expend considerable energy to maintain social bonds throughout their life. Male and female mice show sexually dimorphic behaviors, yet the underlying neural mechanisms of sociability and their dysregulation during social disconnection remain unknown. Dopaminergic neurons in dorsal raphe nucleus (DRNTH) is known to contribute to a loneliness-like state and modulate sociability. We identified that activated subpopulations in DRNTH and nucleus accumbens shell (NAcsh) during 24 hours of social isolation underlie the increase in isolation-induced sociability in male but not in female mice. This effect was reversed by chemogenetically and optogenetically inhibiting the DRNTH-NAcsh circuit. Moreover, synaptic connectivity among the activated neuronal ensembles in this circuit was increased, primarily in D1 receptor-expressing neurons in NAcsh. The increase in synaptic density functionally correlated with elevated dopamine release into NAcsh. Overall, specific synaptic ensembles in DRNTH-NAcsh mediate sex differences in isolation-induced sociability, indicating that sex-dependent circuit dynamics underlie the expression of sexually dimorphic behaviors.

Force Mapping Reveals the Spatial Distribution of Individual Proteins in a Neuron.

Conventional methods for studying the spatial distribution and expression level of proteins within neurons have primarily relied on immunolabeling and/or signal amplification. Here, we present an atomic force microscopy (AFM)-based nanoscale force mapping method, where Anti-LIMK1-tethered AFM probes were used to visualize individual LIMK1 proteins in cultured neurons directly through force measurements. We observed that the number density of LIMK1 decreased in neuronal somas after the cells were depolarized. We also elucidated the spatial distribution of LIMK1 in single spine areas and found that the protein predominantly locates at heads of spines rather than dendritic shafts. The study demonstrates that our method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons without signal amplification or labeling. We expected that this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.

Retinoic acid-induced protein 14 controls dendritic spine dynamics associated with depressive-like behaviors.

Dendritic spines are the central postsynaptic machinery that determines synaptic function. The F-actin within dendritic spines regulates their dynamic formation and elimination. Rai14 is an F-actin-regulating protein with a membrane-shaping function. Here, we identified the roles of Rai14 for the regulation of dendritic spine dynamics associated with stress-induced depressive-like behaviors. Rai14-deficient neurons exhibit reduced dendritic spine density in the Rai14+/- mouse brain, resulting in impaired functional synaptic activity. Rai14 was protected from degradation by complex formation with Tara, and accumulated in the dendritic spine neck, thereby enhancing spine maintenance. Concurrently, Rai14 deficiency in mice altered gene expression profile relevant to depressive conditions and increased depressive-like behaviors. Moreover, Rai14 expression was reduced in the prefrontal cortex of the mouse stress model, which was blocked by antidepressant treatment. Thus, we propose that Rai14-dependent regulation of dendritic spines may underlie the plastic changes of neuronal connections relevant to depressive-like behaviors.

LGI1 governs neuritin-mediated resilience to chronic stress.

Depression is accompanied by neuronal atrophy and decreased neuroplasticity. Leucine-rich glioma-inactivated protein 1 (LGI1), a metastasis suppressor, plays an important role in the development of CNS synapses. We found that LGI1 expression was reduced in the hippocampi of mice that underwent chronic unpredictable stress (CUS), and could be rescued by the antidepressant, fluoxetine. Recombinant soluble neuritin, an endogenous protein previously implicated in antidepressant-like behaviors, elevated hippocampal LGI1 expression in a manner dependent on histone deacetylase 5 (HDAC5) phosphorylation. Accordingly, Nrn1 flox/flox ;Pomc-cre (Nrn1 cOE) mice, which conditionally overexpress neuritin, displayed increases in hippocampal LGI1 level under CUS and exhibited resilience to CUS that were blocked by hippocampal depletion of LGI1. Interestingly, neuritin-mediated LGI1 expression was inhibited by HNMPA-(AM)3, an insulin receptor inhibitor, as was neuritin-mediated HDAC5 phosphorylation. We thus establish hippocampal LGI1 as an effector of neurite outgrowth and stress resilience, and suggest that HDAC5-LGI1 plays a critical role in ameliorating pathological depression.

