cLTP and cLTD protocols

cLTP and cLTD protocols (adapted from protocol provided by Michael Ehlers' lab)


Buffers:

Imaging buffer: NaCl (150mM), KCl (5mM), CaCl₂ (2mM), MgCl₂ (2mM), HEPES (10mM), D-Glucose (30mM), pH=7.34-7.36



Mg²⁺-free buffer: Imaging buffer above, but omit MgCl₂, and add 0.02mM bicuculline
 and 0.001mM strychnine. Bicuculline and strychnine 1000x stocks can be stored at -20 ^oC. pH=7.34-7.36



Mg²⁺ buffer: Mg²⁺-free buffer supplemented with 2mM MgCl_2.

Glycine buffer: Mg²⁺-free buffer plus 0.2mM glycine. Make fresh each time (glycine should be made fresh from powder).

cLTP:

1. Wash the neurons 1x with imaging buffer to get rid of the culture medium. Then 2x with Mg²⁺-free buffer.


2. Change Mg²⁺-free buffer to glycine buffer and incubate the neurons at 37^oC for 15 minutes.



3. Wash the neurons 1x with imaging buffer then incubate the cells in Mg²⁺ buffer (2mM MgCl2) for 30 minutes (37^oC). 

cLTD:

NMDA buffer: Mg²⁺-free buffer plus 20µM NMDA (NMDA stock can be stored at -20 ^oC).



1. Wash neurons 3x with Mg²⁺-free buffer.

2. Incubate neurons in NMDA buffer at 37^oC for 5 minutes.

3. Wash the neurons 1x with imaging buffer then incubate the cells in Mg²⁺ buffer (2mM MgCl2) for 1h (37^oC).

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