Materials & Equipment Checklist
6 items2 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment3
Materials2
Software1
Not specified • Not specified
2 items available from ConductScience
Estimated: $5,946.00
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Protocol Steps
1
Contextual Fear Conditioning
Animals undergo contextual fear conditioning where they learn to associate a context with a fearful stimulus
Not specifiedNot specified
Note: This is the learning phase where engram cells are activated and tagged
View evidence from paper
“learning activates a small ensemble of brain cells, inducing in these cells persistent physical/chemical changes”
2
Engram Cell Tagging
Learning-induced tagging strategy is employed to express channelrhodopsin in the small population of cells activated during contextual fear conditioning
Not specifiedNot specified
Note: Tagging occurs during or immediately after learning to mark engram cells
View evidence from paper
“a learning-induced tagging strategy was employed to express channelrhodopsin in a small population of learning-activated cells”
3
Protein Synthesis Inhibitor Treatment (Optional - for amnesic condition)
Systemic injection of protein synthesis inhibitors within the consolidation window to block late-phase LTP and create amnesic conditions in some animals
Within consolidation window (few hours after learning)Not specified
Note: This treatment creates retrograde amnesia while preserving engram cell connectivity
View evidence from paper
“These changes were blocked by the systemic injection of PSI specifically within the consolidation window”
4
Memory Retrieval Testing
Presentation of retrieval cues to assess memory recall and verify reactivation of tagged engram cells
Not specifiedNot specified
Note: Reactivation of tagged cells upon presentation of retrieval cues is verified
View evidence from paper
“reactivation of tagged cells upon presentation of retrieval cues”
5
Optogenetic Reactivation of Engram Cells
Light-induced artificial reactivation of channelrhodopsin-expressing engram cells to elicit memory recall
Not specifiedNot specified
Note: Can be performed in vivo or ex vivo; demonstrates causal link between engram cell reactivation and memory retrieval
View evidence from paper
“The opsin allowed for the artificial light-induced reactivation of the cellular population labeled during learning and resulted in memory retrieval”
6
Ex Vivo Patch Clamp Recordings
Whole-cell patch clamp recordings from engram cells and non-engram cells to measure synaptic properties including current amplitude, AMPA receptor insertion, spontaneous excitatory post-synaptic currents, and dendritic spine density
Not specifiedNot specified
Note: Engram cells are compared to non-tagged cells to identify synaptic changes associated with memory storage
View evidence from paper
“engram cells were compared to non engram cells (non-tagged cells) by ex vivo patch clamp recordings after contextual fear conditioning”
7
Analysis of Engram Cell Connectivity
Ex vivo examination of preferential connections between engram cells in different brain regions (e.g., dentate gyrus to CA3)
Not specifiedNot specified
Note: Connectivity patterns are examined in both control and PSI-treated amnesic animals
View evidence from paper
“engram cells from the dentate gyrus established preferential connections with engram cells in the downstream hippocampal CA3 region”
8
In Vivo Optogenetic Stimulation and Recording
Optogenetic stimulation of dentate gyrus engram cells in vivo to elicit cellular reactivation patterns in downstream regions (CA3 and amygdala)
Not specifiedNot specified
Note: Performed in both control and amnesic groups to confirm persistence of engram cell connectivity
View evidence from paper
“optogenetic stimulation of DG engram cells in vivo elicited similar cellular reactivation patterns not only in the CA3 region but also in the amygdala”
Subjects / Specimens
- Species
- mouse
- Strain
- Not specified in provided text
- Age
- Not specified in provided text
- Sex
- unknown
- Weight
- Not specified in provided text
Some mice were treated with protein synthesis inhibitors (PSI) within the consolidation window to create amnesic conditions