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Background Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson's, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. Methods We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. Results We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. Conclusion NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Columnar structure is one of the most fundamental morphological features of the cerebral cortex and is thought to be the basis of information processing in higher animals. Yet, how such a topographically precise structure is formed is largely unknown. Formation of columnar projection of layer 4 (L4) axons is preceded by thalamocortical formation, in which type 1 cannabinoid receptors (CB1R) play an important role in shaping barrel-specific targeted projection by operating spike timing-dependent plasticity during development (Itami et al. , J. Neurosci. 36, 7039–7054 [2016]; Kimura & Itami, J. Neurosci. 39, 3784–3791 [2019]). Right after the formation of thalamocortical projections, CB1Rs start to function at L4 axon terminals (Itami & Kimura, J. Neurosci. 32, 15000–15011 [2012]), which coincides with the timing of columnar shaping of L4 axons. Here, we show that the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a crucial role in columnar shaping. We found that L4 axon projections were less organized until P12 and then became columnar after CB1Rs became functional. By contrast, the columnar organization of L4 axons was collapsed in mice genetically lacking diacylglycerol lipase α, the major enzyme for 2-AG synthesis. Intraperitoneally administered CB1R agonists shortened axon length, whereas knockout of CB1R in L4 neurons impaired columnar projection of their axons. Our results suggest that endocannabinoid signaling is crucial for shaping columnar axonal projection in the cerebral cortex.

SummaryNeuronal subnetworks are common features of adult cortical circuits and serve as functional units for information processing and encoding. To determine when neuronal subnetworks are established, we employedin vivocalcium imaging to monitor neuronal activity in the primary somatosensory cortex (S1) of awake mice during early postnatal development. We found several striking developmental trajectories: (1) neuronal activity frequencies and network synchrony were sex-dependent; (2) neuronal subnetworks merged as early as P11; (3) the number of neurons in a subnetwork decreases with increasing age; (4) subnetwork neurons are initially spatially segregated, but become intermingled with other subnetworks neurons with increasing age; (5) neurons within the same subnetwork acquire a more cohesive activity pattern as age increases, and (6) enhancing GABAergic activity at P15 acutely increased female but not male subnetwork coherence. Together, our findings demonstrate that P11-P21 is the critical time for subnetwork assembly and GABAergic inputs strengthen subnetwork coherence.

Introduction: The antidepressant properties of ketamine have been extensively demonstrated in experimental and clinical settings. However, the psychotomimetic side effects still limit its wider use as an antidepressant. It was recently observed that endocannabinoids are inolved in ketamine induced reward properties. As an increase in endocannabinoid signaling induces antidepressant effects, this study aimed to investigate the involvement of cannabinoid type 1 receptors (CB 1R) in the antidepressant and psychostimulant effects induced by ketamine. Methods: We tested the effects of genetic and pharmacological inhibition of CB 1R in the hyperlocomotion and antidepressant-like properties of ketamine. The effects of ketamine (10-20 mg/kg) were assessed in the open-field and the forced swim tests (FSTs) in CB 1R knockout (KO) and wild-type (WT) mice (male and female), and mice pre-treated with rimonabant (CB 1R antagonist, 3-10 mg/kg). Results: We found that the motor hyperactivity elicited by ketamine was impaired in CB 1R male and female KO mice. A similar effect was observed upon pharmacological blockade of CB 1R in WT mice. However, genetic CB 1R deletion did not modify the antidepressant effect of ketamine in male mice submitted to the FST. Surprisingly, pharmacological blockade of CB 1R induced an antidepressant-like effect in both male and female mice, which was not further potentiated by ketamine. Conclusions: Our results support the hypothesis that CB 1R mediate the psychostimulant side effects induced by ketamine, but not its antidepressant properties.

