Source Paper
Yu Zhou, Jaejoon Won, Mikael Guzman Karlsson, Miou Zhou, Thomas Rogerson et al.
Nature Neuroscience • 2009
Objective: Test behavioral specificity of HSV-CREB effects on memory allocation by comparing conditioned taste aversion (CTA) memory in mice with HSV-CREB or HSV-LacZ transfections in lateral amygdala, with reversible inactivation of transfected neurons
This is a Conditioned Taste Aversion protocol using mouse as the model organism. The procedure involves 16 procedural steps, 4 equipment items, 6 materials. Extracted from a 2009 paper published in Nature Neuroscience.
Model and subjects
mouse • Not specified • Not specified • Not specified • Not specified • Not specified for CTA experiment
Study window
~3 day study window | ~97 hours hands-on
Core workflow
Fear conditioning before viral injection • Bilateral microinjection of viral vectors into lateral amygdala • Verify cannula placement
Primary readouts
Key equipment and reagents
Verified items
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Mice are fear conditioned to an auditory tone before viral vector injection to establish baseline fear memory
Note: This is done before (not after) viral injection to test behavioral specificity
“we fear conditioned the mice before (and not after) viral injection”
HSV-CREB or HSV-LacZ viruses are microinjected bilaterally into the lateral amygdala of mice
Note: Approximately 20% of lateral amygdala neurons are transfected with either virus; no significant difference between infection rates (18±3% vs 21±4%)
“These viruses were microinjected into lateral amygdala, which is essential for auditory Pavlovian fear-conditioning”
Confirm bilateral cannula placement in basolateral complex of amygdala using crystal violet staining
Note: Only mice with bilateral placements in basolateral complex included in analysis
“Cannula placement was confirmed with crystal violet staining at the end of each experiment. Only those mice with bilateral placements in the basolateral complex of the amygdala were included in analysis”
Train mice on conditioned taste aversion task 3 days following viral infection
Note: CTA is chosen because it also depends on the amygdala; same mice previously fear conditioned
“we trained the same mice on taste aversion conditioning (CTA) 3 days following viral infection”
Infuse allatostatin (10 µM, 0.5 µl) or saline vehicle bilaterally into lateral amygdala approximately 30 minutes before CTA memory testing
Note: Allatostatin reversibly inactivates HSV-CREB transfected neurons through membrane hyperpolarization
“AL (10 µM, 0.5 µl) or vehicle were infused bilaterally into the lateral amygdala ~30 min before the retrieval of auditory fear memory”
Measure CTA memory by assessing fluid consumption and taste aversion behavior during test session
Note: Total levels of fluid consumption in CTA test were same in different treatment groups
“Importantly, the total levels of fluid consumption in CTA test were same in different treatment groups”
Measure tone fear memory (freezing response) to assess behavioral specificity of HSV-CREB effects
Note: AL infusion should impair CTA memory but not affect tone fear memory if effects are behaviorally specific
“we found that AL infusion impaired CTA memory but had no effect on tone fear memory”
Prepare acute lateral amygdala slices approximately 3 days after virus infusion and perform whole-cell recordings from both GFP+ transfected neurons and GFP- neighboring cells
Note: Recordings performed in both naive and conditioned mice (24 hours post-training)
“we prepared acute lateral amygdala slices ~3 days after virus infusion and performed whole-cell recordings”
Apply allatostatin during whole-cell recordings to measure changes in membrane potential of GFP+ neurons versus GFP- neighboring cells
Note: Allatostatin causes reversible membrane hyperpolarization in GFP+ neurons but not GFP- cells
“AL administration quickly and reversibly caused the membrane potential to become more negative (−5.9 ± 1.2 mV) in GFP+ neurons, but had no effect on GFP− neighboring cells”
During whole-cell recordings, measure spike current threshold and input resistance in response to allatostatin application
Note: AL increases spike current threshold and decreases input resistance in GFP+ neurons only
“AL administration quickly and reversibly increased the spike current threshold (600 ± 210% of the baseline) and decreased the input resistance (51 ± 12% of the baseline) of GFP+ lateral amygdala neurons”
Measure action potential threshold in HSV-CREB transfected neurons versus control neurons using whole-cell recordings
Note: HSV-CREB neurons show significantly lower AP threshold compared to control groups
“it did significantly lower the AP threshold of those neurons (HSV-CREB, −38.8 ± 0.9 mV)”
Apply depolarizing current injections and measure number of action potentials elicited in HSV-CREB neurons versus non-transfected and HSV-LacZ control neurons
Note: HSV-CREB neurons (n=58 from 8 mice) show higher number of action potentials than control neurons
“the number of action potentials triggered by depolarizing current injections was higher than in non-transfected neurons or in neurons transfected with the HSV-LacZ control virus”
Analyze distribution of neuronal firing properties in response to 400 pA, 600 ms current injection to measure spike frequency adaptation
Note: 62% of HSV-CREB neurons fired more than 6 spikes versus only 19% of control neurons
“62% (36/58) of HSV-CREB neurons fired more than 6 spikes in response to a 400 pA, 600 ms current injection, while only 19% (12/64) of control neurons did so”
Measure post-burst AHP of HSV-CREB and control neurons at two time points: at negative peak and 300 ms after end of current pulse
Note: HSV-CREB neurons show significant reduction in AHP amplitude at 300 ms but not at peak amplitude
“HSV-CREB neurons have a significant reduction in the amplitude of the AHP measured at 300 ms after the current pulse, but not at peak amplitude”
Perform whole-cell recordings to measure evoked EPSCs in HSV-CREB neurons and control neurons in both naive and conditioned mice (24 hours post-training)
Note: Fear conditioning potentiates synaptic transmission; HSV-CREB neurons in conditioned mice show enhanced synaptic transmission
“fear conditioning potentiates synaptic transmission and decreases PPF; HSV-CREB neurons in conditioned mice have both enhanced synaptic transmission and reduced PPF”
Measure paired pulse facilitation in thalamo-LA synapses of HSV-CREB neurons versus control neurons in naive and conditioned mice
Note: Fear conditioning decreases PPF; HSV-CREB neurons in conditioned mice show reduced PPF compared to all control groups
“fear conditioning potentiates synaptic transmission and decreases PPF; HSV-CREB neurons in conditioned mice have both enhanced synaptic transmission and reduced PPF”
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.
Test behavioral specificity of HSV-CREB effects on memory allocation by comparing conditioned taste aversion (CTA) memory in mice with HSV-CREB or HSV-LacZ transfections in lateral amygdala, with reversible inactivation of transfected neurons
Objective
Test behavioral specificity of HSV-CREB effects on memory allocation by comparing conditioned taste aversion (CTA) memory in mice with HSV-CREB or HSV-LacZ transfections in lateral amygdala, with reversible inactivation of transfected neurons
Subjects
From papermouse • Not specified • Not specified • Not specified • Not specified
Sample count
From paperNot specified for CTA experiment
Cohort notes
From paperMice previously fear conditioned before viral injection, then trained on CTA 3 days following viral infection
Fear conditioning before viral injection (Not specified)
Bilateral microinjection of viral vectors into lateral amygdala (Approximately 3 days before taste aversion conditioning)
Verify cannula placement (At end of experiment)
Taste aversion conditioning (CTA) (3 days post-viral injection)
CTA memory: fluid consumption and taste aversion behavior during retrieval test
From paperTwo-way ANOVA for fear conditioning effects on synaptic transmission and PPF; One-way Repeated Measures ANOVA for comparing HSV-CREB neurons with control groups; Newman-Keuls Multiple Comparison Test for post-hoc comparisons; Fisher's PLSD for pairwise comparisons; t-tests for comparing treatment groups; statistical significance set at P < 0.05
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Tone fear memory: percentage of time spent freezing during tone presentation (control for behavioral specificity)
From paperTwo-way ANOVA for fear conditioning effects on synaptic transmission and PPF; One-way Repeated Measures ANOVA for comparing HSV-CREB neurons with control groups; Newman-Keuls Multiple Comparison Test for post-hoc comparisons; Fisher's PLSD for pairwise comparisons; t-tests for comparing treatment groups; statistical significance set at P < 0.05
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Membrane potential changes in response to allatostatin
From paperTwo-way ANOVA for fear conditioning effects on synaptic transmission and PPF; One-way Repeated Measures ANOVA for comparing HSV-CREB neurons with control groups; Newman-Keuls Multiple Comparison Test for post-hoc comparisons; Fisher's PLSD for pairwise comparisons; t-tests for comparing treatment groups; statistical significance set at P < 0.05
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Spike current threshold and input resistance changes
From paperTwo-way ANOVA for fear conditioning effects on synaptic transmission and PPF; One-way Repeated Measures ANOVA for comparing HSV-CREB neurons with control groups; Newman-Keuls Multiple Comparison Test for post-hoc comparisons; Fisher's PLSD for pairwise comparisons; t-tests for comparing treatment groups; statistical significance set at P < 0.05
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
CTA memory: fluid consumption and taste aversion behavior during retrieval test
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
Tone fear memory: percentage of time spent freezing during tone presentation (control for behavioral specificity)
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
Membrane potential changes in response to allatostatin
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
Spike current threshold and input resistance changes
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
Two-way ANOVA for fear conditioning effects on synaptic transmission and PPF; One-way Repeated Measures ANOVA for comparing HSV-CREB neurons with control groups; Newman-Keuls Multiple Comparison Test for post-hoc comparisons; Fisher's PLSD for pairwise comparisons; t-tests for comparing treatment groups; statistical significance set at P < 0.05
Scoring or quantification
Quantify the primary readouts for this experiment: CTA memory: fluid consumption and taste aversion behavior during retrieval test; Tone fear memory: percentage of time spent freezing during tone presentation (control for behavioral specificity); Membrane potential changes in response to allatostatin; Spike current threshold and input resistance changes.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for CTA memory: fluid consumption and taste aversion behavior during retrieval test, Tone fear memory: percentage of time spent freezing during tone presentation (control for behavioral specificity), Membrane potential changes in response to allatostatin, Spike current threshold and input resistance changes.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Yu Zhou et al. (2009). CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala. Nature Neuroscience
Fear conditioning before viral injection • Protocol step
“we fear conditioned the mice before (and not after) viral injection”
Bilateral microinjection of viral vectors into lateral amygdala • Protocol step
“These viruses were microinjected into lateral amygdala, which is essential for auditory Pavlovian fear-conditioning”
Verify cannula placement • Protocol step
“Cannula placement was confirmed with crystal violet staining at the end of each experiment. Only those mice with bilateral placements in the basolateral complex of the amygdala were included in analysis”
Taste aversion conditioning (CTA) • Protocol step
“we trained the same mice on taste aversion conditioning (CTA) 3 days following viral infection”
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