Daisuke Takahara

Neuron-Specific Gene Transfer Through Retrograde Transport of Lentiviral Vector Pseudotyped with a Novel Type of Fusion Envelope Glycoprotein

The lentiviral vector system is used extensively in gene therapy trials for various neurological and neurodegenerative disorders. The vector system permits efficient and sustained gene expression in many cell types through integration of the transgene into the host cell genome. However, there is a significant issue concerning the therapeutic use of lentiviral vectors, that transgene insertion may lead to tumorigenesis by altering the expression of proto-oncogenes adjacent to the integration sites. One useful approach for improving safety is to restrict vector transduction to neuronal cells. We have reported the use of human immunodeficiency virus type 1 (HIV-1)-based vectors for efficient retrograde transport by pseudotyping with rabies virus glycoprotein (RV-G) or fusion glycoprotein B type, in which the cytoplasmic domain of RV-G was substituted with the counterpart of vesicular stomatitis virus glycoprotein (VSV-G). Here we developed a novel vector system for neuron-specific retrograde gene transfer (termed NeuRet) by pseudotyping the HIV-1 vector with fusion glycoprotein C type (FuG-C), in which a short C-terminal segment of the extracellular domain and the transmembrane/cytoplasmic domains of RV-G were replaced with the corresponding regions of VSV-G. FuG-C pseudotyping caused efficient gene transfer, mainly through retrograde transport, into neuronal cells in diverse brain regions, whereas the pseudotyping resulted in less efficiency for the transduction of glial and neural stem/progenitor cells. Our NeuRet vector system achieves efficient retrograde gene delivery for therapeutic trials and improves their safety by greatly reducing the risk of gene transduction of dividing cells in the brain.

Motor and non‐motor projections from the cerebellum to rostrocaudally distinct sectors of the dorsal premotor cortex in macaques

In the caudal part of the dorsal premotor cortex of macaques (area F2), both anatomical and physiological studies have identified two rostrocaudally separate sectors. The rostral sector (F2r) is located medial to the genu of the arcuate sulcus, and the caudal sector (F2c) is located lateral to the superior precentral dimple. Here we examined the sites of origin of projections from the cerebellum to F2r and F2c. We applied retrograde transsynaptic transport of a neurotropic virus, CVS‐11 of rabies virus, in macaque monkeys. Three days after rabies injections into F2r or F2c, neuronal labeling was found in the deep cerebellar nuclei mainly of the contralateral hemisphere. After the F2r injection, labeled cells were distributed primarily in the caudoventral portion of the dentate nucleus, whereas cells labeled after the F2c injection were distributed in the rostrodorsal portion of the dentate nucleus, and in the interpositus and fastigial nuclei. Four days after rabies injections, Purkinje cells were densely labeled in the lateral part of the cerebellar cortex. After the F2r injection, Purkinje cell labeling was confined to Crus I and II, whereas the labeling seen after the F2c injection was located broadly from lobules III to VIII, including Crus I and II. These results have revealed that F2c receives inputs from broader areas of the cerebellum than F2r, and that distinct portions of the deep cerebellar nuclei and the cerebellar cortex send major projections to F2r and F2c, suggesting that F2c and F2r may be under specific influences of the cerebellum.

Multisynaptic projections from the ventrolateral prefrontal cortex to the dorsal premotor cortex in macaques – anatomical substrate for conditional visuomotor behavior

Lines of evidence indicate that both the ventrolateral prefrontal cortex (vlPFC) (areas 45/12) and dorsal premotor cortex (PMd) (rostral F2 in area 6) are crucially involved in conditional visuomotor behavior, in which it is required to determine an action based on an associated visual object. However, virtually no direct projections appear to exist between the vlPFC and PMd. In the present study, to elucidate possible multisynaptic networks linking the vlPFC to the PMd, we performed a series of neuroanatomical tract‐tracing experiments in macaque monkeys. First, we identified cortical areas that send projection fibers directly to the PMd by injecting Fast Blue into the PMd. Considerable retrograde labeling occurred in the dorsal prefrontal cortex (dPFC) (areas 46d/9/8B/8Ad), dorsomedial motor cortex (dmMC) (F7 and presupplementary motor area), rostral cingulate motor area, and ventral premotor cortex (F5 and area 44), whereas the vlPFC was virtually devoid of neuronal labeling. Second, we injected the rabies virus, a retrograde transneuronal tracer, into the PMd. At 3 days after the rabies injections, second‐order neurons were labeled in the vlPFC (mainly area 45), indicating that the vlPFC disynaptically projects to the PMd. Finally, to determine areas that connect the vlPFC to the PMd indirectly, we carried out an anterograde/retrograde dual‐labeling experiment in single monkeys. By examining the distribution of axon terminals labeled from the vlPFC and cell bodies labeled from the PMd, we found overlapping labels in the dPFC and dmMC. These results indicate that the vlPFC outflow is directed toward the PMd in a multisynaptic fashion through the dPFC and/or dmMC.

Origins of multisynaptic projections from the basal ganglia to rostrocaudally distinct sectors of the dorsal premotor area in macaques.

