This study investigates the synaptic interactions between genetically modified mouse ventral hippocampus slices and nucleus accumbens neurons. By utilizing calcium imaging techniques, the research explores the effects of nicotine on calcium signaling in these neuronal co-cultures.
Take genetically modified co-cultures of mouse ventral hippocampus slices and nucleus accumbens neurons on a coverslip.
The hippocampal neurons project pre-synaptic axon terminals overexpressing nicotinic acetylcholine receptors, forming synapses with the post-synaptic nucleus accumbens neurons.
Neurons are loaded with a calcium indicator dye for intracellular calcium measurements.
Transfer the coverslip to a confocal microscope chamber perfused with buffer containing calcium ions.
Capture fluorescent images of the hippocampal axonal projections as a control.
Perfuse nicotine, which binds to the nicotinic acetylcholine receptors on hippocampal neurons, triggering calcium influx.
The calcium ions bind to the intracellular dye, increasing fluorescence.
Hippocampal axons release excitatory neurotransmitters, initiating synaptic transmission in nucleus accumbens neurons.
Wash with buffer to remove nicotine.
Over time, nicotinic acetylcholine receptor activation causes calcium release from intracellular stores, leading to increased dye fluorescence and indicating sustained calcium signaling along the ventral hippocampal axons.
Transfer the cultures one at a time to the imaging chamber. Collect the baseline fluorophore fluorescent images of axonal projections from the ventral hippocampal micro-slices as pre-nicotine control.
Next, apply one micromolar of nicotine by rapid perfusion at 2 milliliters per minute for one minute, and then, wash the nicotine away with HBS cocktail. Keep capturing the time-lapse images before, during, and after nicotine application every 10 seconds for 30 minutes.
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