Hi Orla,

Try using this buffer 

10 mM HEPES, 140 mM NaCl, 3 mM KCl, 20 mM glucose, 2 mM CaCl2, 1 mM MgCl2

pH  7.4.Also do you keep your buffer carbonated (5% Co2) and at 37* during your imaging since 24 hours of imaging seems longer. Alternatively you may try using phenol free neurobasal https://www.thermofisher.com/order/catalog/product/12348017

Thanks Karthik,

The buffer we use is not carbonated but maintained at 37* during imaging. We use this buffer routinely in these type of experiments (24 h) in younger cortical neurons with no problems.

Thank you for the details of your buffer - does this one require carbonation or is the HEPES sufficient to mantain the pH? I feel these hippocampal neurons are more actively firing than what I see in younger cortical neurons, so maybe the addition of Mg will help. I am afraid whether Mg will prevent any NMDA mediated effects upon drug treatment though?

We keep our neurons in a Co2 atmosphere for overnight imaging experiments.

what kind of drug are you treating? Also to reduce the network activity of these mature neurons you may want to use tetradotoxin (TTX).

The protein we are going to use has been suggested to alter synaptic activity, thus we are looking at potential changes in intracellular calcium in response to its addition. Yes I was also thinking of TTX to quieten down synaptic transmission, but again worried whether it would hide potential effects of the protein. 

The solution suggested by Karthik works well. If you wish to perform the imaging for 24 hours or so then it is better to bubble the solution and also maintain proper level of humidity;  If you are using Leica microscopes then they have a special chamber to establish required humidity level. In my experience Hippocampal Neurons appears more robust than the cortical neurons and so you should be able to perform the experiments  easily!