Fear response-based prediction for stress susceptibility to PTSD-like phenotypes.

Most individuals undergo traumatic stresses at some points in their life, but only a small proportion develop stress-related disorders such as anxiety diseases and posttraumatic stress disorder (PTSD). Although stress susceptibility is one determinant of mental disorders, the underlying mechanisms and functional implication remain unclear yet. We found that an increased amount of freezing that animals exhibited in the intertrial interval (ITI) of a stress-enhanced fear learning paradigm, predicts ensuing PTSD-like symptoms whereas resilient mice show ITI freezing comparable to that of unstressed mice. To examine the behavioral features, we developed a systematic analytical approach for ITI freezing and stress susceptibility. Thus, we provide a behavioral parameter for prognosis to stress susceptibility of individuals in the development of PTSD-like symptoms as well as a new mathematical means to scrutinize freezing behavior.

Dopaminergic Regulation of Nucleus Accumbens Cholinergic Interneurons Demarcates Susceptibility to Cocaine Addiction.

BACKGROUND: Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) play critical roles in processing information related to reward. However, the contribution of ChINs to the emergence of addiction-like behaviors and its underlying molecular mechanisms remain elusive. METHODS: We employed cocaine self-administration to identify two mouse subpopulations: susceptible and resilient to cocaine seeking. We compared the subpopulations for physiological responses with single-unit recording of NAc ChINs, and for gene expression levels with RNA sequencing of ChINs sorted using fluorescence-activated cell sorting. To provide evidence for a causal relationship, we manipulated the expression level of dopamine D2 receptor (DRD2) in ChINs in a cell type-specific manner. Using optogenetic activation combined with a double whole-cell recording, the effect of ChIN-specific DRD2 manipulation on each synaptic input was assessed in NAc medium spiny neurons in a pathway-specific manner. RESULTS: Susceptible mice showed higher levels of nosepoke responses under a progressive ratio schedule, and impairment in extinction and punishment procedures. DRD2 was highly abundant in the NAc ChINs of susceptible mice. Elevated abundance of DRD2 in NAc ChINs was sufficient and necessary to express high cocaine motivation, putatively through reduction of ChIN activity during cocaine exposure. DRD2 overexpression in ChINs mimicked cocaine-induced effects on the dendritic spine density and the ratios of excitatory inputs between two distinct medium spiny neuron cell types, while DRD2 depletion precluded cocaine-induced synaptic plasticity. CONCLUSIONS: These findings provide a molecular mechanism for dopaminergic control of NAc ChINs that can control the susceptibility to cocaine-seeking behavior.

Dynamic Changes in the Bridging Collaterals of the Basal Ganglia Circuitry Control Stress-Related Behaviors in Mice.

The basal ganglia network has been implicated in the control of adaptive behavior, possibly by integrating motor learning and motivational processes. Both positive and negative reinforcement appear to shape our behavioral adaptation by modulating the function of the basal ganglia. Here, we examined a transgenic mouse line (G2CT) in which synaptic transmissions onto the medium spiny neurons (MSNs) of the basal ganglia are depressed. We found that the level of collaterals from direct pathway MSNs in the external segment of the globus pallidus (GPe) ('bridging collaterals') was decreased in these mice, and this was accompanied by behavioral inhibition under stress. Furthermore, additional manipulations that could further decrease or restore the level of the bridging collaterals resulted in an increase in behavioral inhibition or active behavior in the G2CT mice, respectively. Collectively, our data indicate that the striatum of the basal ganglia network integrates negative emotions and controls appropriate coping responses in which the bridging collateral connections in the GPe play a critical regulatory role.