INTRODUCTION: There is widespread acceptance of cannabis for medical or recreational use across the society, including pregnant women. Concerningly, numerous studies find that the developing central nervous system (CNS) is vulnerable to the detrimental effects of Δ(9)-tetrahydrocannabinol (THC). In contrast, almost nothing on the consequences of perinatal cannabidiol (CBD) exposure. In this study, we used mice to investigate the adult impact of perinatal cannabinoid exposure (PCE) with THC, CBD, or a 1:1 ratio of THC and CBD on behaviors. Furthermore, the lasting impact of PCE on fluoxetine sensitivity in the forced swim test (FST) was evaluated to probe neurochemical pathways interacting with the endocannabinoid system (ECS). METHODS: Pregnant CD1 dams were injected subcutaneously daily with vehicle, 3 mg/kg THC, 3 mg/kg CBD, or 3 mg/kg THC +3 mg/kg CBD from gestational day 5 to postnatal day 10. Mass spectroscopic (MS) analyses were conducted to measure the THC and CBD brain levels in dams and their embryonic progenies. PCE adults were subjected to a battery of behavioral tests: open field arena, sucrose preference test, marble burying test, nestlet shredding test, and FST. RESULTS: MS analysis found substantial levels of THC and CBD in embryonic brains. Our behavioral testing found that PCE females receiving THC or CBD buried significantly more marbles than control mice. Interestingly, PCE males receiving CBD or THC+CBD had significantly increased sucrose preference. While PCE with THC or CBD did not affect FST immobility, PCE with THC or CBD prevented fluoxetine from decreasing immobility in both males and females. Excitingly, fatty acid amide hydrolase (FAAH) inhibition with a dose of URB597 that was behaviorally inactive in the FST rescued fluoxetine efficacy in PCE mice of both sexes. CONCLUSIONS: Our data suggest that PCE with either THC, CBD, or THC+CBD alters repetitive and hedonic behaviors in a phytocannabinoid and sex-dependent manner. In addition, PCE with THC or CBD prevents fluoxetine from enhancing coping behavior. The restoration of fluoxetine responsiveness in THC or CBD PCE adults by inhibition of FAAH suggests that PCE causes a lasting reduction of the ECS and that enhancement of anandamide signaling represents a potential treatment for behavioral deficits following PCE.

Rising opioid use among pregnant women has led to a growing population of neonates exposed to opioids during the prenatal period, but how opioids affect the developing brain remains to be fully understood. Animal models of prenatal opioid exposure have discovered deficits in somatosensory behavioral development that persist into adolescence suggesting opioid exposure induces long lasting neuroadaptations on somatosensory circuitry such as the primary somatosensory cortex (S1). Using a mouse model of prenatal methadone exposure (PME) that displays delays in somatosensory milestone development, we performed an un-biased multi-omics analysis and investigated synaptic functioning in the primary somatosensory cortex (S1), where touch and pain sensory inputs are received in the brain, of early adolescent PME offspring. PME was associated with numerous changes in protein and phosphopeptide abundances that differed considerably between sexes in the S1. Although prominent sex effects were discovered in the multi-omics assessment, functional enrichment analyses revealed the protein and phosphopeptide differences were associated with synapse-related cellular components and synaptic signaling-related biological processes, regardless of sex. Immunohistochemical analysis identified diminished GABAergic synapses in both layer 2/3 and 4 of PME offspring. These immunohistochemical and proteomic alterations were associated with functional consequences as layer 2/3 pyramidal neurons revealed reduced amplitudes and a lengthened decay constant of inhibitory postsynaptic currents. Lastly, in addition to reduced cortical thickness of the S1, cell-type marker analysis revealed reduced microglia density in the upper layer of the S1 that was primarily driven by PME females. Taken together, our studies show the lasting changes on synaptic function and microglia in S1 cortex caused by PME in a sex-dependent manner.

Human brain organoids, 3D brain tissue cultures derived from human pluripotent stem cells, hold promising potential in modeling neuroinflammation for a variety of neurological diseases. However, challenges remain in generating standardized human brain organoids that can recapitulate key physiological features of a human brain. Here, we present tubular organoid-on-a-chip devices to generate better organoids and model neuroinflammation. By employing 3D printed hollow mesh scaffolds, our device can be easily incorporated into multiwell-plates for reliable, scalable, and reproducible generation of tubular organoids. By introducing rocking flows through the tubular device channel, our device can perfuse nutrients and oxygen to minimize organoid necrosis and hypoxia, and incorporate immune cells into organoids to model neuro-immune interactions. Compared with conventional protocols, our method increased neural progenitor proliferation and reduced heterogeneity of human brain organoids. As a proof-of-concept application, we applied this method to model the microglia-mediated neuroinflammation after exposure to an opioid receptor agonist. We found isogenic microglia were activated after exposure to an opioid receptor agonist (DAMGO) and transformed back to the homeostatic status with further treatment by a cannabinoid receptor 2 (CB2) agonist (LY2828360). Importantly, the activated microglia in tubular organoids had stronger cytokine responses compared to those in 2D microglial cultures. Our tubular organoid device is simple, versatile, inexpensive, easy-to-use, and compatible with multiwell-plates, so it can be widely used in common research and clinical laboratory settings. This technology can be broadly used for basic and translational applications in inflammatory diseases including substance use disorders, neural diseases, autoimmune disorders, and infectious diseases.

