A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain.

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids.

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

Processing of visual statistics of naturalistic videos in macaque visual areas V1 and V4.

Natural scenes are characterized by diverse image statistics, including various parameters of the luminance histogram, outputs of Gabor-like filters, and pairwise correlations between the filter outputs of different positions, orientations, and scales (Portilla-Simoncelli statistics). Some of these statistics capture the response properties of visual neurons. However, it remains unclear to what extent such statistics can explain neural responses to natural scenes and how neurons that are tuned to these statistics are distributed across the cortex. Using two-photon calcium imaging and an encoding-model approach, we addressed these issues in macaque visual areas V1 and V4. For each imaged neuron, we constructed an encoding model to mimic its responses to naturalistic videos. By extracting Portilla-Simoncelli statistics through outputs of both filters and filter correlations, and by computing an optimally weighted sum of these outputs, the model successfully reproduced responses in a subpopulation of neurons. We evaluated the selectivities of these neurons by quantifying the contributions of each statistic to visual responses. Neurons whose responses were mainly determined by Gabor-like filter outputs (low-level statistics) were abundant at most imaging sites in V1. In V4, the relative contribution of higher order statistics, such as cross-scale correlation, was increased. Preferred image statistics varied markedly across V4 sites, and the response similarity of two neurons at individual imaging sites gradually declined with increasing cortical distance. The results indicate that natural scene analysis progresses from V1 to V4, and neurons sharing preferred image statistics are locally clustered in V4.

Monosynaptic rabies virus tracing from projection-targeted single neurons.

A single neuron integrates inputs from thousands of presynaptic neurons to generate outputs. Circuit tracing using G-deleted rabies virus (RVΔG) vectors permits the brain-wide labeling of presynaptic inputs to targeted single neurons. However, the experimental procedures are complex, and the success rate of circuit labeling is low because of the lack of validation to increase the accuracy and efficiency of monosynaptic RVΔG tracing from targeted single neurons. We established an efficient RVΔG tracing method from projection target-defined single neurons using TVA950, a transmembrane isoform of TVA receptors, for initial viral infection. Presynaptic neurons were transsynaptically labeled from 80 % of the TVA950-expressing single starter neurons that survived after infection with EnvA-pseudotyped RVΔG in the adult mouse brain. We labeled single neuronal networks in the primary visual cortex (V1) and higher visual areas, namely the posteromedial area (PM) and anteromedial area (AM), as well as the single neuronal networks of PM-projecting V1 single neurons. Monosynaptic RVΔG tracing from projection-targeted single neurons revealed the input-output organization of single neuronal networks. Single-neuron network analysis based on RVΔG tracing will help dissect the heterogeneity of neural circuits and link circuit motifs and large-scale networks across scales, thereby clarifying information processing and circuit computation in the brain.

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.

[Circuit mechanisms of spatial perception and visuomotor integration].

Animals can make appropriate decisions based on sensory information about the environment. Vision is one of the most critical ability for survival in dynamic situations in nature, particularly for mammalian species, such as primates, carnivores, and rodents. Although there is a huge computational cost involved in processing visual information, the brain can perform this task very rapidly using well-organized parallel and hierarchical neural circuits, enabling animals to rapidly sense the environment and, in turn, perform adaptive actions. Physiological, psychophysical, and clinical studies over hundreds of years have delineated the neural circuit mechanisms of the visual system. Artificial intelligence and robotics have also started making progress in this area. However, due to technical limitations, there are still many open questions that elude explanation in understanding the neural mechanism of visuomotor integration. Herein, we initially describe the anatomical structures of occipital cortices related to vision and then provide an overview of the physiological and clinical studies of the dorsal visual pathway related to spatial perception and prediction in non-human primate species. Finally, we introduce recent approaches in which rodents have been used as model species to elucidate the neural circuit mechanism of visually-guided behavior. Uncovering neural implementation of the association between visual-spatial perception and visuomotor function could provide key insights into the engineering of highly active robots and could also contribute to the development of novel therapeutic strategies addressing visual impairment and psychiatric/neurological disorders.