Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention.

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, here we report that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. Our data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.

Evolutionary conservation and diversification of auditory neural circuits that process courtship songs in Drosophila.

Acoustic communication signals diversify even on short evolutionary time scales. To understand how the auditory system underlying acoustic communication could evolve, we conducted a systematic comparison of the early stages of the auditory neural circuit involved in song information processing between closely-related fruit-fly species. Male Drosophila melanogaster and D. simulans produce different sound signals during mating rituals, known as courtship songs. Female flies from these species selectively increase their receptivity when they hear songs with conspecific temporal patterns. Here, we firstly confirmed interspecific differences in temporal pattern preferences; D. simulans preferred pulse songs with longer intervals than D. melanogaster. Primary and secondary song-relay neurons, JO neurons and AMMC-B1 neurons, shared similar morphology and neurotransmitters between species. The temporal pattern preferences of AMMC-B1 neurons were also relatively similar between species, with slight but significant differences in their band-pass properties. Although the shift direction of the response property matched that of the behavior, these differences are not large enough to explain behavioral differences in song preferences. This study enhances our understanding of the conservation and diversification of the architecture of the early-stage neural circuit which processes acoustic communication signals.

Validity and Reliability of Criteria for Plantar Sensation Assessment Using Semmes-Weinstein Monofilament as a Clinically Usable Index.

There is no standard clinically adaptable criterion for assessing plantar sensation for pre- and post-intervention comparisons. Studies using Semmes-Weinstein monofilaments (SWMs) to investigate intervention effects on plantar sensation vary in procedure and do not consider measurement errors. This study aimed to develop a simple criterion using SWMs to assess plantar sensation, determine the measurement error range, and identify areas of low error. Six examiners assessed 87 healthy young adults in Experiment 1, while two examiners assessed 10 participants in Experiment 2. Filaments were graded from 1 to 20 based on increasing diameter. The smallest grade that could be perceived for three sequential stimuli was used as the criterion (smallest perceivable grade, SPG). The SPG was significantly smaller at the hallux and larger at the heel than at other sites. There were no significant differences between the SPG of the repeated tests performed by the same versus different examiners. The interquartile range of the differences was <±3 at all sites. Thus, our criteria were reliable in evaluating the effects of plantar sensation interventions, especially at the heel and the middle of the metatarsal heads and could contribute to the development of more effective treatments for plantar sensations.

Efficient and robust induction of retinal pigment epithelium cells by tankyrase inhibition regardless of the differentiation propensity of human induced pluripotent stem cells.

Transplantation of retinal pigment epithelium (RPE) cells derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) hold great promise as a new therapeutic modality for age-related macular degeneration and Stargardt disease. The development of hESC/hiPSC-derived RPE cells as cell-based therapeutic products requires a robust, scalable production for every hiPSC line congruent for patients. However, individual hESC/hiPSC lines show bias in differentiation. Here we report an efficient, robust method that induces RPE cells regardless of the differentiation propensity of the hiPSC lines. Application of the tankyrase inhibitor IWR-1-endo, which potentially inhibits Wnt signaling, promoted retinal differentiation in dissociated hiPSCs under feeder-free, two-dimensional culture conditions. The other tankyrase inhibitor, XAV939, also promoted retinal differentiation. However, Wnt signaling inhibitors, IWP-2 and iCRT3, that target porcupine and ß-catenin/TCF, respectively, did not. Further treatment with the GSK3ß inhibitor CHIR99021 and FGF receptor inhibitor SU5402 induced hexagonal pigmented cells with phagocytotic ability. Notably, the IWR-1-endo-based differentiation method induced RPE cells even in an hiPSC line that expresses a lower level of the differentiation propensity marker SALL3, which is indicative of resistance to ectoderm differentiation. The present study demonstrated that tankyrase inhibitors cause efficient and robust RPE differentiation, irrespective of the SALL3 expression levels in hiPSC lines. This differentiation method will resolve line-to-line variations of hiPSCs in RPE production and facilitate clinical application and industrialization of RPE cell products for regenerative medicine.

Temporally multiplexed dual-plane imaging of neural activity with four-dimensional precision.

Spatiotemporal patterns of neural activity generate brain functions, such as perception, memory, and behavior. Four-dimensional (4-D: x, y, z, t) analyses of such neural activity will facilitate understanding of brain functions. However, conventional two-photon microscope systems observe single-plane brain tissue alone at a time with cellular resolution. It faces a trade-off between the spatial resolution in the x-, y-, and z-axes and the temporal resolution by a limited point-by-point scan speed. To overcome this trade-off in 4-D imaging, we developed a holographic two-photon microscope for dual-plane imaging. A spatial light modulator (SLM) provided an additional focal plane at a different depth. Temporal multiplexing of split lasers with an optical chopper allowed fast imaging of two different focal planes. We simultaneously recorded the activities of neurons on layers 2/3 and 5 of the cerebral cortex in awake mice in vivo. The present study demonstrated the proof-of-concept of dual-plane two-photon imaging of neural circuits by using the temporally multiplexed SLM-based microscope. The temporally multiplexed holographic microscope, combined with in vivo labeling with genetically encoded probes, enabled 4-D imaging and analysis of neural activities at cellular resolution and physiological timescales. Large-scale 4-D imaging and analysis will facilitate studies of not only the nervous system but also of various biological systems.

