Susan Wonnacott

Presynaptic nicotinic ACh receptors

Nicotinic ACh (nACh) receptors in the CNS are composed of a diverse array of subunits and have a range of pharmacological properties. However, despite the fact that they are ligand-gated cation channels, their physiological functions have not been determined. This has led to increased interest in presynaptic nACh receptors that act to modulate the release of transmitter from presynaptic terminals.

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

Characterization of Nicotinic Receptor-Mediated [3H]Dopamine Release from Synaptosomes Prepared from Mouse Striatum

Abstract: This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose‐dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca2+‐independent, mecamylamine‐insensitive release was also observed. The Ca2+‐dependent nicotine‐evoked release was not blocked by α‐bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.

The paradox of nicotinic acetylcholine receptor upregulation by nicotine

Abstract Chronic exposure to agonist (or conditions that increase the synaptic concentration of the natural transmitter, such as blockade of inactivation mechanisms) results in a downregulation of the target receptor. Conversely, chronic exposure to antagonist (or conditions that decrease the synaptic concentration of transmitter, including denervation) produces an upregulation of receptors.

Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdialysis: comparison of naive and chronic nicotine-treated rats.

Abstract: The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose‐dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine‐evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine‐sensitive, nicotine‐evoked dopamine release was similar in all three brain regions. However, 10−5M tetrodotoxin totally blocked nicotine‐stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg−1 for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg−1 day−1 increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.

Characterisation of the binding of [3H]methyllycaconitine:a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for alpha bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [3H]MLA bound to rat brain membranes with high affinity (Kd = 1.86 +/- 0.31 nM) with a good ratio of specific to non-specific binding. The binding of [3H]MLA was characterised by rapid association (t 1/2 = 2.3 min) and dissociation (t 1/2 = 12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with alpha bungarotoxin-sensitive nAChR. The snake alpha-toxins, alpha bungarotoxin and alpha cobratoxin, displaced [3H]MLA with high affinity (Ki = 1.8 +/- 0.5 and 5.5 +/- 0.9 nM, respectively), whereas nicotine was less potent (Ki = 6.1 +/- 1.1 microM). The distribution of [3H]MLA binding sites in crudely dissected rat brain regions was identical to that of [125I] alpha bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [3H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake alpha toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [3H]MLA, therefore, is a novel radiolabel for characterising alpha 7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled alpha bungarotoxin for rapid and accurate equilibrium binding assays.

Stereoselective Nicotine‐Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Abstract: Using a sensitive perfusion system we have studied the nicotine‐induced release of [3H]dopamine ([3H]DA) from striatal synaptosomes. Nicotine‐evoked release was concentration dependent with an EC50 of 3.8 μM. The response to 1 μM nicotine was comparable to that to 16 mM K+ 10 μM veratridine evoked a larger response. All three samuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 μM (–)‐nicotine (100 μl) at 30‐min intervals resulted in similar levels of [3H]DA release; higher concentrations of (–)‐nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100‐fold higher concentration of (+)‐nicotine was necessary to evoke the same level of response as 1 μM (–)‐nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high‐affinity nicotine binding sites described in mammalian brain.

Precise Localization of α7 Nicotinic Acetylcholine Receptors on Glutamatergic Axon Terminals in the Rat Ventral Tegmental Area

α7 neuronal nicotinic acetylcholine receptors (nAChRs) constitute one of the predominant nAChR subtypes in the mammalian brain. Within the ventral tegmental area (VTA), nicotine application, paired with postsynaptic stimulation, contributes to a form of long-term potentiation, an effect attributed to presynaptic α7 nAChRs on glutamatergic afferents (Mansvelder and McGehee, 2000). The aim of this study was to examine the precise subcellular distribution of α7 nAChRs in the adult rat VTA to establish whether these receptors are indeed present on glutamatergic axon terminals and to determine their relationship with cholinergic afferents. The spatial relationship between α7 nAChRs, labeled using the α7 nAChR-specific antagonist α-bungarotoxin, and the local neurochemical environment was investigated by the application of multiple labeling strategies with antibodies against tyrosine hydroxylase, vesicular glutamate transporters (VGluTs), vesicular acetylcholine transporter, and glial fibrillary acidic protein. α7 nAChRs were localized at both somatodendritic and presynaptic loci within the VTA: on subpopulations of dopaminergic and nondopaminergic neurons and glutamatergic and nonglutamatergic terminals. There was no detectable α7 nAChR expression within astrocytes in the VTA. Most α7 nAChRs were cytoplasmic (82%), and the remainder were associated with the plasma membrane. Most presynaptic receptors (75%) were on glutamatergic axon terminals, with similar levels of α-bungarotoxin binding present on both VGluT1- and VGluT2-immunoreactive boutons. Both preembedding and postembedding electron microscopy revealed that presynaptic α7 nAChRs are often located at extrasynaptic (27%) and perisynaptic (61%) loci. α7 nAChRs were not associated with cholinergic synapses, consistent with their activation by a paracrine mode of acetylcholine or choline delivery.

Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

The ability of methyllycaconitine (MLA) to inhibit the binding of [125I]α‐bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (K i 1.4 × 10−9 M) than for the muscle receptors (K i; 10−5‐10−6 M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their α‐bungarotoxinbinding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.

6-hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3-dependent activation of protein kinase Cδ

6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase Cδ (PKCδ) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKCδ (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKCδ (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.