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Abstract Reactive astrocytes play a pivotal role in the pathogenesis of neurological diseases; however, their functional phenotype and the downstream molecules by which they modify disease pathogenesis remain unclear. Here, we genetically increase P2Y1 receptor (P2Y1R) expression, which is upregulated in reactive astrocytes in several neurological diseases, in astrocytes of male mice to explore its function and the downstream molecule. This astrocyte-specific P2Y1R overexpression causes neuronal hyperexcitability by increasing both astrocytic and neuronal Ca2+ signals. We identify insulin-like growth factor-binding protein 2 (IGFBP2) as a downstream molecule of P2Y1R in astrocytes; IGFBP2 acts as an excitatory signal to cause neuronal excitation. In neurological disease models of epilepsy and stroke, reactive astrocytes upregulate P2Y1R and increase IGFBP2. The present findings identify a mechanism underlying astrocyte-driven neuronal hyperexcitability, which is likely to be shared by several neurological disorders, providing insights that might be relevant for intervention in diverse neurological disorders.

Abstract Lower urinary tract (LUT) function is controlled by the central nervous system, including higher-order cognitive brain regions. The anterior cingulate cortex (ACC) is one of these regions, but the role of its activity in LUT function remains poorly understood. In the present study, we conducted optogenetic experiments to manipulate neural activity in mouse ACC while monitoring bladder pressure to elucidate how the activity of ACC regulates LUT function. Selective optogenetic stimulation of excitatory neurons in ACC induced a sharp increase in bladder pressure, whereas activation of inhibitory neurons in ACC prolonged the interval between bladder contractions. Pharmacological manipulation of ACC also altered bladder contractions, consistent with those observed in optogenetic experiments. Optogenetic mapping of the cortical area responsible for eliciting the increase in bladder pressure revealed that stimulation to ACC showed more potent effects than the neighboring motor cortical areas. These results suggest that ACC plays a crucial role in initiating the bladder pressure change and the micturition reflex. Thus, the balance between excitation and inhibition in ACC may regulate the reflex bidirectionally.

Abstract Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what animals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience-dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two-bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)-positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type-specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory-related nuclei, and especially CeA Sst-positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd-positive neurons instead of Sst-positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience-dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process.

CLICK-III/CaMKIγ is a lipid-anchored neuronal isoform of multifunctional Ca2+/calmodulin-dependent protein kinases, which mediates BDNF-dependent dendritogenesis in cultured cortical neurons. We found that two distinct lipidation states of CaMKIγ, namely, prenylation and palmitoylation, controlled its association with detergent-resistant microdomains in the dendrites and were essential for its dendritogenic activity. However, the impact of each lipid modification on membrane targeting/trafficking and how it specifies functional coupling leading to polarized changes in neuronal morphology are not clear. Here, we show that prenylation induces membrane anchoring of CaMKIγ, permitting access to the Golgi apparatus, and a subsequent palmitoylation facilitates association with cholesterol-enriched lipid microdomains or lipid rafts, in particular at the Golgi. To specifically test the role of palmitoylated CaMKγ in neurite extension, we identified and took advantage of a cell system, PC12, which, unlike neurons, conveniently lacked CaMKIγ and was deficient in the activity-dependent release of a neuritogenic growth factor while possessing the ability to activate polarized rafts signaling for morphogenesis. This system allowed us to rigorously demonstrate that an activity-dependent, lipid rafts-restricted Rac activation leading to neuritogenesis could be functionally rescued by dually lipidated CaMKIγ expression, revealing that not only prenylation but also palmitoylation is essential for CaMKIγ to activate a compartmentalized STEF-Rac1 pathway. These results shed light on the significance of recruiting prenylated and palmitoylated CaMKIγ into the coalescing signalosomes at lipid rafts together with Rac1 and its specific GEF and STEF and forming a compartmentalized Ca2+ signaling pathway that underlies activity-dependent neuritogenesis and morphogenesis during axodendritic polarization critical for brain development and circuitogenesis.

Visualisation of the activity of neural grafts using engineered luciferase provides insights into the integration between the graft and host.

Abstract Several large-scale whole-exome sequencing studies in patients with schizophrenia (SCZ) and autism spectrum disorder (ASD) have identified rare variants with modest or strong effect size as genetic risk factors. Dysregulation of cellular calcium homeostasis might be involved in SCZ/ASD pathogenesis, and genes encoding L-type voltage-gated calcium channel (VGCC) subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), and T-type VGCC subunit Cav3.3 (CACNA1I) recently were identified as risk loci for psychiatric disorders. We performed a screening study, using the Ion Torrent Personal Genome Machine (PGM), of exon regions of these four candidate genes (CACNA1C, CACNA1D, CACNA1S, CACNA1I) in 370 Japanese patients with SCZ and 192 with ASD. Variant filtering was applied to identify biologically relevant mutations that were not registered in the dbSNP database or that have a minor allele frequency of less than 1% in East-Asian samples from databases; and are potentially disruptive, including nonsense, frameshift, canonical splicing site single nucleotide variants (SNVs), and non-synonymous SNVs predicted as damaging by five different in silico analyses. Each of these filtered mutations were confirmed by Sanger sequencing. If parental samples were available, segregation analysis was employed for measuring the inheritance pattern. Using our filter, we discovered one nonsense SNV (p.C1451* in CACNA1D), one de novo SNV (p.A36V in CACNA1C), one rare short deletion (p.E1675del in CACNA1D), and 14 NSstrict SNVs (non-synonymous SNV predicted as damaging by all of five in silico analyses). Neither p.A36V in CACNA1C nor p.C1451* in CACNA1D were found in 1871 SCZ cases, 380 ASD cases, or 1916 healthy controls in the independent sample set, suggesting that these SNVs might be ultra-rare SNVs in the Japanese population. The neuronal splicing isoform of Cav1.2 with the p.A36V mutation, discovered in the present study, showed reduced Ca2+-dependent inhibition, resulting in excessive Ca2+ entry through the mutant channel. These results suggested that this de novo SNV in CACNA1C might predispose to SCZ by affecting Ca2+ homeostasis. Thus, our analysis successfully identified several ultra-rare and potentially disruptive gene variants, lending partial support to the hypothesis that VGCC-encoding genes may contribute to the risk of SCZ/ASD.

CaMKIIα plays a fundamental role in learning and memory and is a key determinant of synaptic plasticity. Its kinase activity is regulated by the binding of Ca2+/CaM and by autophosphorylation that operates in an activity-dependent manner. Though many mutations in CAMK2A were linked to a variety of neurological disorders, the multiplicity of its functional substrates renders the systematic molecular phenotyping challenging. In this study, we report a new case of CAMK2A P212L, a recurrent mutation, in a patient with an intellectual disability. To quantify the effect of this mutation, we developed a FRET-based kinase phenotyping strategy and measured aberrance in Ca2+/CaM-dependent activation dynamics in vitro and in synaptically connected neurons. CaMKIIα P212L revealed a significantly facilitated Ca2+/CaM-dependent activation in vitro. Consistently, this mutant showed faster activation and more delayed inactivation in neurons. More prolonged kinase activation was also accompanied by a leftward shift in the CaMKIIα input frequency tuning curve. In keeping with this, molecular phenotyping of other reported CAMK2A de novo mutations linked to intellectual disability revealed aberrant facilitation of Ca2+/CaM-dependent activation of CaMKIIα in most cases. Finally, the pharmacological reversal of CAMK2A P212L phenotype in neurons was demonstrated using an FDA-approved NMDA receptor antagonist memantine, providing a basis for targeted therapeutics in CAMK2A-linked intellectual disability. Taken together, FRET-based kinase mutation phenotyping sheds light on the biological impact of CAMK2A mutations and provides a selective, sensitive, quantitative, and scalable strategy for gaining novel insights into the molecular etiology of intellectual disability.

Summary: We present a protocol to prepare mouse cranial window implantation for in vivo two-photon wide-field calcium imaging. This protocol uses G-CaMP9a transgenic mice, which express a genetically encoded calcium indicator with high signal-to-noise ratio. We describe in utero electroporation, followed by headplate fixation and cranial window implantation. This protocol can be used for measuring neural activity and is suitable for long-term imaging in large populations. Moreover, this protocol does not require preparation of Flp-expressing transgenic mice.For complete details on the use and execution of this protocol, please refer to Sakamoto et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Genomic defects with large effect size can help elucidate unknown pathologic architecture of mental disorders. We previously reported on a patient with schizophrenia and a balanced translocation between chromosomes 4 and 13 and found that the breakpoint within chromosome 4 is located near the LDB2 gene. We show here that Ldb2 knockout (KO) mice displayed multiple deficits relevant to mental disorders. In particular, Ldb2 KO mice exhibited deficits in the fear‐conditioning paradigm. Analysis of the amygdala suggested that dysregulation of synaptic activities controlled by the immediate early gene Arc is involved in the phenotypes. We show that LDB2 forms protein complexes with known transcription factors. Consistently, ChIP‐seq analyses indicated that LDB2 binds to > 10,000 genomic sites in human neurospheres. We found that many of those sites, including the promoter region of ARC, are occupied by EGR transcription factors. Our previous study showed an association of the EGR family genes with schizophrenia. Collectively, the findings suggest that dysregulation in the gene expression controlled by the LDB2‐EGR axis underlies a pathogenesis of subset of mental disorders.

