Fear conditioning before viral injection

Mice are fear conditioned to an auditory tone before viral vector injection to establish baseline fear memory

Not specifiedNot specified

Note: This is done before (not after) viral injection to test behavioral specificity

View evidence from paper

“we fear conditioned the mice before (and not after) viral injection”

2

Bilateral microinjection of viral vectors into lateral amygdala

HSV-CREB or HSV-LacZ viruses are microinjected bilaterally into the lateral amygdala of mice

Approximately 3 days before taste aversion conditioningNot specified

Note: Approximately 20% of lateral amygdala neurons are transfected with either virus; no significant difference between infection rates (18±3% vs 21±4%)

View evidence from paper

“These viruses were microinjected into lateral amygdala, which is essential for auditory Pavlovian fear-conditioning”

3

Verify cannula placement

Confirm bilateral cannula placement in basolateral complex of amygdala using crystal violet staining

At end of experimentNot specified

Note: Only mice with bilateral placements in basolateral complex included in analysis

View evidence from paper

“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”

4

Taste aversion conditioning (CTA)

Train mice on conditioned taste aversion task 3 days following viral infection

3 days post-viral injectionNot specified

Note: CTA is chosen because it also depends on the amygdala; same mice previously fear conditioned

View evidence from paper

“we trained the same mice on taste aversion conditioning (CTA) 3 days following viral infection”

5

Allatostatin or vehicle infusion before CTA memory retrieval

Infuse allatostatin (10 μM, 0.5 μl) or saline vehicle bilaterally into lateral amygdala approximately 30 minutes before CTA memory testing

30 minutes before retrieval testNot specified

Note: Allatostatin reversibly inactivates HSV-CREB transfected neurons through membrane hyperpolarization

View evidence from paper

“AL (10 μM, 0.5 μl) or vehicle were infused bilaterally into the lateral amygdala ~30 min before the retrieval of auditory fear memory”

6

CTA memory retrieval test

Measure CTA memory by assessing fluid consumption and taste aversion behavior during test session

Not specifiedNot specified

Note: Total levels of fluid consumption in CTA test were same in different treatment groups

View evidence from paper

“Importantly, the total levels of fluid consumption in CTA test were same in different treatment groups”

7

Measure tone fear memory as control

Measure tone fear memory (freezing response) to assess behavioral specificity of HSV-CREB effects

Same session as CTA testingNot specified

Note: AL infusion should impair CTA memory but not affect tone fear memory if effects are behaviorally specific

View evidence from paper

“we found that AL infusion impaired CTA memory but had no effect on tone fear memory”

8

Whole-cell patch clamp recordings from lateral amygdala slices

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

3 days post-viral injectionNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: Recordings performed in both naive and conditioned mice (24 hours post-training)

View evidence from paper

“we prepared acute lateral amygdala slices ~3 days after virus infusion and performed whole-cell recordings”

9

Measure allatostatin effects on membrane potential

Apply allatostatin during whole-cell recordings to measure changes in membrane potential of GFP+ neurons versus GFP- neighboring cells

During recording sessionNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: Allatostatin causes reversible membrane hyperpolarization in GFP+ neurons but not GFP- cells

View evidence from paper

“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”

10

Measure spike current threshold and input resistance

During whole-cell recordings, measure spike current threshold and input resistance in response to allatostatin application

During recording sessionNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: AL increases spike current threshold and decreases input resistance in GFP+ neurons only

View evidence from paper

“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”

11

Measure action potential threshold

Measure action potential threshold in HSV-CREB transfected neurons versus control neurons using whole-cell recordings

During recording sessionNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: HSV-CREB neurons show significantly lower AP threshold compared to control groups

View evidence from paper

“it did significantly lower the AP threshold of those neurons (HSV-CREB, −38.8 ± 0.9 mV)”

12

Measure action potential firing in response to current injection

Apply depolarizing current injections and measure number of action potentials elicited in HSV-CREB neurons versus non-transfected and HSV-LacZ control neurons

During recording sessionNot specified

Note: HSV-CREB neurons (n=58 from 8 mice) show higher number of action potentials than control neurons

View evidence from paper

“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”

13

Analyze spike frequency adaptation

Analyze distribution of neuronal firing properties in response to 400 pA, 600 ms current injection to measure spike frequency adaptation

During recording sessionNot specified

Note: 62% of HSV-CREB neurons fired more than 6 spikes versus only 19% of control neurons

View evidence from paper

“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”

14

Measure afterhyperpolarization (AHP)

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

During recording sessionNot specified

Note: HSV-CREB neurons show significant reduction in AHP amplitude at 300 ms but not at peak amplitude

View evidence from paper

“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”

15

Measure synaptic transmission in thalamo-LA pathway

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)

24 hours post-trainingNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: Fear conditioning potentiates synaptic transmission; HSV-CREB neurons in conditioned mice show enhanced synaptic transmission

View evidence from paper

“fear conditioning potentiates synaptic transmission and decreases PPF; HSV-CREB neurons in conditioned mice have both enhanced synaptic transmission and reduced PPF”

16

Measure paired pulse facilitation (PPF)

Measure paired pulse facilitation in thalamo-LA synapses of HSV-CREB neurons versus control neurons in naive and conditioned mice

24 hours post-trainingNot specified

Note: Fear conditioning decreases PPF; HSV-CREB neurons in conditioned mice show reduced PPF compared to all control groups

View evidence from paper

“fear conditioning potentiates synaptic transmission and decreases PPF; HSV-CREB neurons in conditioned mice have both enhanced synaptic transmission and reduced PPF”

CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala

Conditioned Taste Aversion

Equipment4

Materials6

Software1

Protocol Steps

Fear conditioning before viral injection

Bilateral microinjection of viral vectors into lateral amygdala

Verify cannula placement

Taste aversion conditioning (CTA)

Allatostatin or vehicle infusion before CTA memory retrieval

CTA memory retrieval test

Measure tone fear memory as control

Whole-cell patch clamp recordings from lateral amygdala slices

Measure allatostatin effects on membrane potential

Measure spike current threshold and input resistance

Measure action potential threshold

Measure action potential firing in response to current injection

Analyze spike frequency adaptation

Measure afterhyperpolarization (AHP)

Measure synaptic transmission in thalamo-LA pathway

Measure paired pulse facilitation (PPF)