Research Article

Neuroscience

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons

National Institute of Neurological Disorders and Stroke, NIH, United States
Yale Interdepartmental Neuroscience Program, Yale University, United States
Kansas City University of Medicine and Biosciences, United States
Stony Brook University, United States
National Institute of Environmental Health Sciences, United States
Program in Neuroscience, Stony Brook University, United States
State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, China
PKU-IDG/McGovern Institute for Brain Research, China
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, China
Department of Psychiatry, Yale University, United States

Feb 16, 2024

https://doi.org/10.7554/eLife.86581

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Version of Record: March 11, 2024
Accepted Manuscript: February 16, 2024

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Prithviraj Rajebhosale

Mala R Ananth

Ronald Kim

Richard Crouse

Li Jiang

Gretchen López-Hernández

Chongbo Zhong

Christian Arty

Shaohua Wang

Alice Jone

Niraj S Desai

Yulong Li

Marina R Picciotto

Lorna W Role

David A Talmage

(2024)
Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons

eLife 13:e86581.

https://doi.org/10.7554/eLife.86581
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Additional files

12 figures, 2 tables and 2 additional files

Figures

Figure 1 with 4 supplements

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Acetylcholine (ACh) is released in basolateral amygdala (BLA) during threat recall (see also Figure 1—figure supplements 1–4).

(A) Left: Schematic of strategy for targeting the genetically encoded ACh sensor (GRAB_ACh3.0) to BLA. Right: Image of ACh sensor expression (green). White dotted oval delineates ACh sensor-labeled …

Figure 1—source data 1 Processed ACh release fiber photometry data for Figure 1.: https://cdn.elifesciences.org/articles/86581/elife-86581-fig1-data1-v2.csv
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Figure 1—figure supplement 1

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Exposure to tones during tone–shock pairings does not significantly alter acetylcholine (ACh) release in the basolateral amygdala (BLA) during training.

(A) Left: Schematic of the associative threat-learning protocol employed consisting of three tone + shock pairings during the training period. Right: Quantification of typical freezing behavior …

Figure 1—figure supplement 2

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Repeat tone exposures without foot shock during either a training session or a recall session fail to induce significant changes in acetylcholine (ACh) release in the basolateral amygdala (BLA).

(A) Schematic of tone only training protocol consisting of three 30 s tones on day 1 without shocks followed by presentation of the same tone 24 hr later, on day 2. (B) Average traces of ACh release …

Figure 1—figure supplement 2—source data 1 Processed ACh release fiber photometry data for Figure 1—figure supplement 2.: https://cdn.elifesciences.org/articles/86581/elife-86581-fig1-figsupp2-data1-v2.xlsx
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Figure 1—figure supplement 3

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Shock alone does not sensitize cholinergic tone responses in the basolateral amygdala (BLA).

(A) Top: Schematic of shock only training and recall protocol consisting of three 2 s shocks during the training session (day 1) and a single 30-s tone during the recall session (day 2). …

Figure 1—figure supplement 3—source data 1 Processed ACh release data for Figure 1—figure supplement 3.: https://cdn.elifesciences.org/articles/86581/elife-86581-fig1-figsupp3-data1-v2.xlsx
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Figure 1—figure supplement 4

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Remapping of optic fiber placements.

(A) Relocalization of fiber optic placement mapped across the anterior–posterior axis of the basolateral amygdala (BLA). Top: Atlas images showing fiber tip locations (red squares). (B) Left: …

Figure 2 with 1 supplement

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Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are activated by threat learning and reactivated during threat memory recall (see Figure 2—figure supplement 1).

(A) Strategy for labeling activated NBM/SI_p cholinergic neurons during both training and recall. Chat-IRES-Cre × Fos-tTA/shGFP mice (n = 14) were injected in the NBM/SI_p with ADCD-mCherry virus …

Figure 2—figure supplement 1

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Construction of viral vectors for tagging activated cholinergic neurons.

(A) Top: Diagrams of 1. ADCD and 2. ADCD hM4Di viral constructs. Activity dependence is conferred by the Tet response element (TRE – 7 repeats of the tetO sequence followed by a minimal promoter). …

Figure 3 with 1 supplement

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Re-activation of a subset of nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons is required for threat memory retrieval (see Figure 3—figure supplement 1).

(A) ADCD-hM4Di (AAV9: TRE-DIO-hM4Di.mCherry) was injected into the NBM/SI_p of Chat-IRES-Cre × Fos-tTA/shGFP mice. Two weeks later mice underwent training on regular chow (Dox-chow removed 24 hr …

Figure 3—figure supplement 1

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Time-resolved freezing plot from recall following silencing of training-activated NBM/SI basal forebrain cholinergic neurons (BFCNs).

