Mice with deficiency in Pcdh15, a gene associated with bipolar disorders, exhibit significantly elevated diurnal amplitudes of locomotion and body temperature.

Genetic factors significantly affect the pathogenesis of psychiatric disorders. However, the specific pathogenic mechanisms underlying these effects are not fully understood. Recent extensive genomic studies have implicated the protocadherin-related 15 (PCDH15) gene in the onset of psychiatric disorders, such as bipolar disorder (BD). To further investigate the pathogenesis of these psychiatric disorders, we developed a mouse model lacking Pcdh15. Notably, although PCDH15 is primarily identified as the causative gene of Usher syndrome, which presents with visual and auditory impairments, our mice with Pcdh15 homozygous deletion (Pcdh15-null) did not exhibit observable structural abnormalities in either the retina or the inner ear. The Pcdh15-null mice showed very high levels of spontaneous motor activity which was too disturbed to perform standard behavioral testing. However, the Pcdh15 heterozygous deletion mice (Pcdh15-het) exhibited enhanced spontaneous locomotor activity, reduced prepulse inhibition, and diminished cliff avoidance behavior. These observations agreed with the symptoms observed in patients with various psychiatric disorders and several mouse models of psychiatric diseases. Specifically, the hyperactivity may mirror the manic episodes in BD. To obtain a more physiological, long-term quantification of the hyperactive phenotype, we implanted nano tag® sensor chips in the animals, to enable the continuous monitoring of both activity and body temperature. During the light-off period, Pcdh15-null exhibited elevated activity and body temperature compared with wild-type (WT) mice. However, we observed a decreased body temperature during the light-on period. Comprehensive brain activity was visualized using c-Fos mapping, which was assessed during the activity and temperature peak and trough. There was a stark contrast between the distribution of c-Fos expression in Pcdh15-null and WT brains during both the light-on and light-off periods. These results provide valuable insights into the neural basis of the behavioral and thermal characteristics of Pcdh15-deletion mice. Therefore, Pcdh15-deletion mice can be a novel model for BD with mania and other psychiatric disorders, with a strong genetic component that satisfies both construct and surface validity.

Three-dimensional mouse cochlea imaging based on the modified Sca/eS using confocal microscopy.

The three-dimensional stria vascularis (SV) and cochlear blood vessel structure is essential for inner ear function. Here, modified Sca/eS, a sorbitol-based optical-clearing method, was reported to visualize SV and vascular structure in the intact mouse cochlea. Cochlear macrophages as well as perivascular-resident macrophage-like melanocytes were detected as GFP-positive cells of the CX3CR1+/GFP mice. This study's method was effective in elucidating inner ear function under both physiological and pathological conditions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity. Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

In vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

Precise Temporal Regulation of Molecular Diffusion within Dendritic Spines by Actin Polymers during Structural Plasticity.

The biochemical transduction of excitatory synaptic signals occurs in the cytoplasm within dendritic spines. The associated reaction kinetics are shaped by the mobility of the signaling molecules; however, accurate monitoring of diffusional events within the femtoliter-sized spine structures has not yet been demonstrated. Here, we applied two-photon fluorescence correlation spectroscopy and raster image correlation spectroscopy to monitor protein dynamics within spines, revealing that F-actin restricts the mobility of proteins with a molecular mass of >100 kDa. This restriction is transiently removed during actin remodeling at the initial phase of spine structural plasticity. Photobleaching experiments combined with super-resolution imaging indicate that this increase in mobility facilitates molecular interactions, which may modulate the functions of key postsynaptic signaling molecules, such as Tiam1 and CaMKII. Thus, actin polymers in dendritic spines act as precise temporal regulators of molecular diffusion and modulate signal transduction during synaptic plasticity.

Tumor suppressor protein CYLD regulates morphogenesis of dendrites and spines.

