Source Paper
Mu-ming Poo, Michele Pignatelli, Tomás J. Ryan, Susumu Tonegawa, Tobias Bonhoeffer et al.
BMC Biology • 2016
The mechanism of memory remains one of the great unsolved problems of biology. Grappling with the question more than a hundred years ago, the German zoologist Richard Semon formulated the concept of the engram, lasting connections in the brain that result from simultaneous "excitations", whose precise physical nature and consequences were out of reach of the biology of his day. Neuroscientists now have the knowledge and tools to tackle this question, however, and this Forum brings together leading contemporary views on the mechanisms of memory and what the engram means today.
Objective: To investigate engram cell connectivity and memory storage mechanisms by tagging neurons activated during contextual fear conditioning and examining their synaptic properties and connectivity patterns
This is a Contextual Fear Conditioning protocol using mouse as the model organism. The procedure involves 8 procedural steps, 3 equipment items, 2 materials. Extracted from a 2016 paper published in BMC Biology.
Model and subjects
mouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Study window
Estimated timing pending
Core workflow
Contextual Fear Conditioning • Engram Cell Tagging • Protein Synthesis Inhibitor Treatment (Optional - for amnesic condition)
Primary readouts
Key equipment and reagents
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Animals undergo contextual fear conditioning where they learn to associate a context with a fearful stimulus
Note: This is the learning phase where engram cells are activated and tagged
“learning activates a small ensemble of brain cells, inducing in these cells persistent physical/chemical changes”
Learning-induced tagging strategy is employed to express channelrhodopsin in the small population of cells activated during contextual fear conditioning
Note: Tagging occurs during or immediately after learning to mark engram cells
“a learning-induced tagging strategy was employed to express channelrhodopsin in a small population of learning-activated cells”
Systemic injection of protein synthesis inhibitors within the consolidation window to block late-phase LTP and create amnesic conditions in some animals
Note: This treatment creates retrograde amnesia while preserving engram cell connectivity
“These changes were blocked by the systemic injection of PSI specifically within the consolidation window”
Presentation of retrieval cues to assess memory recall and verify reactivation of tagged engram cells
Note: Reactivation of tagged cells upon presentation of retrieval cues is verified
“reactivation of tagged cells upon presentation of retrieval cues”
Light-induced artificial reactivation of channelrhodopsin-expressing engram cells to elicit memory recall
Note: Can be performed in vivo or ex vivo; demonstrates causal link between engram cell reactivation and memory retrieval
“The opsin allowed for the artificial light-induced reactivation of the cellular population labeled during learning and resulted in memory retrieval”
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
Note: Engram cells are compared to non-tagged cells to identify synaptic changes associated with memory storage
“engram cells were compared to non engram cells (non-tagged cells) by ex vivo patch clamp recordings after contextual fear conditioning”
Ex vivo examination of preferential connections between engram cells in different brain regions (e.g., dentate gyrus to CA3)
Note: Connectivity patterns are examined in both control and PSI-treated amnesic animals
“engram cells from the dentate gyrus established preferential connections with engram cells in the downstream hippocampal CA3 region”
Optogenetic stimulation of dentate gyrus engram cells in vivo to elicit cellular reactivation patterns in downstream regions (CA3 and amygdala)
Note: Performed in both control and amnesic groups to confirm persistence of engram cell connectivity
“optogenetic stimulation of DG engram cells in vivo elicited similar cellular reactivation patterns not only in the CA3 region but also in the amygdala”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To investigate engram cell connectivity and memory storage mechanisms by tagging neurons activated during contextual fear conditioning and examining their synaptic properties and connectivity patterns
Objective
To investigate engram cell connectivity and memory storage mechanisms by tagging neurons activated during contextual fear conditioning and examining their synaptic properties and connectivity patterns
Subjects
From papermouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Cohort notes
From paperSome mice were treated with protein synthesis inhibitors (PSI) within the consolidation window to create amnesic conditions
Contextual Fear Conditioning (Not specified)
Engram Cell Tagging (Not specified)
Protein Synthesis Inhibitor Treatment (Optional - for amnesic condition) (Within consolidation window (few hours after learning))
Memory Retrieval Testing (Not specified)
Memory recall behavior upon retrieval cue presentation
From paperComparison of synaptic properties between engram cells and non-engram cells; analysis of connectivity patterns in control versus PSI-treated amnesic animals; examination of cellular reactivation patterns in response to optogenetic stimulation
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Memory recall upon optogenetic stimulation of engram cells
From paperComparison of synaptic properties between engram cells and non-engram cells; analysis of connectivity patterns in control versus PSI-treated amnesic animals; examination of cellular reactivation patterns in response to optogenetic stimulation
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Synaptic current amplitude in engram cells
From paperComparison of synaptic properties between engram cells and non-engram cells; analysis of connectivity patterns in control versus PSI-treated amnesic animals; examination of cellular reactivation patterns in response to optogenetic stimulation
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
AMPA receptor insertion and conductance
From paperComparison of synaptic properties between engram cells and non-engram cells; analysis of connectivity patterns in control versus PSI-treated amnesic animals; examination of cellular reactivation patterns in response to optogenetic stimulation
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Memory recall behavior upon retrieval cue presentation
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Memory recall upon optogenetic stimulation of engram cells
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Synaptic current amplitude in engram cells
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
AMPA receptor insertion and conductance
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Comparison of synaptic properties between engram cells and non-engram cells; analysis of connectivity patterns in control versus PSI-treated amnesic animals; examination of cellular reactivation patterns in response to optogenetic stimulation
Scoring or quantification
Quantify the primary readouts for this experiment: Memory recall behavior upon retrieval cue presentation; Memory recall upon optogenetic stimulation of engram cells; Synaptic current amplitude in engram cells; AMPA receptor insertion and conductance.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Memory recall behavior upon retrieval cue presentation, Memory recall upon optogenetic stimulation of engram cells, Synaptic current amplitude in engram cells, AMPA receptor insertion and conductance.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Mu-ming Poo et al. (2016). What is memory? The present state of the engram. BMC Biology
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