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.

Rapid processing of threatening faces in the amygdala of nonhuman primates: subcortical inputs and dual roles.

A subcortical pathway through the superior colliculus and pulvinar has been proposed to provide the amygdala with rapid but coarse visual information about emotional faces. However, evidence for short-latency, facial expression-discriminating responses from individual amygdala neurons is lacking; even if such a response exists, how it might contribute to stimulus detection is unclear. Also, no definitive anatomical evidence is available for the assumed pathway. Here we showed that ensemble responses of amygdala neurons in monkeys carried robust information about open-mouthed, presumably threatening, faces within 50 ms after stimulus onset. This short-latency signal was not found in the visual cortex, suggesting a subcortical origin. Temporal analysis revealed that the early response contained excitatory and suppressive components. The excitatory component may be useful for sending rapid signals downstream, while the sharpening of the rising phase of later-arriving inputs (presumably from the cortex) by the suppressive component might improve the processing of facial expressions over time. Injection of a retrograde trans-synaptic tracer into the amygdala revealed presumed monosynaptic labeling in the pulvinar and disynaptic labeling in the superior colliculus, including the retinorecipient layers. We suggest that the early amygdala responses originating from the colliculo-pulvino-amygdalar pathway play dual roles in threat detection.

Laminar Organization of the Entorhinal Cortex in Macaque Monkeys Based on Cell-Type-Specific Markers and Connectivity.

The entorhinal cortex (EC) is a major gateway between the hippocampus and telencephalic structures, and plays a critical role in memory and navigation. Through the use of various molecular markers and genetic tools, neuron types constituting EC are well studied in rodents, and their layer-dependent distributions, connections, and functions have also been characterized. In primates, however, such cell-type-specific understandings are lagging. To bridge the gap between rodents and primates, here we provide the first cell-type-based global map of EC in macaque monkeys. The laminar organization of the monkey EC was systematically examined and compared with that of the rodent EC by using immunohistochemistry for molecular markers which have been well characterized in the rodent EC: reelin, calbindin, and Purkinje cell protein 4 (PCP4). We further employed retrograde neuron labeling from the nucleus accumbens and amygdala to identify the EC output layer. This cell-type-based approach enabled us to apply the latest laminar definition of rodent EC to monkeys. Based on the similarity of the laminar organization, the monkey EC can be divided into two subdivisions: rostral and caudal EC. These subdivisions likely correspond to the lateral and medial EC in rodents, respectively. In addition, we found an overall absence of a clear laminar arrangement of layer V neurons in the rostral EC, unlike rodents. The cell-type-based architectural map provided in this study will accelerate the application of genetic tools in monkeys for better understanding of the role of EC in memory and navigation.

Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections.

For achieving retrograde gene transfer, we have so far developed two types of lentiviral vectors pseudotyped with fusion envelope glycoprotein, termed HiRet vector and NeuRet vector, consisting of distinct combinations of rabies virus and vesicular stomatitis virus glycoproteins. In the present study, we compared the patterns of retrograde transgene expression for the HiRet vs. NeuRet vectors by testing the cortical input system. These vectors were injected into the motor cortex in rats, marmosets, and macaques, and the distributions of retrograde labels were investigated in the cortex and thalamus. Our histological analysis revealed that the NeuRet vector generally exhibits a higher efficiency of retrograde gene transfer than the HiRet vector, though its capacity of retrograde transgene expression in the macaque brain is unexpectedly low, especially in terms of the intracortical connections, as compared to the rat and marmoset brains. It was also demonstrated that the NeuRet but not the HiRet vector displays sufficiently high neuron specificity and causes no marked inflammatory/immune responses at the vector injection sites in the primate (marmoset and macaque) brains. The present results indicate that the retrograde transgene efficiency of the NeuRet vector varies depending not only on the species but also on the input projections.

A note on retrograde gene transfer efficiency and inflammatory response of lentiviral vectors pseudotyped with FuG-E vs. FuG-B2 glycoproteins.

Pseudotyped lentiviral vectors give access to pathway-selective gene manipulation via retrograde transfer. Two types of such lentiviral vectors have been developed. One is the so-called NeuRet vector pseudotyped with fusion glycoprotein type E, which preferentially transduces neurons. The other is the so-called HiRet vector pseudotyped with fusion glycoprotein type B2, which permits gene transfer into both neurons and glial cells at the injection site. Although these vectors have been applied in many studies investigating neural network functions, it remains unclear which vector is more appropriate for retrograde gene delivery in the brain. To compare the gene transfer efficiency and inflammatory response of the NeuRet vs. HiRet vectors, each vector was injected into the striatum in macaque monkeys, common marmosets, and rats. It was revealed that retrograde gene delivery of the NeuRet vector was equal to or greater than that of the HiRet vector. Furthermore, inflammation characterized by microglial and lymphocytic infiltration occurred when the HiRet vector, but not the NeuRet vector, was injected into the primate brain. The present results indicate that the NeuRet vector is more suitable than the HiRet vector for retrograde gene transfer in the primate and rodent brains.

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions.

Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G) enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E), which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E-pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson's disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E-pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson's disease.

Differential regulations of vestibulo-ocular reflex and optokinetic response by ß- and α2-adrenergic receptors in the cerebellar flocculus.

Norepinephrine modulates synaptic plasticity in various brain regions and is implicated in memory formation, consolidation and retrieval. The cerebellum is involved in motor learning, and adaptations of the vestibulo-ocular reflex (VOR) and optokinetic response (OKR) have been studied as models of cerebellum-dependent motor learning. Previous studies showed the involvement of adrenergic systems in the regulation of VOR, OKR and cerebellar synaptic functions. Here, we show differential contributions of ß- and α2-adrenergic receptors in the mouse cerebellar flocculus to VOR and OKR control. Effects of application of ß- or α2-adrenergic agonist or antagonist into the flocculus suggest that the ß-adrenergic receptor activity maintains the VOR gain at high levels and contributes to adaptation of OKR, and that α2-adrenergic receptor counteracts the ß-receptor activity in VOR and OKR control. We also examined effects of norepinephrine application, and the results suggest that norepinephrine regulates VOR and OKR through ß-adrenergic receptor at low concentrations and through α2-receptor at high concentrations.

The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.