Single particle tracking of alpha7 nicotinic AChR in hippocampal neurons reveals regulated confinement at glutamatergic and GABAergic perisynaptic sites.
Alpha7 neuronal nicotinic acetylcholine receptors (alpha7-nAChR) form Ca(2+)-permeable homopentameric channels modulating cortical network activity and cognitive processing. They are located pre- and postsynaptically and are highly abundant in hippocampal GABAergic interneurons. It is unclear how alpha7-nAChRs are positioned in specific membrane microdomains, particularly in cultured neurons which are devoid of cholinergic synapses. To address this issue, we monitored by single particle tracking the lateral mobility of individual alpha7-nAChRs labeled with alpha-bungarotoxin linked to quantum dots in live rat cultured hippocampal interneurons. Quantitative analysis revealed different modes of lateral diffusion of alpha7-nAChR dependent on their subcellular localization. Confined receptors were found in the immediate vicinity of glutamatergic and GABAergic postsynaptic densities, as well as in extrasynaptic clusters of alpha-bungarotoxin labeling on dendrites. alpha7-nAChRs avoided entering postsynaptic densities, but exhibited reduced mobility and long dwell times at perisynaptic locations, indicative of regulated confinement. Their diffusion coefficient was lower, on average, at glutamatergic than at GABAergic perisynaptic sites, suggesting differential, synapse-specific tethering mechanisms. Disruption of the cytoskeleton affected alpha7-nAChR mobility and cell surface expression, but not their ability to form clusters. Finally, using tetrodotoxin to silence network activity, as well as exposure to a selective alpha7-nAChR agonist or antagonist, we observed that alpha7-nAChRs cell surface dynamics is modulated by chronic changes in neuronal activity. Altogether, given their high Ca(2+)-permeability, our results suggest a possible role of alpha7-nAChR on interneurons for activating Ca(2+)-dependent signaling in the vicinity of GABAergic and glutamatergic synapses.
Bürli
Thomas
T
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Baer
Kristin
K
Ewers
Helge
H
Sidler
Corinne
C
Fuhrer
Christian
C
Fritschy
Jean-Marc
JM
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002038:Bungarotoxins / Q000737:chemistry; D002478:Cells, Cultured; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology*; D007150:Immunohistochemistry; D008853:Microscopy; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D011247:Pregnancy; D045663:Quantum Dots; D051381:Rats; D017208:Rats, Wistar; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*; D053778:Thiazolidines / Q000494:pharmacology; D064569:alpha7 Nicotinic Acetylcholine Receptor; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; C579165:Chrna7 protein, rat; D011978:Receptors, Nicotinic; D053778:Thiazolidines; D064569:alpha7 Nicotinic Acetylcholine Receptor; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D015739:Nocodazole; C037067:latrunculin A