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose far-projecting axons are vulnerable to neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring proper axonal ATP levels for axonal transport, a critical function of axons.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting NAD hydrolase sterile alpha and TIR motif-containing protein 1 (SARM1) prevented axonal deficits caused by NMNAT2 loss. Our study used a combination of genetic, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos application.ResultsWe providein vivoevidence that NMNAT2 in cortical glutamatergic neurons is required for axonal survival. Usingin vivoandin vitrostudies we demonstrate that NMNAT2 protects axons by ensuring the proper NAD-redox potential in distal axons of cortical neurons to support glycolysis on vesicular cargos, thus ensuring “onboard” ATP production fueling axonal transport. Exogenous NAD+supplementation to NMNAT2 KO cortical neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate bothin vitroandin vivothat reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Here we show that deleting NMNAT2 from cortical glutamatergic neurons (NMNAT2 cKO) results in progressive axonal loss, neuroinflammation, small hippocampi and enlarged ventricles. Interestingly, dramatic neuroinflammation responses were observed around the long-range axonal tracts of NMNAT2 cKO cortical neurons. In addition to the neurodegenerative-like phenotype, we also found the absence of whisker-representation patterns “barrels” in the primary somatosensory cortex of NMNAT2 cKO mice. These observations suggest that NMNAT2 is required in developing cortical circuits and in maintaining the health of cortical neurons. Unbiased transcriptomic analysis suggests that NMNAT2 loss in cortical neurons after axonal outgrowth phase upregulates mitochondria function while greatly reducing synaptogenesis pathways. Complete loss of Sarm1 function in NMNAT2 cKO mice restores barrel map formation and axonal integrity and abolishes the inflammatory response. Interestingly, reducing Sarm1 function in NMNAT2 cKO mice by deleting only one copy of Sarm1 restores barrel map formation but did not diminish the neurodegenerative-like phenotype. Only complete loss of Sarm1 prevents neurodegeneration and inflammatory responses.

Neuroinflammation plays a central role in the progression of many neurodegenerative diseases such as Alzheimer's disease, and challenges remain in modeling the complex pathological or physiological processes. Here, we report an acoustofluidic method that can rapidly construct 3D neurospheroids and inflammatory microenvironments for modeling microglia-mediated neuroinflammation in Alzheimer's disease. By incorporating a unique contactless and label-free acoustic assembly, this cell culture platform can assemble dissociated embryonic mouse brain cells into hundreds of uniform 3D neurospheroids with controlled cell numbers, composition (e.g. neurons, astrocytes, and microglia), and environmental components (e.g. amyloid-β aggregates) in hydrogel within minutes. Moreover, this platform can maintain and monitor the interaction among neurons, astrocytes, microglia, and amyloid-β aggregates in real-time for several days to weeks, after the integration of a high-throughput, time-lapse cell imaging approach. We demonstrated that our engineered 3D neurospheroids can represent the amyloid-β neurotoxicity, which is one of the main pathological features of Alzheimer's disease. Using this method, we also investigated the microglia migratory behaviors and activation in the engineered 3D inflammatory microenvironment at a high throughput manner, which is not easy to achieve in 2D neuronal cultures or animal models. Along with the simple fabrication and setup, the acoustofluidic technology is compatible with conventional Petri dishes and well-plates, supports the fine-tuning of the cellular and environmental components of 3D neurospheroids, and enables the high-throughput cellular interaction investigation. We believe our technology may be widely used to facilitate 3D in vitro brain models for modeling neurodegenerative diseases, discovering new drugs, and testing neurotoxicity.

Maternal cannabis use during lactation may expose developing infants to cannabinoids (CBs) such as tetrahydrocannabinol (THC) and cannabidiol (CBD). CBs modulate lipid signaling molecules in the central nervous system in age- and cell-dependent ways, but their influence on the lipid composition of breastmilk has yet to be established. This study investigates the effects of THC, CBD, or their combination on milk lipids by analyzing the stomach contents of CD1 mouse pups that have been nursed by dams injected with CBs on postnatal days (PND) 1-10 collected 2 hours after the last injection on PND10. HPLC/MS/MS was used to identify and quantify over 80 endogenous lipid species and cannabinoids in pup stomach contents. We show that CBs differentially accumulate in milk, lead to widespread decreases in free fatty acids, decreases in N-acyl methionine species, increases N-linoleoyl species, as well as modulate levels of endogenous CBs (eCBs) AEA, 2-AG, and their structural congeners. Our data indicate the passage of CBs to pups through breast milk and that maternal CB exposure alters breast milk lipid compositions.