Systematic analysis of expression signatures of neuronal subpopulations in the VTA.

Gene expression profiling across various brain areas at the single-cell resolution enables the identification of molecular markers of neuronal subpopulations and comprehensive characterization of their functional roles. Despite the scientific importance and experimental versatility, systematic methods to analyze such data have not been established yet. To this end, we developed a statistical approach based on in situ hybridization data in the Allen Brain Atlas and thereby identified specific genes for each type of neuron in the ventral tegmental area (VTA). This approach also allowed us to demarcate subregions within the VTA comprising specific neuronal subpopulations. We further identified WW domain-containing oxidoreductase as a molecular marker of a population of VTA neurons that co-express tyrosine hydroxylase and vesicular glutamate transporter 2, and confirmed their region-specific distribution by immunohistochemistry. The results demonstrate the utility of our analytical approach for uncovering expression signatures representing specific cell types and neuronal subpopulations enriched in a given brain area.

Basal Forebrain Cholinergic-induced Activation of Cholecystokinin Inhibitory Neurons in the Basolateral Amygdala.

The basolateral amygdala (BLA) receives dense projections from cholinergic neurons of the basal forebrain. Acetylcholine can contributes to amygdala-dependent behaviors: formation and extinction of fear memory and appetitive instrumental learning. However, the cholinergic mechanism at the circuit level has not been defined yet. We demonstrated that cholinergic-induced di-synaptic inhibition of BLA pyramidal neurons exhibits a retrograde form of short-term synaptic inhibition, depolarization-induced suppression of inhibition (DSI). Activation of nicotinic receptors was sufficient to evoke action potentials in cholecystokinin (CCK)-positive inhibitory neurons, which strongly inhibit pyramidal neurons through their perisomatic synapses. Our cell type-specific monosynaptic retrograde tracing also revealed that CCK neurons are innervated by basal forebrain cholinergic neurons. Therefore, our data indicated that CCK inhibitory neurons mediate the cholinergic-induced di-synaptic inhibition of BLA pyramidal neurons.

Nanoscale imaging reveals miRNA-mediated control of functional states of dendritic spines.

Dendritic spines are major loci of excitatory inputs and undergo activity-dependent structural changes that contribute to synaptic plasticity and memory formation. Despite the existence of various classification types of spines, how they arise and which molecular components trigger their structural plasticity remain elusive. microRNAs (miRNAs) have emerged as critical regulators of synapse development and plasticity via their control of gene expression. Brain-specific miR-134s likely regulate the morphological maturation of spines, but their subcellular distributions and functional impacts have rarely been assessed. Here, we exploited atomic force microscopy to visualize in situ miR-134s, which indicated that they are mainly distributed at nearby dendritic shafts and necks of spines. The abundance of miR-134s varied between morphologically and functionally distinct spine types, and their amounts were inversely correlated with their postulated maturation stages. Moreover, spines exhibited reduced contents of miR-134s when selectively stimulated with beads containing brain-derived neurotropic factor (BDNF). Taken together, in situ visualizations of miRNAs provided unprecedented insights into the "inverse synaptic-tagging" roles of miR-134s that are selective to inactive/irrelevant synapses and potentially a molecular means for modifying synaptic connectivity via structural alteration.

DISC1 Modulates Neuronal Stress Responses by Gate-Keeping ER-Mitochondria Ca2+ Transfer through the MAM.

