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SummaryPrefrontal cortex (PFC) has dramatically expanded in primates, but its organization and interactions with other brain regions are only partially understood. We performed high-resolution connectomic mapping of marmoset PFC and found two contrasting corticocortical and corticostriatal projection patterns: “patchy” projections that formed many columns of submillimeter scale in nearby and distant regions and “diffuse” projections that spread widely across the cortex and striatum. Parcellation-free analyses revealed representations of PFC gradients in these projections’ local and global distribution patterns. We also demonstrated column-scale precision of reciprocal cortico-cortical connectivity, suggesting that PFC contains a mosaic of discrete columns. Diffuse projections showed considerable diversity in the laminar patterns of axonal spread. In mice, columnar projections were much less conspicuous, underscoring the importance of the primate model. Altogether, these fine-grained analyses reveal important principles of local and long-distance PFC circuits in marmosets and provide insights into the functional organization of the primate brain.

Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2−expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

It is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354-372, 2017.

Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity invivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up- and downregulate it for more than 100days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons inlayer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates. Long-term two-photon calcium imaging has been challenging in non-human primates. Sadakane etal. use an inducible expression system to visualize cortical neurons in adult marmosets. They show that the same neuronal population can be followed over 100days and neuronal responses to tactile stimulation can be imaged at subcellular resolution.

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences.

To understand the relationship between the structure and function of primate neocortical areas at a molecular level, we have been screening for genes differentially expressed across macaque neocortical areas by restriction landmark cDNA scanning (RLCS). Here, we report enriched expression of the paraneoplastic antigen-like 5 gene (PNMA5) in association areas but not in primary sensory areas, with the lowest expression level in primary visual cortex. In situ hybridization in the primary sensory areas revealed PNMA5 mRNA expression restricted to layer II. Along the ventral visual pathway, the expression gradually increased in the excitatory neurons from the primary to higher visual areas. This differential expression pattern was very similar to that of retinol-binding protein (RBP) mRNA, another association-area-enriched gene that we reported previously. Additional expression analysis for comparison of other genes in the PNMA gene family, PNMA1, PNMA2, PNMA3, and MOAP1 (PNMA4), showed that they were widely expressed across areas and layers but without the differentiated pattern of PNMA5. In mouse brains, PNMA1 was only faintly expressed and PNMA5 was not detected. Sequence analysis showed divergence of PNMA5 sequences among mammals. These findings suggest that PNMA5 acquired a certain specialized role in the association areas of the neocortex during primate evolution.

Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spinesin vivoin the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)–tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopyin vivoand analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison ofin vivosamples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualizein vivodendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes bothin vitroandin vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex.

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.