BACKGROUND Antagonists of cannabinoid type 1 receptor (CB1) have been shown to promote body weight loss and improve insulin sensitivity. Cannabinoids decrease adiponectin, and CB1 blocker increase adiponectin. However, the mediators of CB1 actions are not well defined. AIM To investigate whether the beneficial effects of CB1 inhibition are, at least in part, mediated by adiponectin. METHODS We compared metabolic and inflammatory phenotypes of wild-type (WT) mice, CB1-null (CB1-/-) and CB1/adiponectin double-knockout (DKO) mice. We assessed the insulin sensitivity using insulin tolerance test and glucose tolerance test, and inflammation using flow cytometry analysis of macrophages. RESULTS CB1 -/- mice exhibited significantly reduced body weight and fat mass when compared to WT mice. While no significance was found in total daily food intake and locomotor activity, CB1-/- mice showed increased energy expenditure, enhanced thermogenesis in brown adipose tissue (BAT), and improved insulin sensitivity compared to WT mice. DKO showed no difference in body weight, adiposity, nor insulin sensitivity; only showed a modestly elevated thermogenesis in BAT compared to CB1-/- mice. The metabolic phenotype of DKO is largely similar to CB1-/- mice, suggesting that adiponectin is not a key mediator of the metabolic effects of CB1. Interestingly, CB1-/- mice showed reduced pro-inflammatory macrophage polarization in both peritoneal macrophages and adipose tissue macrophages compared to WT mice; in contrast, DKO mice exhibited increased pro-inflammatory macrophage polarization in these macrophages compared to CB1-/- mice, suggesting that adiponectin is an important mediator of the inflammatory effect of CB1. CONCLUSION Our findings reveal that CB1 functions through both adiponectin-dependent and adiponectin-independent mechanisms: CB1 regulates energy metabolism in an adiponectin-independent manner, and inflammation in an adiponectin-dependent manner. The differential effects of adiponectin on CB1-mediated metabolic and inflammatory functions should be taken into consideration in CB1 antagonist utilization.

Despite the rising prevalence of methadone treatment in pregnant women with opioid use disorder, the effects of methadone on neurobehavioral development remain unclear. We developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. We investigated the effects of PME on physical development, sensorimotor behavior, and motor neuron properties using a multidisciplinary approach of physical, biochemical, and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging. Methadone accumulated in the placenta and fetal brain, but methadone levels in offspring dropped rapidly at birth which was associated with symptoms and behaviors consistent with neonatal opioid withdrawal. PME produced substantial impairments in offspring physical growth, activity in an open field, and sensorimotor milestone acquisition. Furthermore, these behavioral alterations were associated with reduced neuronal density in the motor cortex and a disruption in motor neuron intrinsic properties and local circuit connectivity. The present study adds to the limited body of work examining PME by providing a comprehensive, translationally relevant characterization of how PME disrupts offspring physical and neurobehavioral development., eLife digest The far-reaching opioid crisis extends to babies born to mothers who take prescription or illicit opioids during pregnancy. Opioids such as oxycodone and methadone can freely cross the placenta from mother to baby. With the rising misuse of and addiction to opioids, the number of babies born physically dependent on opioids has risen sharply over the last decade. Although these infants are only passively exposed to opioids in the womb, they can still experience withdrawal symptoms at birth. This withdrawal is characterized by irritability, excessive crying, body shakes, problems with feeding, fevers and diarrhea. While considerable attention has been given to treating opioid withdrawal in newborn babies, little is known about how these children develop in their first years of life. This is, in part, because it is difficult for researchers to separate drug-related effects from other factors in a child’s home environment that can also disrupt their development. In addition, the biological mechanisms underpinning opioid-related impairments to infant development also remain unclear. Animal models have been used to study the effects of opioid exposure during pregnancy (termed prenatal exposure) on infants. These models, however, could be improved to better replicate the typical pattern of opioid use among pregnant women. Recognizing this gap, Grecco et al. have developed a mouse model of prenatal methadone exposure where female mice that were previously dependent on oxycodone were treated with methadone throughout their pregnancy. Methadone is an opioid drug commonly prescribed for treating opioid use disorder in pregnant women and was found to accumulate at high levels in the fetal brain of mice, which fell quickly after birth. The offspring also experienced withdrawal symptoms. Grecco et al. then examined the physical, behavioral and brain development of mice born to opioid-treated mothers. These included assessments of the animals’ motor skills, sensory reflexes and behavior in their first four weeks of life. Additional experiments tested the properties of nerve cells in the brain to examine cell-level changes. The assessments showed that methadone exposure in the womb impaired the physical growth of offspring and this persisted into ‘adolescence’. Prenatal methadone exposure also delayed progress towards key developmental milestones and led to hyperactivity in three-week-old mice. Moreover, Grecco et al. found that these mice had reduced neuron density and cell-to-cell connectivity in the part of the brain which controls movement. These findings shed light on the potential consequences of prenatal methadone exposure on physical, behavioral and brain development in infants. This model could also be used to study new potential treatments or intervention strategies for offspring exposed to opioids during pregnancy.

