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To study the mechanism of differentiation of sensory neurons and the role of the Bm-3 protein family at the molecular level requires large amounts of a homogeneous population of neuronal cells. While it is relatively easy to dissect the dorsal root ganglia (DRG), the primary cultures produced contain a highly heterogeneous population of cells. To circumvent this problem, attempts have been made to obtain neuronal-derived cell lines. Neuroblastoma cell cultures such as C 1300 (Augusti-Tocco and Sato, 1969) are derived from neural crest precursors of sympathetic neurons that continue to proliferate and show defective differentiation in vivo. By modifying the culture medium in-vitro these cells can be induced to differentiate and display some properties of neuronal cells which are often representative of cells in the central nervous system (CNS). Other cell lines such as the phaechromocytoma cells, PC12, provide a good model for sympathetic neurons since in the presence of nerve growth factor (NGF) these cells readily differentiate from an adrenal medullary cell type to a mature sympathetic neuron (Greene and Tischler, 1976).

However, neither of these cell hnes are suitable models for studying sensory neuronal cell development, characteristics or behaviour. Hence, a number of groups have endeavoured to develop hybrids of immortalized neuroblastoma cells with primary sensory neuronal cells from DRG. Hybrids resulting from this process have been studied and some of these clones, such as the ND7 cells, (Wood et ai, 1990), provide useful models for studying the sensory neurons in vitro, since they can be maintained indefinitely in culture using the appropriate medium but can be induced to differentiate and take up the characteristics and behaviour of sensory neurons.

1.5.1

ND cell lines

The ND (Neuroblastoma + DRG) cell lines were derived by a fusion of neonatal rat

primary DRG and the immortalized mouse neuroblastoma cell line, N18Tg2 (a subclone of the C13(X) cell line) (Wood et a i, 1990). Cloning by limiting dilution allowed the isolation of several ND cell lines that exhibited some properties not displayed by the parental

neuroblastoma cells, such as presence of specific surface markers and neuropeptides present in differentiated DRG neurons. These cells were also characterized in terms of their biochemical and pharmacological responses such as the ability to depolorize in response to bradykinin and capsaicin (tigure 1.3.0) (Wood e ta l, 1990, Wheatley e ta l, 1992).

Some of these cells, ND3, ND7 and ND26 could be induced to differentiate when

transferred to specifically defined medium (Wood et a i, 1990). The cells were able to exit the cell cycle and ceased proliferation while undergoing morphological changes such as the flattening of the cell body and the extension of neurite processes which are characteristic of sensory neurons. Other changes such as the secretion of neuropeptides indicated that some cell hnes were more characteristic of the sensory neuronal parent. In addition, ND3 and ND7 cells could be latently infected by the herpes simplex virus (HS V) in a manner that was similar to the asymptomatic latent infection observed in sensory neurons (Wheatley et

a i, 1991, Latchman, 1990). The ND7 cell line was widely studied and found to be the best model for sensory neuronal cells.

1.5.2

ND7 cells

ND7 cells maintained in medium containing 10% foetal calf serum (referred to as full growth medium, FGM) continue to proliferate but on transfer to low serum medium (0.5% PCS) supplemented with ImM cychc AMP, or into serum free medium, these cells cease to prohferate and by forty eight to seventy two hours assume the morphology of sensory neuronal cells with flat cell bodies and extensive processes (figure 1.5.1). In addition, the electrophysiological responses are similar to that observed in sensory neurons when they were exposed to activators of nociceptive neurons such as bradykinin and capsaicin.

Immunocytochemical studies also show that upon differentiation there is a selective pattem of redistribution of neuropeptides such as neuropeptide Y (NPY) and its C - flanking peptide (CPON), chromogranin A and B, which are associated with secretory granules and synaptophysin which is a marker of synaptic vesicles. Chromogranin A and B as well as NPY and CPON appear to be locahzed to the tips of the cell processes in differentiated ND7 cells (Suboro et a l, 1992). Whereas NPY and CPON are also found in the

differentiated parental N18Tg2 cell line, they are present in the cell body instead of being relocalized to the axons. These lindings, combined with the detection of synaptophysin, a marker of differentiated synapses (Sudhof et a l, 1987), in ND7 cells but not in the N18Tg2 cells reflects the more complex neuronal differentiation in ND7 cells compared with the neuroblastoma cells under similar conditions. Furthermore, these cells have also been shown to transcribe the latency associated transcripts (LATs) which support latent HSV infection by contributing to the failure of transcription of the immediate-early genes necessary for a lytic infection and which are found in sensory neurons but not in the neuroblastoma cell line, C l300 [reviewed in Latchman, 1990(ii)].

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Figure 1.5.1: Morphology of ND7 cells grown in (1) full growth medium containing 10% foetal calf serum, (2) differentiation medium (serum-free) or (3) full growth medium supplemented with cyclic AMP.

Therefore ND7 cells appear to provide a suitable model which parallels the characteristics and responses of DRG sensory neurons. Furthermore, expression of a number of POU domain proteins such as Oct-1, the different isoforms of Oct-2 and Bm-3 proteins which have been identified in sensory neurons have validated the use of this cell hne for studying the modulation of these factors in-vitro (Hatzopoulos et a i, 1990, Wirth et a l, 1990; Latchman et a l, 1992; Lillycrop et a i, 1992). ND7 cells were therefore used for studying the mechanism of sensory neuron differentiation in-vitro and the roles that the Bm-3 proteins may have in this process. The results are discussed in chapters 3 to 6.