A wide range of Ca2+-mediated functions are enabled by the dynamic properties of Ca2+, all of which are dependent on the endoplasmic reticulum (ER) and mitochondria. Disrupted-in-schizophrenia 1 (DISC1) is a scaffold protein that is involved in the function of intracellular organelles and is linked to cognitive and emotional deficits. Here, we demonstrate that DISC1 localizes to the mitochondria-associated ER membrane (MAM). At the MAM, DISC1 interacts with IP3R1 and downregulates its ligand binding, modulating ER-mitochondria Ca2+ transfer through the MAM. The disrupted regulation of Ca2+ transfer caused by DISC1 dysfunction leads to abnormal Ca2+ accumulation in mitochondria following oxidative stress, which impairs mitochondrial functions. DISC1 dysfunction alters corticosterone-induced mitochondrial Ca2+ accumulation in an oxidative stress-dependent manner. Together, these findings link stress-associated neural stimuli with intracellular ER-mitochondria Ca2+ crosstalk via DISC1, providing mechanistic insight into how environmental risk factors can be interpreted by intracellular pathways under the control of genetic components in neurons.

Alterations in Striatal Circuits Underlying Addiction-Like Behaviors.

Drug addiction is a severe psychiatric disorder characterized by the compulsive pursuit of drugs of abuse despite potential adverse consequences. Although several decades of studies have revealed that psychostimulant use can result in extensive alterations of neural circuits and physiology, no effective therapeutic strategies or medicines for drug addiction currently exist. Changes in neuronal connectivity and regulation occurring after repeated drug exposure contribute to addiction-like behaviors in animal models. Among the involved brain areas, including those of the reward system, the striatum is the major area of convergence for glutamate, GABA, and dopamine transmission, and this brain region potentially determines stereotyped behaviors. Although the physiological consequences of striatal neurons after drug exposure have been relatively well documented, it remains to be clarified how changes in striatal connectivity underlie and modulate the expression of addiction-like behaviors. Understanding how striatal circuits contribute to addiction-like behaviors may lead to the development of strategies that successfully attenuate drug-induced behavioral changes. In this review, we summarize the results of recent studies that have examined striatal circuitry and pathway-specific alterations leading to addiction-like behaviors to provide an updated framework for future investigations.

Identification of postsynaptic phosphatidylinositol-4,5-bisphosphate (PIP2) roles for synaptic plasticity using chemically induced dimerization.

Phosphatidylinositol-4,5-bisphosphate (PIP2), one of the key phospholipids, directly interacts with several membrane and cytosolic proteins at neuronal plasma membranes, leading to changes in neuronal properties including the feature and surface expression of ionotropic receptors. Although PIP2 is also concentrated at the dendritic spines, little is known about the direct physiological functions of PIP2 at postsynaptic as opposed to presynaptic sites. Most previous studies used genetic and pharmacological methods to modulate enzymes that alter PIP2 levels, making it difficult to delineate time- or region-specific roles of PIP2. We used chemically-induced dimerization to translocate inositol polyphosphate 5-phosphatase (Inp54p) to plasma membranes in the presence of rapamycin. Upon redistribution of Inp54p, long-term depression (LTD) induced by low-frequency stimulation was blocked in the mouse hippocampal CA3-CA1 pathway, but the catalytically-dead mutant did not affect LTD induction. Collectively, PIP2 is critically required for induction of LTD whereas translocation of Inp54p to plasma membranes has no effect on the intrinsic properties of the neurons, basal synaptic transmission, long-term potentiation or expression of LTD.

Memory and synaptic plasticity are impaired by dysregulated hippocampal O-GlcNAcylation.

O-GlcNAcylated proteins are abundant in the brain and are associated with neuronal functions and neurodegenerative diseases. Although several studies have reported the effects of aberrant regulation of O-GlcNAcylation on brain function, the roles of O-GlcNAcylation in synaptic function remain unclear. To understand the effect of aberrant O-GlcNAcylation on the brain, we used Oga+/- mice which have an increased level of O-GlcNAcylation, and found that Oga+/- mice exhibited impaired spatial learning and memory. Consistent with this result, Oga+/- mice showed a defect in hippocampal synaptic plasticity. Oga heterozygosity causes impairment of both long-term potentiation and long-term depression due to dysregulation of AMPA receptor phosphorylation. These results demonstrate a role for hyper-O-GlcNAcylation in learning and memory.