Abnormal levels of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) have been detected in various neurological disorders. The potent impact of FGF-FGFR in multiple embryonic developmental processes makes it challenging to elucidate their roles in postmitotic neurons. Taking an alternative approach to examine the impact of aberrant FGFR function on glutamatergic neurons, we generated a FGFR gain-of-function (GOF) transgenic mouse, which expresses constitutively activated FGFR3 (FGFR3K650E) in postmitotic glutamatergic neurons. We found that GOF disrupts mitosis of radial-glia neural progenitors (RGCs), inside-out radial migration of post-mitotic glutamatergic neurons, and axonal tract projections. In particular, late-born CUX1-positive neurons are widely dispersed throughout the GOF cortex. Such a cortical migration deficit is likely caused, at least in part, by a significant reduction of the radial processes projecting from RGCs. RNA-sequencing analysis of the GOF embryonic cortex reveals significant alterations in several pathways involved in cell cycle regulation and axonal pathfinding. Collectively, our data suggest that FGFR3 GOF in postmitotic neurons not only alters axonal growth of postmitotic neurons but also impairs RGC neurogenesis and radial glia processes.

Despite the rising prevalence of methadone treatment in pregnant women with opioid use disorder, the effects of methadone on neurobehavioral development remain unclear. We developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. We investigated the effects of PME on physical development, sensorimotor behavior, and motor neuron properties using a multidisciplinary approach of physical, biochemical, and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging. PME produced substantial impairments in offspring physical growth, activity in an open field, and sensorimotor milestone acquisition which were associated with alterations in motor neuron functioning and connectivity. The present study adds to the limited work examining PME by providing a comprehensive, translationally relevant characterization of how PME disrupts offspring development.

The endocannabinoid system (ECS) is a widespread neuromodulatory network involved both in the developing CNS as well as playing a major role in tuning many cognitive and physiological processes. The ECS is composed of endogenous cannabinoids, cannabinoid receptors and the enzymes responsible for the synthesis and degradation of endocannabinoids. In addition to its endogenous roles, cannabinoid receptors are the primary target of Δ(9)-tetrahydrocannabinol (THC), the intoxicating component of cannabis. In this review, we will summarize our current understanding of the ECS. We will start with a description of ECS components and their role in synaptic plasticity and neurodevelopment, and then discuss how phytocannabinoids and other exogenous compounds may perturb the ECS, emphasizing examples relevant to psychosis.

Abnormal levels of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) have been detected in various neurological disorders. The potent impact of FGF-FGFR in multiple embryonic developmental processes makes it challenging to elucidate their roles in post-mitotic neurons. Taking an alternative approach, we directly examined the impact of aberrant FGFR function after neurogenesis by generating a FGFR gain-of-function (GOF) transgenic mouse which expresses constitutively activated FGFR3 (FGFR3K650E) in post-mitotic glutamatergic neurons. We found that enhanced FGFR activity in glutamatergic neurons results in abnormal radial migration and axonal miswiring. Regarding the lamination phenotype in GOF brains, we found later-born Cux1-positive neurons are dispersed throughout the GOF cortex. Such a cortical migration deficit is likely caused, at least in part, by a significant reduction of the radial processes normally projecting from the radial glia cells (RGCs). In addition, FGFR3 GOF also results in the misrouting of several long-range axonal projections, including the corpus callosum, anterior commissure, and postcommissural fornix. RNA-sequencing analysis of the GOF embryonic cortex reveals significant alterations in several pathways involved in cell cycle regulation and axonal pathfinding. Collectively, our results suggest that FGFR hyperfunction in post-mitotic neurons at the late embryonic stage result in cortical dysplasia and circuit miswiring.

Neuroinflammation plays a central role in the progression of many neurodegenerative diseases such as Alzheimer’s disease, and challenges remain in modeling the complex pathological or physiological processes. Here, we report an acoustofluidic 3D cell culture device that can rapidly construct 3D neurospheroids and inflammatory microenvironments for modeling microglia-mediated neuroinflammation in Alzheimer’s disease. By incorporating a unique contactless and label-free acoustic assembly, this cell culture platform can assemble dissociated embryonic mouse brain cells into hundreds of uniform 3D neurospheroids with controlled cell numbers, composition (e.g. neurons, astrocytes, and microglia), and environmental components (e.g. amyloid-β aggregates) in hydrogel within minutes. Moreover, this platform can maintain and monitor the interaction among neurons, astrocytes, microglia, and amyloid-β aggregates in real-time for several days to weeks, after the integration of a high-throughput, time-lapse cell imaging approach. We demonstrated that our engineered 3D neurospheroids can represent the amyloid-β neurotoxicity, which is one of the main pathological features of Alzheimer’s disease. Using this method, we also investigated the microglia migratory behaviors and activation in the engineered 3D inflammatory microenvironment at a high throughput manner, which is not easy to achieve in 2D neuronal cultures or animal models. Along with the simple fabrication and setup, the acoustofluidic technology is compatible with conventional Petri dishes and well-plates, supports the fine-tuning of the cellular and environmental components of 3D neurospheroids, and enables the high-throughput cellular interaction investigation. We believe our technology may be widely used as in vitro brain models for modeling neurodegenerative diseases, discovering new drugs, and testing neurotoxicity.