(A) Freezing behavior during training in sham (black, n = 8 mice) and ADCD-hM4Di injected (red, n = 7 mice). Error bars represent standard error of the mean (SEM). Tone 1 (), Tone 2 (), Tone 3 …

Figure 4

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Basolateral amygdala (BLA)-projecting nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are reactivated by the conditioned tone stimulus.

(A) Left: Strategy for labeling activated NBM/SI_p cholinergic during both training and recall along with mapping of BLA-projecting neurons. Chat-IRES-Cre × Fos-tTA/shGFP mice (n = 7) were injected …

Figure 5 with 2 supplements

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Basolateral amygdala (BLA)-projecting cholinergic neuronal activity is required both during training and during recall for learned threat processing (see Figure 5—figure supplements 1 and 2).

(A) Left: Strategy for retrograde targeting of hM4Di DREADD to BLA-projecting cholinergic neurons. Middle: Re-localization of BLA injection sites (using AAV₉-camk2a-GCaMP6f to mark the injection …

Figure 5—figure supplement 1

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DREADD-induced silencing of basolateral amygdala (BLA)-projecting cholinergic neurons reduces threat induced activation of anterior BLA and CeC neurons.

(A) Top: Strategy for retrograde targeting of hM4Di DREADD to BLA-projecting cholinergic neurons. BLA-projecting cholinergic neurons were silenced by injecting mice with clozapine (CLZ) prior to …

Figure 5—figure supplement 2

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Role of nucleus basalis and posterior substantia innominata (NBM/SI_p) in associative threat memory recall.

(A) Top: NBM/SI_p of Chat-IRES-Cre mice was directly targeted with an AAV₉-DIO-hM4Di.mCherry (DREADD) or AAV₉-DIO-eCFP (sham) and cholinergic neurons were silenced during recall. Bottom: Freezing …

Figure 6 with 2 supplements

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The extent of cholinergic neuronal activation in the anterior nucleus basalis and posterior substantia innominata (NBM/SI_p) co-varies with the behavioral performance during threat memory recall (see Figure 6—figure supplements 1 and 2).

(A) Behavioral performance (freezing) from recall session showing High (black, n = 9) and Low (gray, n = 9) responding mice. High Responders show significantly higher freezing to recall tone whereas …

Figure 6—figure supplement 1

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Reactivation of training-activated cholinergic neurons scales with associative threat learning and with behavioral performance during memory recall.

(A) Left: Strategy for labeling activated nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic during training and recall. Chat-IRES-Cre × Fos-tTA/shGFP mice (n = 14) were …

Figure 6—figure supplement 2

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Recall-activated basal forebrain cholinergic neurons (BFCNs) in nucleus basalis and posterior substantia innominata (NBM/SI_p) of ‘High Responder’ mice are located at a basolateral amygdala (BLA)-projecting locus in the cholinergic basal forebrain.

(A) Representative images of the NBM/SI_p at bregma locations −0.8, 1.0, and 1.3 mm from a Fos-tTA/shGFP High Responder (top) and Low Responder (bottom) mouse sacrificed 2.5 hr after recall. Brain …

Figure 7 with 1 supplement

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Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons show increased intrinsic excitability following threat memory recall (see Figure 7—figure supplement 1).

(A) Schematic of electrophysiological profiling of activated (Fos−shGFP+) vs. non-activated (Fos−shGFP−) neurons from mice following recall or in home cage mice, with post hoc identification of …

Figure 7—figure supplement 1

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Latency and afterhyperpolarization (AHP) amplitudes significantly differed in recall-activated nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons.

(**A–F**) Additional plots of population data on NBM/SI_p cholinergic neurons presented as dot plot + median for the electrophysiological properties of post hoc identified cholinergic neurons from home …

Figure 8 with 1 supplement

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Distinct population of basolateral amygdala (BLA)-projecting cholinergic neurons contribute to innate threat processing (see Figure 8—figure supplement 1).

(A) Fos-tTA/Fos–shGFP mice were placed in chambers containing a gauze pad spotted with either saline or with mountain lion urine (predator odor). Defensive behaviors were monitored for 5 min. Mice …

Figure 8—figure supplement 1

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Predator odor exposure activates VP/SI_a cholinergic neurons.

(A) Left: Fos-tTA/shGFP mice were exposed to predator odor or saline and sacrificed 2.5 hr later. Brain sections were processed for ChAT immunohistochemistry. Representative images of hDB, VP/SI_a, …

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Tables

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus)	Chat-IRES-Cre	The Jackson Laboratory	B6;129S6-Chattm2(cre)Lowl/J	Stock number: 006410
Strain, strain background (Mus musculus)	Fos-tTA, Fos−shGFP	The Jackson Laboratory	TetTag	Stock number: 018306
Strain, strain background (Escherichia coli)	Stellar Competent Cells, HST08	Takara	Cat#636766
Genetic reagent (AAV9)	AAV9-camk2a-GCaMP6f-WPRE-SV40	Penn Vector Core
Genetic reagent (AAV9)	AAV9-DIO-eCFP	This paper, Vector Biolabs		Custom made
Genetic reagent (AAV9)	AAV9-hSyn-GACh4.3	Vigene Biosciences Inc
Genetic reagent (AAV8)	AAV8-DIO-hM4Di-mCherry	Addgene	Cat#44362
Genetic reagent (AAV9)	AAV9-TRE-DIO-oChIEF-mCherry-P2A-tTAH100Y.SV40	This paper	plasmid DNA	See Methods and Figure 2—figure supplement 1, can be obtained from Talmage lab.
Genetic reagent (AAV9)	AAV9-TRE-DIO-hM4Di-mCherry	This paper	Cat#169415	Deposited to Addgene, see methods and Figure 2—figure supplement 1, can be obtained from Talmage lab.
Genetic reagent (CAV2)	CAV2-DIO-hM4Di-mCherry	Dr. EJ Kremer, Institut de Génétique Moléculaire de Montpellier, France
Antibody	anti-ChAT (Goat polyclonal)	Millipore	Cat# AB144P; RRID:AB_2079751	IHC (1:500)
Antibody	anti-GFP (Rabbit polyclonal)	Thermo Fisher Scientific	Cat#: A-11122; RRID:AB_221569	IHC (1:1000)
Antibody	anti-GFP (Rabbit polyclonal)	Abcam	Cat#: ab13970; RRID:AB_300798	IHC (1:500)
Antibody	anti-mCherry (Mouse monoclonal)	Takara	Cat#: 632543; RRID:AB_2307319	IHC (1:500)
Antibody	anti-DsRed (Rabbit polyclonal)	Takara	Cat#: 632496; RRID:AB_10013483	IHC (1:500)
Antibody	anti-c-Fos (Rabbit polyclonal)	Synaptic Systems	Cat#: 226003; RRID:AB_2231974	IHC (1:500)
Antibody	anti-Rabbit IgG (H+L)-AlexaFluor 488 (Donkey polyclonal)	Thermo Fisher	Cat#: A32790; RRID:AB_2762833	IHC (1:1000)
Antibody	anti-Rabbit IgG (H+L)-Rhodamine Red-X (Donkey polyclonal)	Jackson Immunoresearch	Cat#: 711-295-152; RRID:AB_2340613	IHC (1:1000)
Antibody	anti-Goat IgG (H+L)- AlexaFluor 594 (Donkey polyclonal)	Thermo Fisher	Cat#: A-11058; RRID:AB_142540	IHC (1:1000)
Antibody	anti-Chicken IgY-Cy2 (Donkey polyclonal)	Gift from Dr.Shaoyu Ge, Stony Brook University NY		IHC (1:1000)
Chemical compound	NeuroTrace 435/455 Blue Fluorescent Nissl Stain	Thermo Fisher	Cat#: N21479	IHC (1:500)
Recombinant DNA reagent	pAAV-hSyn-DIO-hM4D(Gi)-mCherry (plasmid)	Addgene	Cat#44362
Recombinant DNA reagent	pV2SGE (plasmid)	This paper	Gift from Dr.Shaoyu Ge, Stony Brook University NY	Used in the construction of reagent #25
Recombinant DNA reagent	pAAV-TRE-DIO-oChIEF-mCherry-P2A-tTAH100Y.SV40 (plasmid)	This paper	Deposited to Addgene	Addgene Cat# 169414
Recombinant DNA reagent	pAAV-TRE-DIO-hM4Di-mCherry (plasmid)	This paper	Deposited to Addgene	Addgene Cat# 169415
Sequence-based reagent	chat_F	IDT	PCR primers	TCTGGCAACTTCGTCGGA
Sequence-based reagent	chat_R	IDT	PCR primers	CTCCTGGGCTGTTACGCAC
Sequence-based reagent	pV2.1 – Gene Block 7xTetO-LoxP-Lox2272-tTAH100Y.SV40	IDT	Gene block, custom
Sequence-based reagent	pV2.2 – Gene Block oChIEF-LoxP-Lox2272	IDT	Gene block, custom
Commercial assay or kit	In-Fusion HD Cloning Plus	Takara/Clontech	Cat#: 638920
Commercial assay or kit	High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems	Cat#: 4368814
Peptide, recombinant protein	T4 DNA Ligase	NEB	Cat#M0202S
Peptide, recombinant protein	BglII	NEB	Cat#R0144S
Peptide, recombinant protein	AscI	NEB	Cat#R0558S
Peptide, recombinant protein	BamHI-HF	NEB	Cat#R3136S
Peptide, recombinant protein	PmlI	NEB	Cat#R0532S
Peptide, recombinant protein	Phusion High-Fidelity DNA Polymerase	NEB	Cat#M0530S
Chemical compound, drug	Clozapine	Sigma-Aldrich	Cat#C6305-25MG
Chemical compound, drug	Fast Blue	Polysciences Inc	Cat#17740-1
Other	Mt.Lion Urine	Maine outdoor solutions LLC		Obtained from https://predatorpeestore.com/.
Software, algorithm	Prism	GraphPad Software Inc	RRID:SCR_002798
Software, algorithm	Sigmaplot 12.5	Systat Software Inc	RRID:SCR_003210
Software, algorithm	OriginPro 9.1	Origin Lab Corporation	RRID:SCR_014212
Software, algorithm	Fiji is just imagej	Fiji	RRID:SCR_002285
Software, algorithm	FreezeFrame v3	Actimetrics	RRID:SCR_014429
Software, algorithm	MATLAB	Mathworks	RRID:SCR_001622
Software, algorithm	Pre-processing analysis MATLAB Script for FiberPhotometry	Doric
Software, algorithm	ACh sensor analysis MATLAB script	Crouse, Richard B., et al. Elife 9 (2020): e57335