Cylindromatosis tumor suppressor protein (CYLD) was initially identified as a tumor suppressor deubiquitylating protein in familial cylindromatosis patients. Proteomic analyses using rodent brain samples revealed enrichment of CYLD in purified postsynaptic density fractions. Here, we report that CYLD regulates dendritic growth and postsynaptic differentiation in mouse hippocampal neurons. CYLD showed diffuse localization in rapidly growing dendrites, but was gradually concentrated in spines. Overexpression and knockdown of CYLD in the early stage of cultured neurons demonstrated that CYLD positively regulated dendritic growth. Phenotypes in dendritic morphogenesis induced by CYLD overexpression and knockdown could be reversed by manipulation of the critical acetylation site of α-tubulin, suggesting tubulin acetylation is a downstream pathway of CYLD-dependent dendritic growth. Overexpression and knockdown of CYLD in the later stage of cultured neurons revealed that CYLD promoted formation of postsynaptic spines. Influence of CYLD on spines was not affected by co-expression of acetylation mutant forms of α-tubulin, indicating that CYLD regulates dendritic growth and spine formation through different molecular mechanisms. Analyses with the truncated and mutated forms of CYLD demonstrated that the first microtubule-binding domain of CYLD was critical for spine formation. These results suggest important roles of CYLD in sequential promotion of dendritic growth and postsynaptic spine maturation.

Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics.

Optogenetics has revolutionized the experimental interrogation of neural circuits and holds promise for the treatment of neurological disorders. It is limited, however, because visible light cannot penetrate deep inside brain tissue. Upconversion nanoparticles (UCNPs) absorb tissue-penetrating near-infrared (NIR) light and emit wavelength-specific visible light. Here, we demonstrate that molecularly tailored UCNPs can serve as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons. Transcranial NIR UCNP-mediated optogenetics evoked dopamine release from genetically tagged neurons in the ventral tegmental area, induced brain oscillations through activation of inhibitory neurons in the medial septum, silenced seizure by inhibition of hippocampal excitatory cells, and triggered memory recall. UCNP technology will enable less-invasive optical neuronal activity manipulation with the potential for remote therapy.

LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

Enhancement of sciatic nerve regeneration by adenovirus-mediated expression of dominant negative RhoA and Rac1.

It is known that Rho family small GTPases activate a number of signal transduction pathways involved in cell cycle progression, gene expression, and cell survival. These small G proteins play an important role in neuronal survival and axon regeneration in neural injury. In this study, we tested whether the activity of RhoA or Rac1 regulates neurite extension in dorsal root ganglia (DRGs) in vitro and nerve regeneration in injured sciatic nerves. Regeneration of neurites from explanted DRGs was accelerated by combined suppression of RhoA and Rac1 activity using adenoviruses expressing dominant negative (DN) forms of both RhoA and Rac1 (Ad-Rho/RacDN) in vitro. Rat sciatic nerves were cut and Ad-Rho/RacDN was injected into the proximal stumps. After bridge grafting with chitosan mesh tubes, muscle evoked potentials induced by transcranial electrical stimulation were recorded eight weeks postoperatively. The terminal latencies were shorter in the Ad-Rho/RacDN group than in the control group. Histological analysis revealed extensive regrowth of neurofilament-positive and myelinated axons within the tubes in the group that received Ad-Rho/RacDN. These findings suggest that combined regulation of RhoA and Rac1 using DN adenoviral transgenic methods has the potential to modify injured peripheral nerve tissues directly.

Critical involvement of Rho GTPase activity in the efficient transplantation of neural stem cells into the injured spinal cord.

BACKGROUND: Transplantation of neural stem/progenitor cells is a promising approach toward functional restoration of the damaged neural tissue, but the injured spinal cord has been shown to be an adverse environment for the survival, migration, and differentiation of the donor cells. To improve the efficiency of cell replacement therapy, cell autonomous factors in the donor cells should be optimized. In light of recent findings that Rho family GTPases regulate stem cell functions, genetic manipulation of Rho GTPases can potentially control phenotypes of transplanted cells. Therefore we expressed mutant forms of Rho GTPases, Rac, Rho, and Cdc42, in the neural stem/progenitor cells and examined their survival and migration after transplantation. RESULTS: Manipulation of the individual Rho GTPases showed differential effects on survival, with little variation in their migratory route and predominant differentiation into the oligodendroglial lineage. Combined suppression of both Rac and Rho activity had a prominent effect on promoting survival, consistent with its highly protective effect on drug-induced apoptosis in culture. CONCLUSION: Manipulation of Rac and Rho activities fully rescued suppression of cell survival induced by the spinal cord injury. Our results indicate that precise regulation of cell autonomous factors within the donor cells can ameliorate the detrimental environment created by the injury.

