Viral vector preparation and microinjection

Prepare HSV-CREB or HSV-LacZ viral vectors. Microinject viruses bilaterally into lateral amygdala of mice approximately 3 days before tone fear conditioning.

~3 days before conditioningNot specified

Note: Approximately 20% of lateral amygdala neurons were transfected with either virus. No significant difference in infection rates between HSV-CREB (18±3%) and HSV-LacZ (21±4%, n=4 mice per group, P>0.05)

View evidence from paper

“These viruses were microinjected into lateral amygdala, which is essential for auditory Pavlovian fear-conditioning. Histological staining with a GFP antibody revealed that ~20% of lateral amygdala neurons were transfected with either virus”

2

Cannula placement verification

Confirm bilateral cannula placements in basolateral complex of amygdala using crystal violet staining. Only include mice with correct bilateral placements in analysis.

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”

3

Verify CREB expression increase

Perform western blot analysis to confirm that HSV-CREB viral transfection increased CREB levels in amygdala and that HSV-LacZ had no detectable effect on CREB expression.

Not specifiedNot specified

Note: HSV-CREB transfection increased CREB levels; HSV-LacZ had no detectable effect

View evidence from paper

“western blot analyses showed that HSV-CREB viral transfection increased CREB levels in the amygdala; Transfection with HSV-LacZ had no detectable effect on CREB expression”

4

Verify selective neuronal silencing by allatostatin

Perform whole-cell patch clamp recordings in visually identified GFP+ neurons and GFP− neighboring cells from HSV-CREB or HSV-LacZ mice. Administer allatostatin (AL) and measure membrane potential changes, spike current threshold, and input resistance.

Not specifiedNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: AL administration caused membrane potential to become more negative in GFP+ neurons (-5.9±1.2 mV, t=3.73, P<0.01) but had no effect on GFP− cells (-0.3±0.9 mV). AL increased spike current threshold (600±210% of baseline, t=2.33, P<0.05) and decreased input resistance (51±12% of baseline, t=3.96, P<0.01) in GFP+ neurons only.

View evidence from paper

“AL administration quickly and reversibly caused the membrane potential to become more negative (−5.9 ± 1.2 mV, t = 3.73, P < 0.01) in GFP + neurons, but had no effect on GFP − neighboring cells”

5

Auditory fear conditioning training

Conduct tone fear conditioning approximately 3 days after viral injection. Expose mice to tone-shock pairings during training session.

Not specifiedNot specified

Note: Training occurs ~3 days after viral microinjection

View evidence from paper

“both virus preparations were micro-infused bilaterally into the lateral amygdala ~3 days before tone fear conditioning”

6

Memory retrieval testing - 24 hour timepoint

Conduct memory retrieval test 24 hours after conditioning. Measure percentage of time mice spend freezing during tone presentation. Infuse allatostatin (10 μM, 0.5 μl) or vehicle bilaterally into lateral amygdala approximately 30 minutes before testing.

24 hours post-conditioningNot specified

Note: HSV-CREB mice treated with AL showed less freezing (41.4%±3.7%, t=2.58, P<0.05) than HSV-CREB mice treated with saline (65.3%±8.5%). AL had no effect on HSV-LacZ mice freezing.

View evidence from paper

“Memory for tone conditioning was assessed by measuring the percentage of time the mice spent freezing during a tone presentation conducted 24 h after conditioning. To test the impact of inactivating transfected neurons, AL (10 μM, 0.5 μl) or vehicle were infused bilaterally into the lateral amygdala ~30 min before the retrieval of auditory fear memory”

7

Reversibility control test - 4 hour delay

In separate groups, infuse allatostatin or vehicle into lateral amygdala and wait 4 hours (instead of 30 minutes) before testing tone conditioning to determine duration of AL effects.

4 hours post-infusionNot specified

Note: AL infusion 4 hours before testing had no effect on retrieval, indicating AL effects last at least 30 minutes but no longer than 4 hours

View evidence from paper

“we also infused AL or vehicle into the lateral amygdala of separate groups of mice and waited 4 h (instead of 30 min) before testing tone conditioning. Our results showed that AL infusion 4 h before testing had no effect on retrieval”

8

Reversibility test - second retrieval session

Repeat memory retrieval test 24 hours after first test. Reverse treatments so mice that received AL in first test receive vehicle in second test and vice versa.

24 hours after first testNot specified

Note: HSV-CREB mice treated with AL in test 1 (50.7%±5.7% freezing) showed normal freezing in test 2 (67.1%±5.1%, t=2.14, n=15, P<0.05) when treated with saline. HSV-CREB mice treated with saline in test 1 (71.1%±4.8%) showed memory impairment in test 2 (47.3%±5.8%, t=3.17, n=17, P<0.01) when treated with AL.

View evidence from paper

“we repeated the experiment just described, but this time added a second test 24 h after the first test. In the second test we reversed the treatments so that mice that got AL in the first test got VEH in the second test”

9

AL manipulation during both training and testing

Conduct tone fear conditioning with allatostatin or vehicle infusion during training. Test memory retrieval with allatostatin or vehicle infusion during testing. Compare four conditions: VEH/VEH, AL/VEH, VEH/AL, AL/AL.