Abstract Autism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders and needs new therapeutic approaches. Recently, oxytocin (OXT) showed potential as the first anti-ASD drug. Many reports have described the efficacy of intranasal OXT therapy to improve the core symptoms of patients with ASD; however, the underlying neurobiological mechanism remains unknown. The OXT/oxytocin receptor (OXTR) system, through the lateral septum (LS), contributes to social behavior, which is disrupted in ASD. Therefore, we selectively express hM3Dq in OXTR-expressing (OXTR+) neurons in the LS to investigate this effect in ASD mouse models developed by environmental and genetic cues. In mice that received valproic acid (environmental cue), we demonstrated successful recovery of impaired social memory with three-chamber test after OXTR+ neuron activation in the LS. Application of a similar strategy to Nl3 R451C knock-in mice (genetic cue) also caused successful recovery of impaired social memory in single field test. OXTR+ neurons in the LS, which are activated by social stimuli, are projected to the CA1 region of the hippocampus. This study identified a candidate mechanism for improving core symptoms of ASD by artificial activation of DREADDs, as a simulation of OXT administration to activate OXTR+ neurons in the LS.

The central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST) are the two major nuclei of the central extended amygdala that plays essential roles in threat processing, responsible for emotional states such as fear and anxiety. While some studies suggested functional differences between these nuclei, others showed anatomical and neurochemical similarities. Despite their complex subnuclear organization, subnuclei-specific functional impact on behavior and their underlying molecular profiles remain obscure. We here constitutively inhibited neurotransmission of protein kinase C-δ-positive (PKCδ+) neurons—a major cell type of the lateral subdivision of the CeA (CeL) and the oval nucleus of the BNST (BNSTov)—and found striking subnuclei-specific effects on fear- and anxiety-related behaviors, respectively. To obtain molecular clues for this dissociation, we conducted RNA sequencing in subnuclei-targeted micropunch samples. The CeL and the BNSTov displayed similar gene expression profiles at the basal level; however, both displayed differential gene expression when animals were exposed to fear-related stimuli, with a more robust expression change in the CeL. These findings provide novel insights into the molecular makeup and differential engagement of distinct subnuclei of the extended amygdala, critical for regulation of threat processing.

Neuropilin 2 (Nrp2) is expressed by a subset of mitral cells in the postero-ventral olfactory bulb in mice. Here the authors show that Nrp2 is sufficient to instruct mitral cell targeting to the anterior part of medial amygdala that modulates olfactory-driven attractive social behaviour.

Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. To disambiguate orexin-dependent and -independent physiological functions of orexin neurons, we generated a novel Orexin-flippase (Flp) knock-in mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was knock-out in homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, such activation increased the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons.

Summary: Neural network remodeling underpins the ability to remember life experiences, but little is known about the long-term plasticity of neural populations. To study how the brain encodes episodic events, we used time-lapse two-photon microscopy and a fluorescent reporter of neural plasticity based on an enhanced form of the synaptic activity-responsive element (E-SARE) within the Arc promoter to track thousands of CA1 hippocampal pyramidal cells over weeks in mice that repeatedly encountered different environments. Each environment evokes characteristic patterns of ensemble neural plasticity, but with each encounter, the set of activated cells gradually evolves. After repeated exposures, the plasticity patterns evoked by an individual environment progressively stabilize. Compared with young adults, plasticity patterns in aged mice are less specific to individual environments and less stable across repeat experiences. Long-term consolidation of hippocampal plasticity patterns may support long-term memory formation, whereas weaker consolidation in aged subjects might reflect declining memory function. : Attardo et al. use a fluorescent reporter of neural plasticity to image ensemble plasticity patterns in hippocampal neurons of live mice. These patterns turn over but progressively stabilize across repeated explorations of an enriched environment. In aged mice, plasticity patterns do not stabilize and are less specific to individual environments. Keywords: hippocampus, plasticity, immediate-early genes, two-photon imaging, representations, aging

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR.

In vivo experimental evidence indicates that acute neuronal activation increases Aβ release from presynaptic terminals, whereas long-term effects of chronic synaptic activation on Aβ pathology remain unclear. To address this issue, we adopted optogenetics and transduced stabilized step-function opsin, a channelrhodopsin engineered to elicit a long-lasting neuronal hyperexcitability, into the hippocampal perforant pathway of APP transgenic mice. In vivo microdialysis revealed a ∼24% increase in the hippocampal interstitial fluid Aβ42 levels immediately after acute light activation. Five months of chronic optogenetic stimulation increased Aβ burden specifically in the projection area of the perforant pathway (i.e., outer molecular layer of the dentate gyrus) of the stimulated side by ∼2.5-fold compared with that in the contralateral side. Epileptic seizures were observed during the course of chronic stimulation, which might have partly contributed to the Aβ pathology. These findings implicate functional abnormalities of specific neuronal circuitry in Aβ pathology and Alzheimer disease.