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking.

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)-dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference-mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.

Cre complementation with variable dimerizers for inducible expression in neurons.

Cre complementation is a process of reconstitution of the activity of DNA recombinase by noncovalent association of multiple segments of Cre recombinase, which are enzymatically inactive by themselves. Cre complementation is potentially useful in restriction of Cre activity in a specific subset of cells, with temporal regulation, by limiting overlap in expression of Cre fragments. We analyzed the efficiency of Cre complementation using three different dimerizing modules in the context of non-neuronal cells and found differential Cre complementation efficiency. We further tested the efficiency of Cre complementation in primary hippocampal neurons derived from transgenic mice harboring a reporter gene flanked by loxP sites and confirmed differential activity of dimerization modules in Cre-dependent recombination of the transgene. These results suggest possible application of dimerizer-based Cre complementation in inducible expression/inactivation of target genes in a specific subset of neurons in the complex environment of nervous tissue in vivo.

[Visualization of synapse-glia dynamics].

Increasing evidence indicates the importance of neuron-astrocyte interaction in synaptic function. However, structural evidence is scarce compared to abundant information from electrophysiological studies. Meticulous studies using serial electron microscopic technique in hippocampal CAI and cerebellum provided the earliest knowledge about three-dimensional close relationship between synapses and glial processes. Nevertheless, morphological observation of synapse-glia interaction in live tissues is important to support the idea of astrocytic effects on synaptic transmission. Recently several methods enabled live imaging of astrocytes as well as dendritic spines in acute slices and tissues cultures. The techniques to visualize live astrocytes in brain tissues include transgenic mice (GFAP promoter-GFP), sulforhodamine 101 (SR101) application to the surface of neocortex in vivo, ballistic labeling with EGFP plasmid and recombinant viruses (Semliki Forest virus A7 or adenovirus expressing EGFP). Live astrocytes in brain tissues showed higher motility than neuronal structures in the vicinity of dendrites. Astrocytes extend or retract their numerous fine processes and change their volume or shape in a complex manner. Simultaneous observation of filopodia/spines and astrocytes revealed that filopodia/spines often contact with astrocytic processes and that they showed coordinated morphological dynamics in adult and developmental stage, suggesting possible functions of synapse-astrocyte contacts. Indeed, the local regulation of filopodial stabilization and maturation into spines by astrocytic contacts was reported. In the next step any astrocytic structural changes around mature synapses correlated with plastic change of synaptic efficacy, such as long-term potentiation, should be investigated. Structural relationship between axon terminals and astrocytic processes should also be revealed. Furthermore, in vivo time-lapse imaging of synapseastrocyte pairs will soon be accomplished, as techniques of in vivo two-photon imaging showed remarkable progress recently.

Myosin-Va facilitates the accumulation of mRNA/protein complex in dendritic spines.

mRNA localization has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion, and allowing the local control of protein synthesis in neurons. In neuronal dendrites, the localization and translocation of mRNA is considered as one of the molecular bases of synaptic plasticity. Recent imaging and functional studies revealed that several RNA-binding proteins form a large messenger ribonucleoprotein (mRNP) complex that is involved in transport and translation of mRNA in dendrites. However, the mechanism of mRNA translocation into dendritic spines is unknown. Here, we show that an actin-based motor, myosin-Va, plays a significant role in mRNP transport in neuronal dendrites and spines. Myosin-Va was Ca2+-dependently associated with TLS, an RNA-binding protein, and its target RNA Nd1-L, an actin stabilizer. A dominant-negative mutant or RNAi of myosin-Va in neurons suppressed TLS accumulation in spines and further impaired TLS dynamics upon activation of mGluRs. The TLS translocation into spines was impeded also in neurons prepared from myosin-Va-null dilute-lethal (dl) mice, which exhibit neurological defects. Our results demonstrate that myosin-Va facilitates the transport of TLS-containing mRNP complexes in spines and may function in synaptic plasticity through Ca2+ signaling.