MicroRNAs (miRs) are 18~23 nucleotides long non-coding RNAs that regulate gene expression. To explore whether miR alterations in tauopathy contribute to pathological conditions, we first determined which hippocampal miRs are altered at the presymptomatic and symptomatic stages of tauopathy using rTg4510 mice (Tau mice), a well-characterized tauopathy model. miR-RNA pairing analysis using QIAGEN Ingenuity Pathway Analysis (IPA) revealed 401 genes that can be regulated by 71 miRs altered in Tau hippocampi at the presymptomatic stage. Among several miRs confirmed with real-time qPCR, miR142 (−3p and −5p) in Tau hippocampi were significantly upregulated by two-weeks of age and onward. Transcriptome studies by RNAseq and IPA revealed several overlapping biological and disease associated pathways affected by either Tau or miR142 overexpression, including Signal Transducer and Activator of Transcription 3 (Stat3) and Tumor Necrosis Factor Receptor 2 (Tnfr2) signaling pathways. Similar to what was observed in Tau brains, overexpressing miR142 in wildtype cortical neurons augments mRNA levels of Glial Fibrillary Acidic Protein (Gfap) and Colony Stimulating Factor 1 (Csf1), accompanied by a significant increase in microglia and reactive astrocyte numbers. Taken together, our study suggests that miR alterations by Tau overexpression may contribute to the neuroinflammation observed in Tau brains.

Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known for their potent effects on cell proliferation/differentiation and cortical patterning in the developing brain. However, little is known regarding the roles of FGFs/FGFRs in cortical circuit formation. Here we show thatFgfr1/2/3 andFgf7/9/10/22mRNAs are expressed in the developing primary somatosensory (S1) barrel cortex. Barrel cortex layer IV spiny stellate cells (bSCs) are the primary recipients of ascending sensory information via thalamocortical axons (TCAs). Detail quantification revealed distinctive phases for bSC dendritogenesis: orienting dendrites toward TCAs, addingde novodendritic segments, and elongating dendritic length, while maintaining dendritic patterns. DeletingFgfr1/2/3in bSCs had minimal impact on dendritic polarity but transiently increased the number of dendritic segments. However, 6 d later, FGFR1/2/3 loss of function reduced dendritic branch numbers. These data suggest that FGFs/FGFRs have a role in stabilizing dendritic patterning. Depolarization of cultured mouse cortical neurons upregulated the levels of severalFgf/FgfrmRNAs within 2 h.In vivo, within 6 h of systemic kainic acid administration at postnatal day 6, mRNA levels ofFgf9,Fgf10,Fgfr2c, andFgfr3bin S1 cortices were enhanced, and this was accompanied by exuberant dendritogenesis of bSCs by 24 h. DeletingFgfr1/2/3abolished kainic acid-induced bSC dendritic overgrowth. Finally, FGF9/10 gain of function also resulted in extensive dendritogenesis. Together, our data suggest that FGFs/FGFRs can be regulated by glutamate transmission to modulate/stabilize bSC dendritic complexity. Both male and female mice were used for our study.SIGNIFICANCE STATEMENTGlutamatergic transmission plays critical roles in cortical circuit formation. Its dysregulation has been proposed as a core factor in the etiology of many neurological diseases. We found that excessive glutamate transmission upregulated mRNA expression ofFgfrsand their ligandsFgfs. DeletingFgfr1/2/3not only impaired bSC dendritogenesis but also abolished glutamate transmission-induced dendritic overgrowth. Overexpressing FGF9 or FGF10 in cortical glutamatergic neurons results in excessive dendritic outgrowth within 24 h, resembling the changes induced by excessive glutamate transmission. Our findings provide strong evidence for the physiological role of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in establishing and maintaining cortical circuits. Perturbing the expression levels of FGFs/FGFRs by excessive glutamatergic neurotransmission could lead to abnormal neuronal circuits, which may contribute to neurological and psychiatric disease.