The activity-regulated cytoskeleton associated protein Arc is strongly and quickly upregulated by neuronal activity, synaptic potentiation and learning. Arc entry in the synapse is followed by the endocytosis of glutamatergic AMPA receptors (AMPARs), and its nuclear accumulation has been shown in vitro to result in a small decline in the transcription of the GluA1 subunit of AMPARs. Since these effects result in a decline in synaptic strength, we asked whether a change in Arc dynamics may temporally correlate with sleep-dependent GluA1 down-regulation. We measured the ratio of nuclear to cytoplasmic Arc expression (Arc Nuc/Cyto) in the cerebral cortex of EGFP-Arc transgenic mice that were awake most of the night and then perfused immediately before lights on (W mice), or were awake most of the night and then allowed to sleep (S mice) or sleep deprived (SD mice) for the first 2 h of the light phase. In primary motor cortex (M1), neurons with high levels of nuclear Arc (High Arc cells) were present in all mice, but in these cells Arc Nuc/Cyto was higher in S mice than in W mice and, importantly, ~15% higher in S mice than in SD mice collected at the same time of day, ruling out circadian effects. Greater Arc Nuc/Cyto with sleep was observed in the superficial layers of M1, but not in the deep layers. In High Arc cells, Arc Nuc/Cyto was also ~15%–30% higher in S mice than in W and SD mice in the superficial layers of primary somatosensory cortex (S1) and cingulate cortex area 1 (Cg1). In High Arc Cells of Cg1, Arc Nuc/Cyto and cytoplasmic levels of GluA1 immunoreactivities in the soma were also negatively correlated, independent of behavioral state. Thus, Arc moves to the nucleus during both sleep and wake, but its nuclear to cytoplasmic ratio increases with sleep in the superficial layers of several cortical areas. It remains to be determined whether the relative increase in nuclear Arc contributes significantly to the overall decline in the strength of excitatory synapses that occurs during sleep. Similarly, it remains to be determined whether the entry of Arc into specific synapses is gated by sleep.

How information encoded in glutamate release rates at individual synapses is converted into biochemical activation patterns of postsynaptic enzymes remains unexplored. To address this, we developed a dual fluorescence resonance energy transfer (FRET) imaging platform and recorded CaMKIIα and calcineurin activities in hippocampal neurons while varying glutamate uncaging frequencies. With little spine morphological change, 5 Hz spine glutamate uncaging strongly stimulated calcineurin, but not CaMKIIα. In contrast, 20 Hz spine glutamate uncaging, which induced spine growth, activated both CaMKIIα and calcineurin with distinct spatiotemporal kinetics. Higher temporal resolution recording in the soma revealed that CaMKIIα activity summed supralinearly and sensed both higher frequency and input number, thus acting as an input frequency/number decoder. In contrast, calcineurin activity summated sublinearly with increasing input number and showed little frequency dependence, thus functioning as an input number counter. These results provide evidence that CaMKIIα and calcineurin are fine-tuned to unique bandwidths and compute input variables in an asymmetric manner.

We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA) and the transgene under the TET Responsive Element promoter (TRE) on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP) or turboFP635 (RFP) in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.

CaMKIIa plays a fundamental role in learning and memory and is a key determinant of synaptic plasticity. Its kinase activity is regulated by the binding of Ca2+/CaM and by autophosphorylation that operates in an activity-dependent manner. Though many mutations in CAMK2A were linked to a variety of neurological disorders, the multiplicity of its functional substrates renders the systematic molecular phenotyping challenging. In this study, we report a new case of CAMK2A P212L, a recurrent mutation, in a patient with an intellectual disability. To quantify the effect of this mutation, we developed a FRET-based kinase phenotyping strategy and measured aberrance in Ca2+/CaM-dependent activation dynamics in vitro and in synaptically connected neurons. CaMKIIa P212L revealed a significantly facilitated Ca2+/CaM-dependent activation in vitro. Consistently, this mutant showed faster activation and more delayed inactivation in neurons. More prolonged kinase activation was also accompanied by a leftward shift in the CaMKIIa input frequency tuning curve. In keeping with this, molecular phenotyping of other reported CAMK2A de novo mutations linked to intellectual disability revealed aberrant facilitation of Ca2+/CaM-dependent activation of CaMKIIa in most cases. Finally, the pharmacological reversal of CAMK2A P212L phenotype in neurons was demonstrated using an FDA-approved NMDA receptor antagonist memantine, providing a basis for targeted therapeutics in CAMK2A-linked intellectual disability. Taken together, FRET-based kinase mutation phenotyping sheds light on the biological impact of CAMK2A mutations and provides a selective, sensitive, quantitative, and scalable strategy for gaining novel insights into the molecular etiology of intellectual disability. [ABSTRACT FROM AUTHOR]