The site and regulation of neurotrophic factor release from neurons is poorly understood. We used a combination of model cell lines and primary culture systems to study the polarity of BDNF sorting and the regulation of its release from hippocampal neurons. Transfection and expression of a human BDNF cDNA in a mouse pituitary cell line, AtT20, resulted in the colocalization of BDNF with the secretory granule marker, chromogranin A. Furthermore, stimulation of these cells with 56 mM KCl or with 5 mM 8-bromo-cAMP increased the release of BDNF approximately 10-to 15-fold within 30 min. To study BDNF release from primary cultures of hippocampal neurons, cells were infected with a defective Herpes Simplex Viral (HSV) vector expressing human BDNF. Depolarizing conditions increased the release of BDNF 5-fold from these cells, further verifying that secretion is regulated. Immunocytochemical analysis using highly specific antibodies determined that endogenous BDNF was predominantly localized to the somatodentritic domain of hippocampal neurons. These findings support the view that BDNF functions as a target-derived signal for afferents to hippocampal pyramidal cells and that it may serve as a regulator of hippocampal plasticity.

Tau protein hyperphosphorylation and aggregation into neurofibrillary tangles are characteristic features of several neurodegenerative disorders referred to as tauopathies. Among them, frontotemporal dementia and Parkinsonism linked to chromosome 17

Vectors based on herpes simplex virus type 1 (HSV-1) show promise for gene transfer into mammalian cells because of their wide host range, efficient infection and ability to deliver genes to nondividing cells. Defective HSV-1 vectors, or amplicons, are plasmid vectors which are unable to propagate on their own but contain specific HSV-1 sequences that, in the presence of helper virus, support DNA replication and subsequent packaging into virus particles. We compared three replication-incompetent HSV-1 mutants (KOS strain 5dll.2, strain 17 D30EBA, KOS strain dl20) as the helper virus for packaging the prototype defective HSV-1 vector, pHSVlac, which uses the HSV-1 immediate-early (IE) 4/5 promoter to regulate expression of the Escherichia coli lacZ gene. Use of 5dl1.2, which contains a deletion in the IE 2 gene, consistently produced virus stocks that contained a high level of vector, undetectable levels of wild-type HSV-1 and a ratio of vector to helper greater than 1. Virus stocks prepared using 5dl1.2 w...

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.