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Role of nucleus basalis magnocellularis cholinergic neurons in contextual fear memory

Addgene #44362-AAV8

Data Submitted By
Brianna Shultz , Negin Mohammadmirzaei and Dayan Knox
Submission Date
July 27, 2019
Publication Date
July 27, 2019 (modified June 15, 2023)
DOI
https://doi.org/10.57733/addgene.88hyvn
Funding
NIGMS 1P20GM103653
Abstract
To examine the potential role of NBM cholinergic neurons in contextual fear memory we conducted an experiment in rats expressing cre in cholinergic neurons whereby the inhibitory DREADD (hM4Di) was expressed in NBM cholinergic neurons (n = 2) or a control viral vector expressing mCherry was expressed in another group of rats (n = 4). CNO (3.0mg/kg) was administered to all rats 1-hr prior to contextual fear conditioning (five pure tones that co-terminated with a 1s, 0.5mA footshock) and one day later all animals were tested for contextual fear. All rats acquired contextual fear in an equivalent manner during fear conditioning, but rats expressing hM4Di showed lower levels of contextual fear during the memory test. These results raise the possibility that NBM cholinergic neurons could be critical for contextual fear memory formation.
Citation
Role of nucleus basalis magnocellularis cholinergic neurons in contextual fear memory. Shultz B, Mohammadmirzaei N, Knox D 2019. Addgene Report, https://doi.org/10.57733/addgene.88hyvn
Temporary inhibition of cholinergic neurons in the nucleus basalis (chNBM) during contextual fear conditioning disrupts contextual fear memory. A) ChAT::cre rats were either infused with vector constructs that express hM4Di (n = 2) or mCherry only (n = 4). Rats were then subjected to contextual fear conditioning by presenting a 1mA, 0.5s foot-shock UCS five times (60s ISIs). Testing of fear memory occurred one day later in an 8-min session. B) Cells expressing either ChAT alone (asterisk) or mCherry and ChAT (arrows) in NBM (20X images). C) Administration of CNO prior to fear conditioning had no effect on freezing during fear conditioning, but disrupted contextual fear conditioned freezing, in rats expressing hM4Di, during the fear memory test (p < .05).
Caption: Temporary inhibition of cholinergic neurons in the nucleus basalis (chNBM) during contextual fear conditioning disrupts contextual fear memory. A) ChAT::cre rats were either infused with vector constructs that express hM4Di (n = 2) or mCherry only (n = 4). Rats were then subjected to contextual fear conditioning by presenting a 1mA, 0.5s foot-shock UCS five times (60s ISIs). Testing of fear memory occurred one day later in an 8-min session. B) Cells expressing either ChAT alone (asterisk) or mCherry and ChAT (arrows) in NBM (20X images). C) Administration of CNO prior to fear conditioning had no effect on freezing during fear conditioning, but disrupted contextual fear conditioned freezing, in rats expressing hM4Di, during the fear memory test (p < .05).
Image attribution: This image was acquired by the Knox Neurobiology Lab

Any further use of this image requires permission from the lab who submitted the data.

Vector Used
pAAV-hSyn-DIO-hM4D(Gi)-mCherry
Addgene #44362-AAV8

Virus & Injection

Virus

Virus Name
pAAV-hSyn-DIO-hM4D(Gi)-mCherry
Serotype
AAV8
Source
Addgene
Catalog Number
44362-AAV8
Virus Lot Number
v24795
Promoter
Synapsin
Cargo Type
Chemogenetics
Injection Titer / Dose
2.5 x 10e13 vg/mL
Injection Volume
0.5 μl
Injection Rate
Bolus
Injector Material
Hamilton syringe (22s gauge needle)
Tip Diameter
22
Coatings
None used
Injection Time
5 minutes
Injection Site / Route
basal nucleus, Neuronames ID#: 275
Additional Details
Experiments were done in MedAssociate boxes and freezing scored using Any-maze. No dilution was used.
View in Addgene Catalog

Other Details

Species
Rat
Strain or Cell Line
ChAT::Cre
Source
RRRC
Other Information
Rats are Long-Evans rats that have been genetically engineered to express cre recombinase.
Age at Injection
3 months
Sex
Male
Time After Injection
30 days
Detection Method
Double Labeling Immunohistochemistry
Assay & Results
Cholinergic neuron cell bodies and axons originating from NBM. Double labeling IHC was used to detect mCherry (axons and cell bodies) as well as mCherry and ChAT in cell bodies. Results demonstrate that all mCherry neurons expressed ChAT as well.
Functional Testing
The vector expressed the inhibitory DREADD hM4Di. Behavioral data suggested the inhibition worked.
Other Information
Did not observe any non-specific damage
Tips & Comments
Actual inhibition produced by CNO or C21 injection should be examined. We have a protocol for this, but was running short on time, so could not implement it.