RATIONALE: A role of group I metabotropic glutamate receptor 5 (mGlu5) in regulating spontaneous locomotion and psychostimulant-induced hyperactivity has been proposed. OBJECTIVES: This study aims to determine if mGlu5 in GABAergic neurons regulates spontaneous or psychostimulant-induced locomotion. METHODS: We generated mice specifically lacking mGlu5 in forebrain GABAergic neuron by crossing DLX-Cre mice with mGlu5(flox/flox) mice to generate DLX-mGlu5 KO mice. The locomotion of adult mice was examined in the open field assay (OFA) and home cage setting. The effects of the mGlu5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP), cocaine and methylphenidate on acute motor behaviors in DLX-mGlu5 KO and littermate control mice were assessed in OFA. Striatal synaptic plasticity of these mice was examined with field potential electrophysiological recordings. RESULTS: Deleting mGlu5 from forebrain GABAergic neurons results in failure to induce long-term depression (LTD) in the dorsal striatum and absence of habituated locomotion in both novel and familiar settings. In a familiar environment (home cage), DLX-mGlu5 KO mice were hyperactive. In the OFA, DLX-mGlu5 KO mice exhibited initial hypo-activity, and then gradually increased their locomotion with time, resulting in no habituation response. DLX-mGlu5 KO mice exhibited almost no locomotor response to MPEP (40 mg/kg), while the same dose elicited hyper-locomotion in control mice. The DLX-mGlu5 KO mice also showed reduced hyperactivity response to cocaine, while they retained normal hyperactivity response to methylphenidate, albeit with delayed onset. CONCLUSION: mGlu5 in forebrain GABAergic neurons is critical to trigger habituation upon the initiation of locomotion as well as to mediate MPEP-induced hyper-locomotion and modulate psychostimulant-induced hyperactivity.

The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Δ 9 -tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs. SIGNIFICANCE STATEMENT It has been shown that neural activity is required for initial synapse formation of thalamocortical axons with cortical cells, but precisely what sort of activities in presynaptic and postsynaptic cells are required is not yet clear. In addition, how activity is further translated into structural changes is unclear. We show here that the period during which spike timing-dependent long-term potentiation and depression (t-LTP, t-LTD) can be induced closely matches the time course of synapse formation and retraction, respectively, at the thalamocortical synapse. Moreover, administration of cannabinoid agonists, which mimic t-LTD, caused TCA retraction, suggesting that cannabinoids translate physiological changes into morphological consequences.

The endocannabinoid system (ECS) is a widespread neuromodulatory system that plays important roles in central nervous system (CNS) development, synaptic plasticity, and the response to endogenous and environmental insults. The ECS is comprised of cannabinoid receptors, endogenous cannabinoids (endocannabinoids), and the enzymes responsible for the synthesis and degradation of the endocannabinoids. The most abundant cannabinoid receptor is the CB1 cannabinoid receptors, however CB2 cannabinoid receptors, transient receptor potential (TRP) channels, and peroxisome proliferator activated receptors (PPAR’s) are also engaged by some cannabinoids. Exogenous cannabinoids, such as tetrahydrocannabinol, produce their biological effects through their interactions with cannabinoid receptors. 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide) are the best-studied endogenous cannabinoids. Despite similarities in chemical structure, 2-AG and anandamide are synthesized and degraded by distinct enzymatic pathways, which impart fundamentally different physiological and pathophysiological roles to these two endocannabinoids. Because of the pervasive social use of cannabis and the involvement of endocannabinoids in a multitude of biological processes, much has been learned about the physiological and pathophysiological roles of the ECS. This review will provide an introduction to the ECS with an emphasis on its role in synaptic plasticity and how the ECS is perturbed in schizophrenia.

Significant advancements have been made in unraveling and understanding the non-coding elements of the human genome. New insights into the structure and function of noncoding RNAs have emerged. Their relevance in the context of both physiological cellular homeostasis and human diseases is getting appreciated. As a result, exploration of noncoding RNAs, in particular microRNAs (miRs), as therapeutic agents or targets of therapeutic strategies is under way. This review summarizes and discusses in depth the current literature on the role of miRs in neurodegenerative diseases.

The age-dependent progression of tau pathology is a major characteristic of tauopathies, including Alzheimer's disease (AD), and plays an important role in the behavioral phenotypes of AD, including memory deficits. Despite extensive molecular and cellular studies on tau pathology, it remains to be determined how it alters the neural circuit functions underlying learning and memoryin vivo. In rTg4510 mice, a Tau-P301L tauopathy model, hippocampal place fields that support spatial memories are abnormal at old age (7–9 months) when tau tangles and neurodegeneration are extensive. However, it is unclear how the abnormality in the hippocampal circuit function arises and progresses with the age-dependent progression of tau pathology. Here we show that in young (2–4 months of age) rTg4510 mice, place fields of hippocampal CA1 cells are largely normal, with only subtle differences from those of age-matched wild-type control mice. Second, high-frequency ripple oscillations of local field potentials in the hippocampal CA1 area are significantly reduced in young rTg4510 mice, and even further deteriorated in old rTg4510 mice. The ripple reduction is associated with less bursty firing and altered synchrony of CA1 cells. Together, the data indicate that deficits in ripples and neuronal synchronization occur before overt deficits in place fields in these mice. The results reveal a tau-pathology-induced progression of hippocampal functional changesin vivo.

