Use of TH-EGFP transgenic mice as a source of identified dopaminergic neurons for physiological studies in postnatal cell culture.
The physiological and pharmacological properties of dopaminergic neurons in the brain are of major interest. Although much has been learned from cell culture studies, the physiological properties of these neurons remain difficult to study in such models because they are usually in minority and are difficult to distinguish from other non-dopaminergic neurons. Here we have taken advantage of a recently engineered transgenic mouse model expressing enhanced green fluorescence protein (EGFP) under the control of the tyrosine hydroxylase promoter to establish a more effective dopaminergic neuron cell culture model. We first evaluated the specificity of the EGFP expression. Although ectopic expression of EGFP was found in cultures derived from postnatal day 0 pups, this decreased over time in culture such that after 2 weeks, approximately 70% of EGFP-expressing neurons were dopaminergic. We next sought to validate this dopaminergic neuron culture model. We evaluated whether EGFP-expressing dopaminergic neurons displayed some of the well-established properties of dopaminergic neurons. Autoreceptor stimulation inhibited the activity of dopaminergic neurons while neurotensin receptor activation produced the opposite effect. Confocal imaging of the synaptic vesicle optical tracer FM4-64 in EGFP-expressing dopaminergic neurons demonstrated the feasibility of high resolution monitoring of the activity of single terminals established by these neurons. Together, this work provides evidence that primary cultures of postnatal TH-EGFP mice currently represent an excellent model to study the properties of these cells in culture.
Jomphe
C
C
Department of Pharmacology, Faculty of Medicine, Centre de Recherche en Sciences Neurologiques, Université de Montréal, P.O. Box 6128, Succursale Centre-Ville, Montréal, Que., Canada H3C 3J7.
Bourque
M-J
MJ
Fortin
G D
GD
St-Gelais
F
F
Okano
H
H
Kobayashi
K
K
Trudeau
L-E
LE
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D017660:Autoreceptors / Q000187:drug effects / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D018491:Dopamine Agonists / Q000494:pharmacology; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009496:Neurotensin / Q000378:metabolism / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D017448:Receptors, Dopamine D2 / Q000187:drug effects / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000187:drug effects / Q000378:metabolism; D013378:Substantia Nigra / Q000187:drug effects / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*; D017557:Ventral Tegmental Area / Q000187:drug effects / Q000378:metabolism
D017660:Autoreceptors; D018491:Dopamine Agonists; C092350:FM 4-64; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D017448:Receptors, Dopamine D2; D018028:Receptors, Neurotensin; D049452:Green Fluorescent Proteins; D009496:Neurotensin; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine