Jeremy Findlay

ω-Agatoxin IVA identifies a single calcium channel subtype which contributes to the potassium-induced release of acetylcholine, 5-hydroxytryptamine, dopamine, γ-aminobutyric acid and glutamate from rat brain slices

The voltage-dependent calcium channels (VDCCs) involved in K+-induced transmitter release have been studied. A maximally effective concentration of the N-type VDCC inhibitor, ω-conotoxin GVIA (GVIA) blocked the release of 5-HT (30%), DA (30%) and ACh (60%) but not that of GABA or glutamate. The O, P and Q-type VDCC inhibitor, ω-agatoxin IVA (Aga IVA, 1 μM), blocked 100% of GABA and glutamate, 70% of DA and about 50% of 5-HT and ACh release. The slopes of the inhibition curves indicate that it acts on the same, single type of VDCC in all cases. ω-Conotoxin MVIIC (MVIIC) completely inhibited the release of all the transmitters. It is concluded that a single GVIA-insensitive type of VDCC is involved in the K+-induced release of all the transmitters and, in addition, N-type VDCCs, with a higher affinity for GVIA than MVIIC, are required for the release of 5-HT, DA and ACh. The non-N-type VDCC is not the O-type as it is not blocked by low (< 10 nM) concentrations of MVIIC. Further resolution of this VDCC into P or Q-type requires more selective antagonists.

Effect of voltage-sensitive calcium channel antagonists on the release of 5-hydroxytryptamine from rat hippocampus in vivo.

Abstract: The effect of calcium channel antagonists on the release of 5‐hydroxytryptamine from the hippocampus of the chloral hydrate‐anaesthetised rat was studied using the technique of intracerebral microdialysis. As the basal concentration of 5‐hydroxytryptamine was close to the limit of detection of the HPLC method (8 fmol), the 5‐hydroxytryptamine reuptake inhibitor, fluoxetine (10 μM], was included in the perfusion fluid. The l‐type voltage‐sensitive calcium channel antagonists, PN200‐110, diltiazem, and verapamil, all passed through the dialysis membrane, giving a recovery of 20‐30%. The N‐type voltage‐sensitive calcium channel antagonist, ω‐conotoxin, penetrated less readily (12% recovery). The dihydropyridine, PN200‐110, adhered to the probe, resulting in an effective concentration at the membrane 30% of that in the perfusion fluid. The concentration of 5‐hydroxytryptamine in the dialysate samples was reduced by 60% in the absence of calcium. The L channel antagonists had little effect on the release of 5‐hydroxytryptamine, which was inhibited, in a dose‐dependent manner, to a maximum of 40% by w‐conotoxin. It is concluded that, under physiological conditions, the release of 5‐hydroxytryptamine from the rat hippocampus is dependent on the entry of calcium through N‐type voltage‐sensitive calcium channels, although another calcium channel may also be involved.

An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors:

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer’s disease and schizophrenia.

A high-throughput model for investigating neuronal function and synaptic transmission in cultured neuronal networks

Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer’s disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.