We examined the organization of multisynaptic projections from the basal ganglia (BG) to the dorsal premotor area in macaques. After injection of the rabies virus into the rostral sector of the caudal aspect of the dorsal premotor area (F2r) and the caudal sector of the caudal aspect of the dorsal premotor area (F2c), second‐order neuron labeling occurred in the internal segment of the globus pallidus (GPi) and the substantia nigra pars reticulata (SNr). Labeled GPi neurons were found in the caudoventral portion after F2c injection, and in the dorsal portion at the rostrocaudal middle level after F2r injection. In the SNr, F2c and F2r injections led to labeling in the caudal or rostral part, respectively. Subsequently, third‐order neuron labeling was observed in the external segment of the globus pallidus (GPe), the subthalamic nucleus (STN), and the striatum. After F2c injection, labeled neurons were observed over a broad territory in the GPe, whereas after F2r injection, labeled neurons tended to be restricted to the rostral and dorsal portions. In the STN, F2c injection resulted in extensive labeling over the nucleus, whereas F2r injection resulted in labeling in the ventral portion only. After both F2r and F2c injections, labeled neurons in the striatum were widely observed in the striatal cell bridge region and neighboring areas, as well as in the ventral striatum. The present results revealed that the origins of multisynaptic projections to F2c and F2r in the BG are segregated in the output stations of the BG, whereas intermingling rather than segregation is evident with respect to their input station.

Dorsal Area 46 Is a Major Target of Disynaptic Projections From the Medial Temporal Lobe

The medial temporal lobe (MTL) is responsible for various mnemonic functions, such as association/conjunction memory. The lateral prefrontal cortex (LPFC) also plays crucial roles in mnemonic functions and memory-based cognitive behaviors, for example, decision-making. Therefore, it is considered that the MTL and LPFC connect with each other and cooperate for the control of cognitive behaviors. However, there exist very weak, if any, direct inputs from the MTL to the LPFC. Employing retrograde transsynaptic transport of rabies virus, we investigated the organization of disynaptic bottom-up pathways connecting the MTL and the inferotemporal cortex to the LPFC in macaques. Three days after rabies injections into dorsal area 46, a large number of labeled neurons were observed in the MTL, such as the hippocampal formation (including the entorhinal cortex), the perirhinal cortex, and the parahippocampal cortex. In contrast, a majority of the labeled neurons were located in the inferotemporal cortex following rabies injections into ventral area 46 and lateral area 12. Rabies injections into lateral area 9/area 8B labeled only a small number of neurons in the MTL and the inferotemporal cortex. The present results indicate that, among the LPFC, dorsal area 46 is the main target of disynaptic inputs from the MTL.

Neuron-specific gene transfer system through retrograde transport of lentiviral vector for the study of structure and function of brain neural circuit

P4-u20 Neuron-specific gene transfer system through retrograde transport of lentiviral vector for the study of structure and function of brain neural circuit Masahito Kuramochi 1 , Shigeki Kato 1, Kenta Kobayashi 1, Kenji Takasumi 1, Daisuke Takahara 2, Kenichi Inoue 2, Takashi Shimada 3, Masahiko Takada 2, Kazuto Kobayashi 1 1 Dep. of Mol. Genetics, Inst. of Biomed. Sci., Fukushima Med. University, Fukushima, Japan 2 Systems Neurosci. Section, Primate Res. Inst., Kyoto University, Inuyama, Japan 3 Dep. of Biochem. & Mol. Bio., Nippon Medical School, Tokyo, Japan

Multisynaptic inputs from the basal ganglia (BG) to rostrocaudally distinct sectors of the dorsal premotor cortex (PMd) in macaques

P2-g12 Multisynaptic inputs from the basal ganglia (BG) to rostrocaudally distinct sectors of the dorsal premotor cortex (PMd) in macaques Eiji Hoshi 1 , Yosuke Saga 1, Daisuke Takahara 2,3,4, Yoshihiro Hirata 2,3, Kenichi Inoue 2,3, Shigehiro Miyachi 5, Atsushi Nambu 4, Jun Tanji 1, Masahiko Takada 2,3 1 Tamagawa Univ Brain Sci Inst 2 Dept System Neurosci, Tokyo Metropolitan Inst for Neurosci 3 Systems Neurosci Sect, Primate Res Inst, Kyoto Univ 4 Div System Neurophysiol, National Inst for Physiological Sci 5 Cognitive Neurosci Sect, Primate Res Inst, Kyoto Univ

Multisynaptic inputs from the cerebellum to the dorsal premotor area (PMd) of macaques

The aim of this study was to identify the origin of multisynaptic inputs from the GPi to two sectors of the PMd. We injected rabies virus into the rostral (F2r) or caudal part (F2c) of the PMd in macaque monkeys. The virus was transported across synapses from postsynaptic to presynaptic neurons. After a 3-day postinjection period that allows for the second-order neuron labeling, the GPi contained labeled neurons. There was a difference in the distribution area of neuronal labeling between the two injection cases. After the F2r injection, labeled GPi neurons were found in the associative territory. By contrast, the F2c injection led to labeling of the motor territory. The present results revealed that F2r and F2c participate in the associative or motor loop, respectively, suggesting a distinct involvement of these regions in motor planning versus execution.

Frontal lobe inputs to the shoulder region of the dorsal premotor area (PMd)

Single unit activity of striatal neurons was recorded in monkeys during the performance of a reaching task with delay, and their responses were examined after electrical stimulation of the primary motor cortex and supplementary motor area. In addition to projection neurons, fast active neurons (FANs) that were considered interneurons as characterized by higher spontaneous activity and short-latency strong excitation were identified in response to the cortical stimulation. FANs also showed movement-related activity, but their specificity to task events and target locations was not so marked as that of projection neurons. Possible functional roles of FANs will be discussed in terms of motor control.