Silencing of fragile X mental retardation 1 (FMR1) gene and loss of fragile X mental retardation protein (FMRP) cause fragile X syndrome (FXS), a genetic disorder characterized by intellectual disability and autistic behaviors. FMRP is an mRNA-binding protein regulating neuronal translation of target mRNAs. Abnormalities in actin-rich dendritic spines are major neuronal features in FXS, but the molecular mechanism and identity of FMRP targets mediating this phenotype remain largely unknown. Cytoplasmic FMR1-interacting protein 2 (Cyfip2) was identified as an interactor of FMRP, and its mRNA is a highly ranked FMRP target in mouse brain. Importantly, Cyfip2 is a component of WAVE regulatory complex, a key regulator of actin cytoskeleton, suggesting that Cyfip2 could be implicated in the dendritic spine phenotype of FXS. Here, we generated and characterized Cyfip2-mutant (Cyfip2(+/-)) mice. We found that Cyfip2(+/-) mice exhibited behavioral phenotypes similar to Fmr1-null (Fmr1(-/y)) mice, an animal model of FXS. Synaptic plasticity and dendritic spines were normal in Cyfip2(+/-) hippocampus. However, dendritic spines were altered in Cyfip2(+/-) cortex, and the dendritic spine phenotype of Fmr1(-/y) cortex was aggravated in Fmr1(-/y); Cyfip2(+/-) double-mutant mice. In addition to the spine changes at basal state, metabotropic glutamate receptor (mGluR)-induced dendritic spine regulation was impaired in both Fmr1(-/y) and Cyfip2(+/-) cortical neurons. Mechanistically, mGluR activation induced mRNA translation-dependent increase of Cyfip2 in wild-type cortical neurons, but not in Fmr1(-/y) or Cyfip2(+/-) neurons. These results suggest that misregulation of Cyfip2 function and its mGluR-induced expression contribute to the neurobehavioral phenotypes of FXS.

Impaired neurogenesis in the adult hippocampus has been implicated in AD pathogenesis. Here we reveal that the APP plays an important role in the neural progenitor proliferation and newborn neuron maturation in the mouse dentate gyrus. APP controls adult neurogenesis through a noncell-autonomous mechanism by GABAergic neurons, as selective deletion of GABAergic, but not glutamatergic, APP disrupts adult hippocampal neurogenesis. APP, highly expressed in the majority of GABAergic neurons in the dentate gyrus, enhances the inhibitory tone to granule cells. By regulating both tonic and phasic GABAergic inputs to dentate granule cells, APP maintains excitatory–inhibitory balance and preserves cognitive functions. Our studies uncover an indispensable role of APP in the GABAergic system for controlling adult hippocampal neurogenesis, and our findings indicate that APP dysfunction may contribute to impaired neurogenesis and cognitive decline associated with AD.

Neurons receive and integrate synaptic inputs at their dendrites, thus dendritic patterning shapes neural connectivity and behavior. Aberrant dendritogenesis is present in neurodevelopmental disorders such as Down's syndrome and autism. Abnormal glutamatergic signaling has been observed in these diseases, as has dysfunction of the metabotropic glutamate receptor 5 (mGluR5). Deleting mGluR5 in cortical glutamatergic neurons disrupted their coordinated dendritic outgrowth toward thalamocortical axons and perturbed somatosensory circuits. Here we show that mGluR5 loss-of-function disrupts dendritogenesis of cortical neurons by increasing mRNA levels of nerve growth factor (NGF) and fibroblast growth factor 10 (FGF10), in part through calcium-permeable AMPA receptors (CP-AMPARs), as the whisker-barrel map is forming. Postnatal NGF and FGF10 expression in cortical layer IV spiny stellate neurons differentially impacted dendritic patterns. Remarkably, NGF-expressing neurons exhibited dendritic patterns resembling mGluR5 knockout neurons: increased total dendritic length/complexity and reduced polarity. Furthermore, suppressing the kinase activity of TrkA, a major NGF receptor, prevents aberrant dendritic patterning in barrel cortex of mGluR5 knockout neurons. These results reveal novel roles for NGF-TrkA signaling and CP-AMPARs for proper dendritic development of cortical neurons. This is the first in vivo demonstration that cortical neuronal NGF expression modulates dendritic patterning during postnatal brain development.

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer's, Huntington's, Parkinson's diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons.

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer’s, Huntington’s, Parkinson’s diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons.