CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala methods
Aim. Evidence-backed execution summary for CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala methods from CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Surgery and virus infusion
reagent used in the protocol.
- Use
- Mice were pretreated with atropine sulfate (0.1 mg kg -1, i.p.), anesthetized with chloral hydrate (400 mg kg -1, i.p.), and placed in a stereotaxic frame. The skin was retracted and holes drilled in the skull bilaterally above the LA (AP = -1.3, ML = ± 3.3, V = - 4.8 mm from bregma) a...
AL administration
reagent used in the protocol.
- Use
- The peptide allatostatin (AL) (New England) was dissolved in ddH 2 O to make a 2.5 mM stock solution. Saline was added to dilute it to the proper final concentration for each specific experiment. AL solution (0.5 µl) or same amount of vehicle (saline) was delivered bilaterally to the LA of awake animals at a fl...
Histology
reagent used in the protocol.
- Use
- After the behavioral experiments, mice were perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Brains were sliced coronally (40 µm). The cannula tip locations were confirmed by crystal violet staining at the end of each experiment. Only those mice with bilateral placements in the...
Histology
reagent used in the protocol.
- Use
- Quantitative analyses of infection levels were performed using the NIH Image processing system. Before confocal imaging we used transmission light microscopy and the thalamo-lateral amygdala fibers (ic) and the cortico-lateral amygdale fibers (oc) to locate the dorsal and lateral boundary of LA. The total number of...
Electrophysiology
reagent used in the protocol.
- Use
- Two to three days after virus infusion, brains were rapidly removed from adult mice (3 to 4 months old) and placed in ice cold artificial cerebral spinal fluid (ACSF) containing (in mM): 120 NaCl, 20 NaHCO 3, 3.5 KCl, 1.25 NaH 2 PO 4, 2.5 CaCl 2, 1.3 MgSO 4, and 10 d-glucose. Coronal slices (400 µm thick) c...
Electrophysiology
The thalamo-LA pathway was stimulated with a bipolar platinum electrode. The distance between the recording and stimulating sites was between 150 and 450µm. 100us stimuli were delivered at 10 s intervals. For the basal synaptic transmission studies, the input-output curve was constructed by varying stimulus int...
- Use
- The thalamo-LA pathway was stimulated with a bipolar platinum electrode. The distance between the recording and stimulating sites was between 150 and 450µm. 100us stimuli were delivered at 10 s intervals. For the basal synaptic transmission studies, the input-output curve was constructed by varying stimulus int...
Surgery and virus infusion
Mice were pretreated with atropine sulfate (0.1 mg kg -1, i.p.), anesthetized with chloral hydrate (400 mg kg -1, i.p.), and placed in a stereotaxic frame. The skin was retracted and holes drilled in the skull bilaterally above the LA (AP = -1.3, ML = ± 3.3, V = - 4.8 mm from bregma) a...
- Use
- Mice were pretreated with atropine sulfate (0.1 mg kg -1, i.p.), anesthetized with chloral hydrate (400 mg kg -1, i.p.), and placed in a stereotaxic frame. The skin was retracted and holes drilled in the skull bilaterally above the LA (AP = -1.3, ML = ± 3.3, V = - 4.8 mm from bregma) a...
Auditory (tone) fear conditioning
Training consisted of placing the mice in a conditioning chamber (Context A) and 2 min later presenting a tone (2800 Hz, 85 dB, 30 sec) that co-terminated with a shock (2 sec, 0.5 mA). Mice remained in the chamber for an additional 1 min. Test for auditory fear conditioning occurred either 30 min or 24 h later. Mice...
- Use
- Training consisted of placing the mice in a conditioning chamber (Context A) and 2 min later presenting a tone (2800 Hz, 85 dB, 30 sec) that co-terminated with a shock (2 sec, 0.5 mA). Mice remained in the chamber for an additional 1 min. Test for auditory fear conditioning occurred either 30 min or 24 h later. Mice...
Histology
Quantitative analyses of infection levels were performed using the NIH Image processing system. Before confocal imaging we used transmission light microscopy and the thalamo-lateral amygdala fibers (ic) and the cortico-lateral amygdale fibers (oc) to locate the dorsal and lateral boundary of LA. The total number of...
- Use
- Quantitative analyses of infection levels were performed using the NIH Image processing system. Before confocal imaging we used transmission light microscopy and the thalamo-lateral amygdala fibers (ic) and the cortico-lateral amygdale fibers (oc) to locate the dorsal and lateral boundary of LA. The total number of...
Electrophysiology
Two to three days after virus infusion, brains were rapidly removed from adult mice (3 to 4 months old) and placed in ice cold artificial cerebral spinal fluid (ACSF) containing (in mM): 120 NaCl, 20 NaHCO 3, 3.5 KCl, 1.25 NaH 2 PO 4, 2.5 CaCl 2, 1.3 MgSO 4, and 10 d-glucose. Coronal slices (400 µm thick) c...
- Use
- Two to three days after virus infusion, brains were rapidly removed from adult mice (3 to 4 months old) and placed in ice cold artificial cerebral spinal fluid (ACSF) containing (in mM): 120 NaCl, 20 NaHCO 3, 3.5 KCl, 1.25 NaH 2 PO 4, 2.5 CaCl 2, 1.3 MgSO 4, and 10 d-glucose. Coronal slices (400 µm thick) c...
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HSV-CREB neurons show increased neuronal excitability
How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling proteins that increase the excitability of these neurons -, thereby increasing the probability that they are recruited during learning. To test this hypothesis, 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 (, HSV-CREB, -38.8 ± 0.9 mV, one-way ANOVA, P < 0.05 compared to the other three control groups, -35.4 ± 1.0 mV~ -35.7 ± 1.0 mV). In addition, HSV-CREB caused a r...
Surgery and virus infusion
Mice were pretreated with atropine sulfate (0.1 mg kg -1, i.p.), anesthetized with chloral hydrate (400 mg kg -1, i.p.), and placed in a stereotaxic frame. The skin was retracted and holes drilled in the skull bilaterally above the LA (AP = -1.3, ML = ± 3.3, V = - 4.8 mm from bregma) according to Paxinos and The Franklin mouse brain atlas. For behavioral experiments, a stainless steel outer cannula (Plastic One) was implanted to LA and fixed on the skull with dental cement. After cannula implantation, mice were single-housed and handled every day. Virus infusion was delayed until seven days after surgery to ensure physical recovery. A virus solution (1.3 µl; bilateral) was delivered to lateral amygdala at a flow rate of 0.065 µl/min through inner injection cannula (Plastic one, 22 gauge) attached by polyethylene tubing to Hamilton microsyringe...
AL administration
The peptide allatostatin (AL) (New England) was dissolved in ddH 2 O to make a 2.5 mM stock solution. Saline was added to dilute it to the proper final concentration for each specific experiment. AL solution (0.5 µl) or same amount of vehicle (saline) was delivered bilaterally to the LA of awake animals at a flow rate of 0.25 µl/min through inner injection cannula. The cannula was left in place for 2 min post-injection. Behavioral procedures were started ~30 min after injection.
Auditory (tone) fear conditioning
Training consisted of placing the mice in a conditioning chamber (Context A) and 2 min later presenting a tone (2800 Hz, 85 dB, 30 sec) that co-terminated with a shock (2 sec, 0.5 mA). Mice remained in the chamber for an additional 1 min. Test for auditory fear conditioning occurred either 30 min or 24 h later. Mice were placed in a novel chamber (Context B) and 2 min later the tone CS was presented (for 1 min). Our index of memory, freezing (the cessation of all movement except for respiration), was assessed via an automated scoring system (Med Associates Inc.) with a 30 frames/sec sampling; the animals needed to freeze continuously for at least 1 sec before freezing could be counted. The same training protocol was also used in the electrophysiological studies. Slices were cut 24 h after training.
Conditioned taste aversion (CTA)
CTA training took place in the light part of the cycle 7 days following surgery. Mice were water deprived for 24 h and then pretrained for 5 days to get their daily water ration within 40 min per day from two tubes (10 ml). Three hours after the 2 nd day pretraining, HSV virus was infused into LA on both sides. Conditioning day started 3 days after virus injection.
Conditioned taste aversion (CTA)
On the conditioning day, the two tubes were filled with 0.2% saccharin sodium salt (w/v, the taste CS) instead of water. The CS was presented for 20 min and 20 min later, mice were treated with the malaise inducing agent lithium chloride (LiCl; 0.06 M, 2% body weight i.p.).
Conditioned taste aversion (CTA)
Testing for aversion to saccharin occurred 24 h later. AL/vehicle was infused 30min before testing. Two tubes (one contains water and the other contains saccharin) were presented for 40 min. The intake of each fluid was measured and the aversion index (AI) was defined as follows: [milliliters of water consumed/(milliliters of water + milliliters of saccharin consumed)] × 100%. 50% AI is equal preference level, and the higher the AI, the more the mice prefer water to saccharin).
Histology
Quantitative analyses of infection levels were performed using the NIH Image processing system. Before confocal imaging we used transmission light microscopy and the thalamo-lateral amygdala fibers (ic) and the cortico-lateral amygdale fibers (oc) to locate the dorsal and lateral boundary of LA. The total number of GFP-positive cells in the LA was counted bilaterally with a fixed sample window (0.1 mm 2 ) across at least six sections from comparable anteroposterior levels from each mouse. To assess the number of nuclei in the areas counted, the slices were re-stained using 4',6'-diamidino-2-phenylindole (DAPI) after washout of FITC-conjugated GFP primary antibody. The percentage of GFP positive cells was calculated as the percentage of GFP-positive cell/total DAPI-labeled nuclei in the LA. After the immunofluorescent studies, brain slices were counter stained with crystal violet to co...
Measurement outputs
What raw and processed outputs should exist?
Importantly, we found that HSV-CREB neurons in conditioned mice (conditioned HSV-CREB) have both enhanced synaptic transmission and reduced PPF when compared with the other thre...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling prote...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Previous studies showed that both changes in synaptic strength (LTP) and changes in intrinsic excitability could lead to a left shift of the E-S curve -. Our whole-cell r...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Two vectors were used, HSV-CREB-AlstR (HSV-CREB vector) and HSV-LacZ-AlstR (Control vector). Genes of interest ( Creb1, LacZ and AlstR ) were cloned into a bicistronic HSV ampl...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Previous studies in thalamo-LA synapses following tone fear conditioning revealed increases in synaptic transmission and decreases in paired pulse facilitation (PPF),.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Importantly, we found that HSV-CREB neurons in conditioned mice (conditioned HSV-CREB) have both enhanced synaptic transmission and reduced PPF when compared with the other thre...; How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling prote...; Previous studies showed that both changes in synaptic strength (LTP) and changes in intrinsic excitability could lead to a left shift of the E-S curve -. Our whole-cell r...; Two vectors were used, HSV-CREB-AlstR (HSV-CREB vector) and HSV-LacZ-AlstR (Control vector). Genes of interest ( Creb1, LacZ and AlstR ) were cloned into a bicistronic HSV ampl....
from paperStatistical comparison
Previous studies in thalamo-LA synapses following tone fear conditioning revealed increases in synaptic transmission and decreases in paired pulse facilitation (PPF),. To test...; Importantly, we found that HSV-CREB neurons in conditioned mice (conditioned HSV-CREB) have both enhanced synaptic transmission and reduced PPF when compared with the other thre...; How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling prote...; Results are expressed as mean ± SEM. ANOVA analyses or t -tests were used for statistical comparisons between groups as described in the context. P < 0.05 means significant...
from paperReporting output
Report representative outputs alongside summary comparisons for Importantly, we found that HSV-CREB neurons in conditioned mice (conditioned HSV-CREB) have both enhanced synaptic transmission and reduced PPF when compared with the other thre..., How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling prote..., Previous studies showed that both changes in synaptic strength (LTP) and changes in intrinsic excitability could lead to a left shift of the E-S curve -. Our whole-cell r..., Two vectors were used, HSV-CREB-AlstR (HSV-CREB vector) and HSV-LacZ-AlstR (Control vector). Genes of interest ( Creb1, LacZ and AlstR ) were cloned into a bicistronic HSV ampl....
inferred from protocolStructured statistical methods
Previous studies in thalamo-LA synapses following tone fear conditioning revealed increases in synaptic transmission and decreases in paired pulse facilitation (PPF),. To test...; Importantly, we found that HSV-CREB neurons in conditioned mice (conditioned HSV-CREB) have both enhanced synaptic transmission and reduced PPF when compared with the other thre...; How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling prote...; Results are expressed as mean ± SEM. ANOVA analyses or t -tests were used for statistical comparisons between groups as described in the context. P < 0.05 means significant...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling proteins that increase the excitability of these neurons -, thereby increasing the probability that they are recruited during learning. To test this hypothesis, 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 (, HSV-CREB, -38.8 ± 0.9 mV, one-way ANOVA, P < 0.05 compared to the other three control groups, -35.4 ± 1.0 mV~ -35.7 ± 1.0 mV). In addition, HSV-CREB caused a robust increase in the number of action potentials elicited by depolarizing current injections: in HSV-CREB neurons ( n = 58, from 8 mice), the number of action potentials triggered by depolarizing current injections was higher than in non-transfected neurons or in neurons transfected with the HSV-La...
Mice were pretreated with atropine sulfate (0.1 mg kg -1, i.p.), anesthetized with chloral hydrate (400 mg kg -1, i.p.), and placed in a stereotaxic frame. The skin was retracted and holes drilled in the skull bilaterally above the LA (AP = -1.3, ML = ± 3.3, V = - 4.8 mm from bregma) according to Paxinos and The Franklin mouse brain atlas. For behavioral experiments, a stainless steel outer cannula (Plastic One) was implanted to LA and fixed on the skull with dental cement. After cannula implantation, mice were single-housed and handled every day. Virus infusion was delayed until seven days after surgery to ensure physical recovery. A virus solution (1.3 µl; bilateral) was delivered to lateral amygdala at a flow rate of 0.065 µl/min through inner injection cannula (Plastic one, 22 gauge) attached by polyethylene tubing to Hamilton microsyringes mounted in an infusion pump (Harvard Instruments). The infusion cannula was left in place an additional 10 min to ensure diffusion of the vector. For electrophysiological studies, virus solution (1.3 µl) was delivered to LA over 5 min through glass micropipettes (Sutter Instrument) immediatel...
The peptide allatostatin (AL) (New England) was dissolved in ddH 2 O to make a 2.5 mM stock solution. Saline was added to dilute it to the proper final concentration for each specific experiment. AL solution (0.5 µl) or same amount of vehicle (saline) was delivered bilaterally to the LA of awake animals at a flow rate of 0.25 µl/min through inner injection cannula. The cannula was left in place for 2 min post-injection. Behavioral procedures were started ~30 min after injection.
Training consisted of placing the mice in a conditioning chamber (Context A) and 2 min later presenting a tone (2800 Hz, 85 dB, 30 sec) that co-terminated with a shock (2 sec, 0.5 mA). Mice remained in the chamber for an additional 1 min. Test for auditory fear conditioning occurred either 30 min or 24 h later. Mice were placed in a novel chamber (Context B) and 2 min later the tone CS was presented (for 1 min). Our index of memory, freezing (the cessation of all movement except for respiration), was assessed via an automated scoring system (Med Associates Inc.) with a 30 frames/sec sampling; the animals needed to freeze continuously for at least 1 sec before freezing could be counted. The same training protocol was also used in the electrophysiological studies. Slices were cut 24 h after training.
CTA training took place in the light part of the cycle 7 days following surgery. Mice were water deprived for 24 h and then pretrained for 5 days to get their daily water ration within 40 min per day from two tubes (10 ml). Three hours after the 2 nd day pretraining, HSV virus was infused into LA on both sides. Conditioning day started 3 days after virus injection.
On the conditioning day, the two tubes were filled with 0.2% saccharin sodium salt (w/v, the taste CS) instead of water. The CS was presented for 20 min and 20 min later, mice were treated with the malaise inducing agent lithium chloride (LiCl; 0.06 M, 2% body weight i.p.).
Testing for aversion to saccharin occurred 24 h later. AL/vehicle was infused 30min before testing. Two tubes (one contains water and the other contains saccharin) were presented for 40 min. The intake of each fluid was measured and the aversion index (AI) was defined as follows: [milliliters of water consumed/(milliliters of water + milliliters of saccharin consumed)] × 100%. 50% AI is equal preference level, and the higher the AI, the more the mice prefer water to saccharin).
Quantitative analyses of infection levels were performed using the NIH Image processing system. Before confocal imaging we used transmission light microscopy and the thalamo-lateral amygdala fibers (ic) and the cortico-lateral amygdale fibers (oc) to locate the dorsal and lateral boundary of LA. The total number of GFP-positive cells in the LA was counted bilaterally with a fixed sample window (0.1 mm 2 ) across at least six sections from comparable anteroposterior levels from each mouse. To assess the number of nuclei in the areas counted, the slices were re-stained using 4',6'-diamidino-2-phenylindole (DAPI) after washout of FITC-conjugated GFP primary antibody. The percentage of GFP positive cells was calculated as the percentage of GFP-positive cell/total DAPI-labeled nuclei in the LA. After the immunofluorescent studies, brain slices were counter stained with crystal violet to confirm the position of the infusion cannula.
Machine-readable layer
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"text": "How could neurons with higher CREB levels bias memory allocation? It is possible that higher levels of CREB lead to increased expression of specific channels and signaling proteins that increase the excitability of these neurons -, thereby increasing the probability that they are recruited during learning. To test this hypothesis, 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 (, HSV-CREB, -38.8 ± 0.9 mV, one-way ANOVA, P < 0.05 compared to the other three control groups, -35.4 ± 1.0 mV~ -35.7 ± 1.0 mV). In addition, HSV-CREB caused a r..."
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