Author response table 1

Max Firing Rate	0.0032
HC vs. Fos-	0.3206
HC vs. Fos+	0.003
Fos- vs. Fos+	0.0034

Additional files

Supplementary file 1 Statistical reporting table. This file contains exact sample sizes for each group, group median, 95% confidence interval of the median, actual confidence interval, statistical test used, p-values, and test statistic for each of the reported plots in the manuscript.: https://cdn.elifesciences.org/articles/86581/elife-86581-supp1-v2.xlsx
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MDAR checklist: https://cdn.elifesciences.org/articles/86581/elife-86581-mdarchecklist1-v2.pdf
Download elife-86581-mdarchecklist1-v2.pdf

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Figures

Acetylcholine (ACh) is released in basolateral amygdala (BLA) during threat recall (see also Figure 1—figure supplements 1–4).

Figure 1—source data 1

Exposure to tones during tone–shock pairings does not significantly alter acetylcholine (ACh) release in the basolateral amygdala (BLA) during training.

Repeat tone exposures without foot shock during either a training session or a recall session fail to induce significant changes in acetylcholine (ACh) release in the basolateral amygdala (BLA).

Figure 1—figure supplement 2—source data 1

Shock alone does not sensitize cholinergic tone responses in the basolateral amygdala (BLA).

Figure 1—figure supplement 3—source data 1

Remapping of optic fiber placements.

Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are activated by threat learning and reactivated during threat memory recall (see Figure 2—figure supplement 1).

Construction of viral vectors for tagging activated cholinergic neurons.

Re-activation of a subset of nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons is required for threat memory retrieval (see Figure 3—figure supplement 1).

Time-resolved freezing plot from recall following silencing of training-activated NBM/SI basal forebrain cholinergic neurons (BFCNs).

Basolateral amygdala (BLA)-projecting nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are reactivated by the conditioned tone stimulus.

Basolateral amygdala (BLA)-projecting cholinergic neuronal activity is required both during training and during recall for learned threat processing (see Figure 5—figure supplements 1 and 2).

DREADD-induced silencing of basolateral amygdala (BLA)-projecting cholinergic neurons reduces threat induced activation of anterior BLA and CeC neurons.

Role of nucleus basalis and posterior substantia innominata (NBM/SI_p) in associative threat memory recall.

The extent of cholinergic neuronal activation in the anterior nucleus basalis and posterior substantia innominata (NBM/SI_p) co-varies with the behavioral performance during threat memory recall (see Figure 6—figure supplements 1 and 2).

Reactivation of training-activated cholinergic neurons scales with associative threat learning and with behavioral performance during memory recall.

Recall-activated basal forebrain cholinergic neurons (BFCNs) in nucleus basalis and posterior substantia innominata (NBM/SI_p) of ‘High Responder’ mice are located at a basolateral amygdala (BLA)-projecting locus in the cholinergic basal forebrain.

Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons show increased intrinsic excitability following threat memory recall (see Figure 7—figure supplement 1).