Cell type- and layer-specific convergence in core and shell neurons of the dorsal lateral geniculate nucleus.

Over 40 distinct types of retinal ganglion cells (RGCs) generate parallel processing pathways in the visual system. In mice, two subdivisions of the dorsal lateral geniculate nucleus (dLGN), the core and the shell, organize distinct parallel channels to transmit visual information from the retina to the primary visual cortex (V1). To investigate how the dLGN core and shell differentially integrate visual information and other modalities, we mapped synaptic input sources to each dLGN subdivision at the cell-type level with G-deleted rabies viral vectors. The monosynaptic circuit tracing revealed that dLGN core neurons received inputs from alpha-RGCs, Layer 6 neurons of the V1, the superficial and intermediate layers of the superior colliculus (SC), the internal ventral LGN, the lower layer of the external ventral LGN (vLGNe), the intergeniculate leaf, the thalamic reticular nucleus (TRN), and the pretectal nucleus (PT). Conversely, shell neurons received inputs from alpha-RGCs and direction-selective ganglion cells of the retina, Layer 6 neurons of the V1, the superficial layer of the SC, the superficial and lower layers of the vLGNe, the TRN, the PT, and the parabigeminal nucleus. The present study provides anatomical evidence of the cell type- and layer-specific convergence in dLGN core and shell neurons. These findings suggest that dLGN core neurons integrate and process more multimodal information along with visual information than shell neurons and that LGN core and shell neurons integrate different types of information, send their own convergent information to discrete populations of the V1, and differentially contribute to visual perception and behavior.

Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

Transplantation of retinal pigment epithelial (RPE) sheets derived from human induced pluripotent cells (hiPSC) is a promising cell therapy for RPE degeneration, such as in age-related macular degeneration. Current RPE replacement therapies, however, face major challenges. They require a tedious manual process of selecting differentiated RPE from hiPSC-derived cells, and despite wide variation in quality of RPE sheets, there exists no efficient process for distinguishing functional RPE sheets from those unsuitable for transplantation. To overcome these issues, we developed methods for the generation of RPE sheets from hiPSC, and image-based evaluation. We found that stepwise treatment with six signaling pathway inhibitors along with nicotinamide increased RPE differentiation efficiency (RPE6iN), enabling the RPE sheet generation at high purity without manual selection. Machine learning models were developed based on cellular morphological features of F-actin-labeled RPE images for predicting transepithelial electrical resistance values, an indicator of RPE sheet function. Our model was effective at identifying low-quality RPE sheets for elimination, even when using label-free images. The RPE6iN-based RPE sheet generation combined with the non-destructive image-based prediction offers a comprehensive new solution for the large-scale production of pure RPE sheets with lot-to-lot variations and should facilitate the further development of RPE replacement therapies.

Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors.

Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.

Associations of maternal and neonatal serum trace element concentrations with neonatal birth weight.

BACKGROUND: Trace elements play important nutritional roles in neonates. Our objective was to examine whether there are differences in maternal/neonatal serum trace element concentrations between mature infants and premature infants. METHODS: During 2012, 44 infants born at National Center for Global Health and Medicine, Tokyo, Japan, were enrolled. Serum samples were collected to measure serum iron, zinc, copper, and selenium concentrations 5 days after birth. Maternal serum samples were obtained before delivery and cord blood was taken at delivery to measure the same trace elements. We compared the results between term group whose birth weight were ≥2500 g and gestational age were ≥37 weeks and premature group whose birth weight were <2500 g or gestational age were <37 weeks. Variables significantly different between two groups were included in linear regression models to identify significant predictors of birth weight. Values of P<0.05 were considered statistically significant. RESULTS: Serum selenium concentrations were lower in premature group than in term group (43.3±7.0 µg/L vs. 52.0±8.9 µg/L, P = 0.001). Maternal serum selenium concentrations were also significantly lower in the mothers of premature group than in the mothers of term group (79.3±19.3 µg/L vs. 94.1±18.1 µg/L, P = 0.032). There were no significant differences in neonatal or maternal iron, zinc, or copper concentrations between two groups. Multivariate linear regression analysis showed that, except for gestational age, only maternal serum selenium was significantly associated with birth weight (P = 0.015). CONCLUSIONS: Serum selenium concentrations were lower in premature group and their mothers compared with the term group. The maternal serum selenium concentration was positively correlated with birth weight. These results suggest that maternal serum selenium concentration may influence neonatal birth weight.