Simultaneous release of glutamate and acetylcholine from single magnocellular "cholinergic" basal forebrain neurons

Basal forebrain (BF) neurons provide the principal cholinergic drive to the hippocampus and cortex. Their degeneration is associated with the cognitive defects of Alzheimer's disease. Immunohistochemical studies suggest that some of these neurons contain glutamate, so might also release it. To test this, we made microisland cultures of single BF neurons from 12- to 14-d-old rats. Over 1-8 weeks in culture, neuronal processes made autaptic connections onto the neuron. In 34 of 36 cells tested, a somatically generated action potential was followed by a short-latency EPSC that was blocked by 1 mM kynurenic acid, showing that they released glutamate. To test whether the same neuron also released acetylcholine, we placed a voltage-clamped rat myoball expressing nicotinic receptors in contact with a neurite. In six of six neurons tested, the glutamatergic EPSC was accompanied by a nicotinic (hexamethonium-sensitive) myoball current. Stimulation of the M-2-muscarinic presynaptic receptors ( characterized using tripitramine and pirenzepine) produced a parallel inhibition of autaptic glutamatergic and myoball nicotinic responses; metabotropic glutamate receptor stimulation produced similar but less consistent and weaker effects. Atropine enhanced the glutamatergic EPSCs during repetitive stimulation by 25 +/- 6%; the anti-cholinesterase neostigmine reduced the train EPSCs by 37 +/- 6%. Hence, synaptically released acetylcholine exerted a negative-feedback inhibition of coreleased glutamate. We conclude that most cholinergic basal forebrain neurons are capable of releasing glutamate as a cotransmitter and that the release of both transmitters is subject to simultaneous feedback inhibition by synaptically released acetylcholine. This has implications for BF neuron function and for the use of cholinesterase inhibitors in Alzheimer's disease

The oxidation of cysteine by aqueous ferricyanide: A kinetic study using boron doped diamond electrode voltammetry

The oxidation of cysteine by ferricyanide is well established. However, electrochemical studies of the mechanism have to date been underdeveloped due to the lack of resolution between the oxidation waves of the ferrocyanide and cysteine on most electrode materials. It is shown that on boron doped diamond electrode, unlike for example platinum electrodes, the voltammetric responses of the ferrocyanide wave and the targets are sufficiently different, with the former at a lower oxidizing potential, to permit examination of the kinetics of the homogeneous oxidation via voltammetric methods. Accordingly conventional cyclic voltammetry using a BDD electrode is used to show that an EC′ mechanism occurs and rate constants deduced. Both the protonated and de-protonated forms of cysteine (pKa = 10.5) are shown to undergo oxidation by ferricyanide. The cyclic voltammetry data obtained is corroborated by use of channel flow cell experiments with excellent agreement shown between the two techniques for deduced values of the homogeneous electrocatalytic rate constants: Channel electrodes are seen to provide a convenient means to allow quantitative mechanistic hydrodynamic voltammetry at BDD electrods