Elsevier

Neuropharmacology

Volume 39, Issue 13, December 2000, Pages 2663-2672
Neuropharmacology

Assessment of nicotinic acetylcholine receptor-mediated release of [3H]-norepinephrine from rat brain slices using a new 96-well format assay

https://doi.org/10.1016/S0028-3908(00)00143-XGet rights and content

Abstract

The study of the modulatory effects of nicotinic acetylcholine receptor (nAChR) agonists on neurotransmitter release from tissue slices has been hampered by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced comparable results to previously published data, yet expanded throughput to permit more complete pharmacological characterization. Rat brain slices, preloaded with [3H]-norepinephrine ([3H]-NE), were distributed onto 96-well filter plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (−)-Nicotine (NIC) and 1,1-dimethyl-4-phenylpiperazine (DMPP) evoked release of [3H]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippocampus. Concentration–response curves revealed a rank order of potency of (±)-epibatidine>>anatoxin-a>A-85380>DMPP=NIC=(−)-cytisine in the hippocampus, thalamus, and frontal cortex. EC50 values were approximately 0.005, 0.2, 1, 5, 5 and 5 μM, respectively. Concentration–inhibition curves of nicotine evoked [3H]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine>hexamethonium>d-tubocurare>DHβE. Schild analysis revealed apparent noncompetitive antagonism of [3H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHβE. In contrast, DHβE antagonism of [3H]-dopamine release from striatal slices using a similar methodology was competitive.

Introduction

The modulation of neurotransmitter release by presynaptic nicotinic acetylcholine receptors (nAChRs) has long been the subject of studies in both the peripheral (Boehm and Huck, 1997) and the central nervous systems (McGehee and Role, 1995, Wonnacott, 1997, Vizi, 1999). Activation of nAChRs has been demonstrated to be involved in the release of norepinephrine, dopamine, serotonin, and acetylcholine as well as glutamate and GABA (MacDermott et al., 1999). In the early 1970s, nicotine was shown to evoke release of [3H]-norepinephrine ([3H]-NE) from brain slices (Hall and Turner, 1972, Westfall, 1974). Since that time, additional studies have further characterized the involvement of nAChRs in the modulation of neurotransmitter release from brain slices (Arqueros et al., 1978, Sacaan et al., 1995, Sershen et al., 1997) and synaptosomes (Rapier et al., 1990, Clarke and Reuben, 1996). These studies have primarily concentrated on nicotine-evoked [3H]-NE release from the hippocampus since this region yields the most robust response. More recently, microdialysis studies have expanded the scope of NE release investigations (Fu et al., 1998, Fu et al., 1999).

The present study introduces a new methodology for the study of neurotransmitter release from tissue slices. In place of the laborious commonly used superfusion method, a 96-well plate format has been developed. This new method to study neurotransmitter release utilizes a support membrane, which allows for vacuum filtration or centrifugation of eluate containing released neurotransmitter. The release of [3H]-NE evoked by nicotinic compounds from several brain regions has been evaluated using this new methodology. Results are compared with previous results using superfusion methodology. Other brain regions not previously studied, as well as areas of the spinal cord, have also been surveyed for release of [3H]-NE in response to nicotinic agonists. Furthermore, the present study utilized selected brain regions to characterize the pharmacology of responses to six nicotinic agonists and to four nicotinic antagonists.

Section snippets

Materials

l-[ring-2,5,6-3H]-Norepinephrine ([3H]-NE, 50–55 Ci/mmol) and 3,4-[7-3H]-dihydroxyphenylethylamine ([3H]-DA, 27.5 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). (−)-Nicotine bitartrate (NIC), 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), (+)-anatoxin-a (AnTX), (−)-cytisine (CYT), mecamylamine (MEC), hexamethonium bromide (HEX), d-tubocurare (d-TC), desipramine, pargyline, nisoxetine and ascorbic acid were purchased from Sigma (St Louis, MO). A-85380,

Results

In a survey of rat brain regions and the spinal cord, 100 μM (−)-nicotine and 100 μM DMPP elicited a measurable release of [3H]-NE in all areas that were examined (Table 1). The greatest responses to both NIC and DMPP occurred in the hippocampus. As shown in Table 1, the rank order of responses in the various brain regions, as well as the responses relative to hippocampus, were different for NIC and DMPP. A much wider range of responses was observed with NIC than with DMPP. In spinal cord

Discussion

A new methodology was developed to study the release of neurotransmitters from tissue slices. By employing a 96-well filtration plate equipped with a fine pore membrane, effluent from basal and stimulated conditions could be rapidly collected and assayed. The throughput is much greater with this new method than with conventional superfusion techniques. A typical superfusion apparatus has 12–20 chambers, which severely limits the number of controls and replicates. Also, fraction collection in

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