Latency and afterhyperpolarization (AHP) amplitudes significantly differed in recall-activated nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons.

Distinct population of basolateral amygdala (BLA)-projecting cholinergic neurons contribute to innate threat processing (see Figure 8—figure supplement 1).

Predator odor exposure activates VP/SI_a cholinergic neurons.

Tables

Additional files

Supplementary file 1

MDAR checklist

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Acetylcholine (ACh) is released in basolateral amygdala (BLA) during threat recall (see also Figure 1—figure supplements 1–4).

Figure 1—source data 1

Exposure to tones during tone–shock pairings does not significantly alter acetylcholine (ACh) release in the basolateral amygdala (BLA) during training.

Repeat tone exposures without foot shock during either a training session or a recall session fail to induce significant changes in acetylcholine (ACh) release in the basolateral amygdala (BLA).

Figure 1—figure supplement 2—source data 1

Shock alone does not sensitize cholinergic tone responses in the basolateral amygdala (BLA).

Figure 1—figure supplement 3—source data 1

Remapping of optic fiber placements.

Nucleus basalis and posterior substantia innominata (NBM/SIp) cholinergic neurons are activated by threat learning and reactivated during threat memory recall (see Figure 2—figure supplement 1).

Construction of viral vectors for tagging activated cholinergic neurons.

Re-activation of a subset of nucleus basalis and posterior substantia innominata (NBM/SIp) cholinergic neurons is required for threat memory retrieval (see Figure 3—figure supplement 1).

Time-resolved freezing plot from recall following silencing of training-activated NBM/SI basal forebrain cholinergic neurons (BFCNs).

Basolateral amygdala (BLA)-projecting nucleus basalis and posterior substantia innominata (NBM/SIp) cholinergic neurons are reactivated by the conditioned tone stimulus.

Basolateral amygdala (BLA)-projecting cholinergic neuronal activity is required both during training and during recall for learned threat processing (see Figure 5—figure supplements 1 and 2).

DREADD-induced silencing of basolateral amygdala (BLA)-projecting cholinergic neurons reduces threat induced activation of anterior BLA and CeC neurons.

Role of nucleus basalis and posterior substantia innominata (NBM/SIp) in associative threat memory recall.

The extent of cholinergic neuronal activation in the anterior nucleus basalis and posterior substantia innominata (NBM/SIp) co-varies with the behavioral performance during threat memory recall (see Figure 6—figure supplements 1 and 2).

Reactivation of training-activated cholinergic neurons scales with associative threat learning and with behavioral performance during memory recall.

Recall-activated basal forebrain cholinergic neurons (BFCNs) in nucleus basalis and posterior substantia innominata (NBM/SIp) of ‘High Responder’ mice are located at a basolateral amygdala (BLA)-projecting locus in the cholinergic basal forebrain.

Nucleus basalis and posterior substantia innominata (NBM/SIp) cholinergic neurons show increased intrinsic excitability following threat memory recall (see Figure 7—figure supplement 1).

Latency and afterhyperpolarization (AHP) amplitudes significantly differed in recall-activated nucleus basalis and posterior substantia innominata (NBM/SIp) cholinergic neurons.

Distinct population of basolateral amygdala (BLA)-projecting cholinergic neurons contribute to innate threat processing (see Figure 8—figure supplement 1).

Predator odor exposure activates VP/SIa cholinergic neurons.

Supplementary file 1

MDAR checklist

Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are activated by threat learning and reactivated during threat memory recall (see Figure 2—figure supplement 1).

Re-activation of a subset of nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons is required for threat memory retrieval (see Figure 3—figure supplement 1).

Basolateral amygdala (BLA)-projecting nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons are reactivated by the conditioned tone stimulus.

Role of nucleus basalis and posterior substantia innominata (NBM/SI_p) in associative threat memory recall.

The extent of cholinergic neuronal activation in the anterior nucleus basalis and posterior substantia innominata (NBM/SI_p) co-varies with the behavioral performance during threat memory recall (see Figure 6—figure supplements 1 and 2).

Recall-activated basal forebrain cholinergic neurons (BFCNs) in nucleus basalis and posterior substantia innominata (NBM/SI_p) of ‘High Responder’ mice are located at a basolateral amygdala (BLA)-projecting locus in the cholinergic basal forebrain.

Nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons show increased intrinsic excitability following threat memory recall (see Figure 7—figure supplement 1).

Latency and afterhyperpolarization (AHP) amplitudes significantly differed in recall-activated nucleus basalis and posterior substantia innominata (NBM/SI_p) cholinergic neurons.

Predator odor exposure activates VP/SI_a cholinergic neurons.