Training and testing separated by 24 hoursNot specified

Note: VEH/VEH: 70.0%±5.5% freezing (n=12); AL/VEH: 47.4%±6.4% freezing (n=12); VEH/AL: disrupted freezing; AL/AL: 49.5%±6.6% freezing (n=12), not significantly different from AL/VEH (P>0.05)

View evidence from paper

“we included an AL manipulation during both training and testing. Consistent with CREB's role in memory consolidation, we found that activation of the HSV-CREB neurons during training enhanced tone-associated fear memory (VEH/VEH, 70.0% ± 5.5%, n = 12); while inactivation of the HSV-CREB neurons with AL during training reduced tone-associated fear memory to control levels”

10

Early memory allocation test - 30 minute timepoint

Test memory allocation 30 minutes after training (before memory consolidation occurs). Infuse allatostatin or vehicle immediately after training and test freezing 30 minutes post-training.

30 minutes post-trainingNot specified

Note: HSV-CREB mice did not show higher freezing than HSV-LacZ mice at 30 min post-training. AL administration immediately after training disrupted 30-min memory in HSV-CREB mice (HSV-CREB/VEH: 50.9%±5.5%, HSV-CREB/AL: 28.9%±5.2%, F3,44=6.93, P<0.05, n=12 per group) but not in HSV-LacZ mice (HSV-LacZ/VEH: 48.1%±7.5%, HSV-LacZ/AL: 52.9%±5.7%)

View evidence from paper

“we tested memory allocation 30 min after training, a time point known to precede memory consolidation. Indeed, when tested 30 min post-training, HSV-CREB mice did not show higher freezing than HSV-LacZ mice”

11

Behavioral specificity test - taste aversion conditioning

Fear condition mice before (not after) viral injection. Three days after viral infection, train same mice on conditioned taste aversion (CTA). Test CTA memory with allatostatin or vehicle infusion.

3 days post-viral injectionNot specified

Note: AL impaired CTA memory (HSV-CREB/VEH: 71.9%±7.7%, HSV-CREB/AL: 50.0%±5.9%, t=2.31, P<0.05) but had no effect on tone fear memory (HSV-CREB/VEH: 50.6%±8.6%, HSV-CREB/AL: 49.9%±9.8%, P>0.05). Total fluid consumption in CTA test was same in different treatment groups.

View evidence from paper

“we fear conditioned the mice before (and not after) viral injection. Then, we trained the same mice on taste aversion conditioning (CTA) 3 days following viral infection”

12

Synaptic transmission and paired pulse facilitation measurements

Perform whole-cell recordings from thalamo-LA synapses in naive and conditioned mice (24 hours post-training). Measure evoked EPSCs and paired pulse facilitation (PPF) in HSV-CREB neurons and non-transfected neighboring cells.

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 (F1,37=7.68, P<0.01) and decreases PPF (F1,82=26.35, P<0.001). HSV-CREB neurons in conditioned mice show enhanced synaptic transmission and reduced PPF compared to three control groups (P<0.001).

View evidence from paper

“we studied synaptic transmission and PPF in HSV-CREB neurons and non-transfected neighboring cells in the same slices in both naive and conditioned mice (24 hrs post-training)”

13

Neuronal excitability measurements

Prepare acute lateral amygdala slices approximately 3 days after virus infusion. Perform whole-cell recordings to measure action potential threshold, number of action potentials elicited by depolarizing current injections, spike frequency adaptation, and afterhyperpolarization (AHP).

~3 days post-viral injectionNot specified

Blood Vessel Clamps_ SS Micro Clamps

Matches: Whole-cell patch clamp recording setup

$46View

Note: HSV-CREB neurons show: lower AP threshold (-38.8±0.9 mV vs -35.4±1.0 to -35.7±1.0 mV, P<0.05); increased number of action potentials (P<0.05); reduced spike frequency adaptation (62% of HSV-CREB neurons fired >6 spikes vs 19% of control neurons); reduced AHP amplitude at 300 ms after current pulse

View evidence from paper

“we prepared acute lateral amygdala slices ~3 days after virus infusion and performed whole-cell recordings. We found that although HSV-CREB did not affect the resting membrane potential, input resistance, spike amplitude or the spike half-width of transfected lateral amygdala pyramidal neurons, it did significantly lower the AP threshold of those neurons”

14

Input-output function analysis

Measure relationship between EPSP slope and spike probability following different synaptic stimulation intensities in HSV-CREB and control neurons.

Not specifiedNot specified

Note: Determines whether intrinsic excitability changes alter input-output function critical for information processing during learning

View evidence from paper

“We measured the relationship between the EPSP slope and spike probability following different synaptic stimulation intensities”

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

Auditory Fear Conditioning

Equipment7

Materials6

Software1

Protocol Steps

Viral vector preparation and microinjection

Cannula placement verification

Verify CREB expression increase

Verify selective neuronal silencing by allatostatin

Auditory fear conditioning training

Memory retrieval testing - 24 hour timepoint

Reversibility control test - 4 hour delay

Reversibility test - second retrieval session

AL manipulation during both training and testing

Early memory allocation test - 30 minute timepoint

Behavioral specificity test - taste aversion conditioning

Synaptic transmission and paired pulse facilitation measurements

Neuronal excitability measurements

Input-output function analysis