Presence of alpha7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective alpha-neurotoxins in histochemistry.
In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.
Shelukhina
Irina V
IV
Laboratory of Neuropeptide Receptors, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia. ivshelukhina@mail.ru
Kryukova
Elena V
EV
Lips
Katrin Susanne
KS
Tsetlin
Victor I
VI
Kummer
Wolfgang
W
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism / Q000633:toxicity; D003039:Cobra Neurotoxin Proteins / Q000633:toxicity; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D005838:Genotype; D006651:Histocytochemistry; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000294:innervation / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000633:toxicity*; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579166:Chrna7 protein, mouse; D003039:Cobra Neurotoxin Proteins; D007457:Iodine Radioisotopes; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor