An ability to transfect neurons in the spinal cord with an efficient high throughput system would hold many advantages for studying gene function. This could be achieved by using an appropriate culture system and a biolistic method for the introduction of DNA constructs (Arnold & Heintz, 1997). To this end, organotypic spinal slice cultures were assessed electrophysiologically and morphologically for their suitability as a model system for the study of nociceptive processing. In addition, cultures were subjected to biolistic transfection with an enhanced green fluorescent protein DNA construct with a cytomegalovirus promoter (CMV-EGFP) and the level of transfection assessed.

Wistar rats aged 8 days (15-22 g) were terminally anaesthetised with urethane (2 g kg^-1 I.P.). The spinal cord was removed and 350 mm transverse slices were cultured by a modified interface method adapted from Stoppini et al. (1991). Whole-cell patch-clamp recordings were made from the superficial dorsal horn in the presence of bicuculline (30 mM) and strychnine (10 mM). Biocytin (0.1 %) was included in the electrode for recovery and visualisation of the neuron. Graded focal stimulation with a bipolar electrode evoked EPSCs of increasing amplitude at a constant latency indicative of monosynaptic events. The selective AMPA antagonist CNQX (20 mM) completely inhibited EPSCs (cells held at -60 mV) and the m-opioid agonist DAMGO (1 mM) partially inhibited EPSCs. Recovery of biocytin-filled neurons revealed preservation of an extensive dendritic network in the majority of cells. Immunostaining for neuron-specific nuclear protein (NeuN) confirmed a dense cell population throughout the dorsal horn. In CMV-EGFP transfection studies, slice cultures were shot with a PDS-1000/He particle delivery system (Biorad) with a vacuum evacuated to 91 kPa (27ÔÔ Hg) and under a pressure of 7584 kPa (1100 p.s.i.). To facilitate neuronal survival the slices were placed on an ice-cold sterile agar plate and covered with a nylon baffle. Cultures were assessed at > 40 h post-shooting and a number of transfected cells in superficial and deep laminae were evident. Double-labelling for glial fibrillary acidic protein compared with NeuN was used to evaluate relative levels of transfection in different cell populations. However, as no co-localisation with the cellular markers GFAP or NeuN was evident, the identity of the transfected population remains to be established.

These findings show that organotypic culture combined with an optimised biolistic protocol could provide a useful model system for the study of genes relevant to nociceptive processing in the spinal cord.

This work was supported by the BBSRC and University of Leeds Research Fund.

All procedures accord with current UK legislation.