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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 ofin vitroNHP brain system. Here, we report anin vitroNHP 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 neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs showed mature neural activity with an unsynchronized pattern. The marmoset assembloid system will provide anin vitroplatform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

The purpose of sensory processing in the brain involves guiding animals to appropriate actions by estimating the causes of sensory changes in the environment. Encoding prediction error computed from the mismatch between actual input and predictions based on experience is considered an effective strategy for sensory perception in the brain. However, it remains unknown how predictive computation is implemented in the hierarchy of neural circuits in cortical areas. Here, we monitored dorsal cortex-wide neural activity using a wide-field Ca2+ imaging technique when mice encountered visuomotor mismatches in virtual reality. Posterior medial cortical areas sequentially responded to the visuomotor mismatches. The magnitude of these responses was associated with the predictability of the mismatch events. Granger causality and anatomical analysis demonstrated experience-dependent information flow structure underlying the mismatch signal transmission across the cortex. Our results reveal that the subnetworks in posteromedial cortical areas process the prediction errors in a hierarchical manner.

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.

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.

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.