Antipsychotic medication and cognitive function in schizophrenia.

Antipsychotic polypharmacy and excessive dosing still prevail worldwide in the treatment of schizophrenia, while their possible association with cognitive function has not well been examined. We examined whether the "non-standard" use of antipsychotics (defined as antipsychotic polypharmacy or dosage >1,000 mg/day of chlorpromazine equivalents) is associated with cognitive function. Furthermore, we compared cognitive function between patients taking only atypical antipsychotics and those taking only conventionals. Neurocognitive functions were assessed in 67 patients with chronic schizophrenia and 92 controls using the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale-Revised (WAIS-R), the Wisconsin Card Sorting Test (WCST), and the Advanced Trail Making Test (ATMT). Patients showed markedly poorer performance than controls on all these tests. Patients on non-standard antipsychotic medication demonstrated poorer performance than those on standard medication on visual memory, delayed recall, performance IQ, and executive function. Patients taking atypical antipsychotics showed better performance than those taking conventionals on visual memory, delayed recall, and executive function. Clinical characteristics such as duration of medication, number of hospitalizations, and concomitant antiparkinsonian drugs were different between the treatment groups (both dichotomies of standard/non-standard and conventional/atypical). These results provide evidence for an association between antipsychotic medication and cognitive function. This association between antipsychotic medication and cognitive function may be due to differential illness severity (e.g., non-standard treatment for severely ill patients who have severe cognitive impairment). Alternatively, poorer cognitive function may be due in part to polypharmacy or excessive dosing. Further investigations are required to draw any conclusions.

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology.

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.

High-purity lineage selection of embryonic stem cell-derived neurons.

The derivation of somatic cell types from pluripotent and self-renewing embryonic stem (ES) cells offers attractive prospects for basic research, compound development, and regenerative medicine. A key prerequisite for biomedical applications of ES cells is the ability to differentiate and isolate defined somatic cell populations at high purity. In this study, we explore the potential of the Talpha1- enhanced green fluorescent protein (EGFP) transgene and polysialic acid (PSA)-neural cell adhesion molecule (NCAM) as lineage selection markers for the derivation of ES cell-derived neurons. Upon controlled in vitro differentiation, ES cells engineered to express EGFP under control of the Talpha1-tubulin promoter exhibited exclusive transgene expression in neurons. Similarly, PSA-NCAM expression during the early stages of ES cell differentiation was restricted to neuronal progeny. Talpha1- EGFP- and PSA-NCAM-positive neurons comprised both inhibitory and excitatory phenotypes. Compared to Talpha1-EGFP, the expression of PSA-NCAM was initiated at slightly earlier stages of neural differentiation. FACSorting of Talpha1-EGFP-positive cells and immunopanning of PSA-NCAMexpressing cells yielded neuronal populations at purities up to 99.6% and 96.9%, respectively. These findings depict Talpha1-EGFP and PSA-NCAM as suitable markers for high-purity selection of early ES cell-derived neurons.

Electroporation-mediated gene transfer system applied to cultured CNS neurons.

Electroporation is effective in transferring foreign genes into immature neurons in intact brain tissue. We utilized this approach to transfect genes into developing rodent hippocampi. Transfected hippocampi were subsequently dissociated and allowed to differentiate in culture. By optimizing developmental stage of the hippocampus, promoters to drive the marker cDNA, and culture conditions, neurons kept strong expression of multiple marker genes for more than two weeks after electroporation. We could also induce transient expression in mature neurons by combining electroporation of plasmids containing loxP-flanked stopper sequences and infection of Cre-producing recombinant adenoviruses. The system described here is useful in analyzing biological roles of multiple genes in specific stages of neuronal development.

p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor signaling.

N-methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRepsilon2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRepsilon2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.