Adrian T. Rogers

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

Abstract High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [ 3 H]nicotine or [ 3 H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [ 3 H]QNB were present at a density of 0.75 pmol/mg protein. [ 125 I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B max of 128 fmol/mg protein and a K d of 0.6 nM; cortical cultures expressed five times fewer [ 125 I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10 4 and 2 × 10 4 binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [ 125 I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.

Differential effects of chronic drug treatment on α3* and α7 nicotinic receptor binding sites, in hippocampal neurones and SH‐SY5Y cells

The aim of this study was to compare the effects of chronic treatment (for 4 or 7 days) with nicotinic drugs and 20 mM KCl on numbers of surface α7 nicotinic AChR, identified by [125I]‐α bungarotoxin (α‐Bgt) binding, in primary hippocampal cultures and SH‐SY5Y cells. Numbers of α3* nicotinic AChR were also examined in SH‐SY5Y cells, using [3H]‐epibatidine, which is predicted to label the total cellular population of predominantly α3β2* nicotinic AChR under the conditions used. All the nicotinic agonists examined, the antagonists d‐tubocurarine and methyllycaconitine, and KCl, upregulated [125I]‐α Bgt binding sites by 20–60% in hippocampal neurones and, where examined, SH‐SY5Y cells. Upregulation of [125I]‐α‐Bgt binding sites by KCl was prevented by co‐incubation with the L‐type Ca2+ channel blocker verapamil or the Ca2+‐calmodulin dependent kinase II (CaM‐kinase II) inhibitor KN‐62. Upregulation of [125I]‐α‐Bgt binding sites by nicotine or 3,[(4‐dimethylamino) cinnamylidene] anabaseine maleate (DMAC) was insensitive to these agents. [3H]‐Epibatidine binding sites in SH‐SY5Y cells were not affected by KCl but were upregulated in a verapamil‐insensitive manner by nicotine and DMAC. KN‐62 itself provoked a 2 fold increase in [3H]‐epibatidine binding. The inactive analogue KN‐04 had no effect, suggesting that CaM‐kinase II plays a role in regulating numbers of α3* nicotinic AChR. These data indicate that numbers of α3* and α7 nicotinic AChR are modulated differently. Nicotinic agonists and KCl upregulate α7 nicotinic AChR through distinct cellular mechanisms, the latter involving L‐type Ca2+ channels and CaM‐kinase II. In contrast, α3* nicotinic AChR are not upregulated by KCl. This difference may reflect the distinct physiological roles proposed for α7 nicotinic AChR.

Development of a neuronal pressure sensor

Neuronal sensory systems are capable of performing very complex signal processing functions. Reconstruction of such sensory systems in vitro should enable whole-cell biological sensors to be generated that possess inherent signal processing capabilities. In this paper, the results of preliminary investigations to produce a mechanosensory neuronal network are presented. An in vitro network of rat dorsal root ganglion neurons has been produced on a microelectrode plate revealing an interesting rhythmical pattern of spontaneous discharges. This periodic activity has been shown to be disrupted following the application of a static pressure to the cell culture. These results indicate that neuronal networks represent a practical system that may be used for the development of intelligent, whole-cell, biological sensors.

Neuronotrophic activities of normal and motor neurone disease patient skeletal muscle extract on cultured rat spinal neurones

Cultured foetal rat spinal neurones, grown in serum-free medium from day 3, respond to neuronotrophic factors present in adult human skeletal muscle extracts. Levels of total protein, neurofilament protein and choline acetyltransferase (ChAT) activity are all increased approx. 2 to 3-fold at 200 micrograms extract/ml medium. Culture survival is also extended from an average of 21 days to more than 30 days. Extracts prepared from skeletal muscle biopsies of motor neurone disease (MND) patients do not differ significantly from non-MND patient muscle extracts in their ability to boost the parameter levels or survival of the cultures. The results show no evidence that neuronotrophic factors are lacking in the skeletal muscle of MND patients.