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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. [ABSTRACT FROM AUTHOR]

Abstract: Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway. [Copyright &y& Elsevier]

Lamina formation in the developing cerebral cortex requires precisely regulated generation and migration of the cortical progenitor cells. To test the possible involvement of brain-derived neurotrophic factor (BDNF) in the formation of the cortical lamina, we investigated the effects of BDNF protein and anti-BDNF antibody separately administered into the telencephalic ventricular space of 13.5-d-old mouse embryos. BDNF altered the position, gene-expression properties, and projections of neurons otherwise destined for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex, whereas anti-BDNF antibody changed some of those of neurons of upper layers II/III. Additional analysis revealed that BDNF altered the laminar fate of neurons only if their parent progenitor cells were exposed to it at approximately S-phase and that it hastened the timing of the withdrawal of their daughter neurons from the ventricular proliferating pool by accelerating the completion of S-phase, downregulation of the Pax6 (paired box gene 6) expression, an essential transcription factor for generation of the upper layer neurons, and interkinetic nuclear migration of cortical progenitors in the ventricular zone. These observations suggest that BDNF participates in the processes forming the neuronal laminas in the developing cerebral cortex. BDNF can therefore be counted as one of the key extrinsic factors that regulate the laminar fate of cortical neurons. [ABSTRACT FROM AUTHOR]

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under the control of enhanced synaptic activity-responsive element (E-SARE), a potent neuronal activity-dependent synthetic promoter, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we found that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI. Visualisation of the activity of neural grafts using engineered luciferase provides insights into the integration between the graft and host. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]