Cholinergic basal forebrain neurons regulate fear extinction consolidation through p75 neurotrophin receptor signaling - PubMed (original) (raw)
Cholinergic basal forebrain neurons regulate fear extinction consolidation through p75 neurotrophin receptor signaling
Zoran Boskovic et al. Transl Psychiatry. 2018.
Abstract
Cholinergic basal forebrain (cBF)-derived neurotransmission plays a crucial role in regulating neuronal function throughout the cortex, yet the mechanisms controlling cholinergic innervation to downstream targets have not been elucidated. Here we report that removing the p75 neurotrophin receptor (p75NTR) from cBF neurons induces a significant impairment in fear extinction consolidation. We demonstrate that this is achieved through alterations in synaptic connectivity and functional activity within the medial prefrontal cortex. These deficits revert back to wild-type levels upon re-expression of the active domain of p75NTR in adult animals. These findings demonstrate a novel role for cholinergic neurons in fear extinction consolidation and suggest that neurotrophic signaling is a key regulator of cholinergic-cortical innervation and function.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Fig. 1. Fear extinction consolidation is impaired in p75NTR-deficient animals.
a The experimental paradigm for assessing fear extinction consolidation consisted of a four-day protocol. On the first day (Conditioning) animals were conditioned by pairing 3 conditioned stimuli (CS) to a unconditioned stimulus (US) in context A. On the second day (Extinction) all animals, were moved to a different context (context B) and, except for the No-EXT group, exposed to 30 CS without a US pairing. Twenty-four hours later, fear extinction consolidation was tested in context B (Test 1) with 2 CS presentations. On the final day, animals were returned to context A and exposed to 2 CS presentations to test context discrimination (Test 2). b Average fear response of control (WT No-EXT = wild-type animals that did not undergo extinction training; p75fl/fl = transgenic controls; p75fl/wt = transgenic heterozygous controls; ChAT-cre p75in/wt = heterozygous mutants) and mutant (ChAT-cre p75in/in) animals during conditioning training. There were no significant differences in conditioning between any of the groups. c Average fear response of animals on day 3 of behavioral testing. Fear extinction consolidation was assessed prior to the first CS presentation (PreCS) and as an average freezing time during the 2 CS presentations (Average CS). All mice displayed significant freezing on CS presentation compared to PreCS levels. Average CS assessment revealed that ChAT-cre p75in/in animals displayed significantly higher freezing than transgenic control groups (two-way ANOVA comparing preCS to CS as well as genotype, *p < 0.05; **p < 0.01, ***p < 0.001) but their degree of freezing was not significantly different from the WT No-EXT control). Data are represented as mean±SEM). d Fear assessment of mice when they were returned to the conditioning context on day 4 of behavioral testing. There were no significant differences between any of the transgenic groups, indicating that context discrimination was intact in all of these mice. e Average freezing response of ChAT-cre p75in/in mice that received control (Tau-GFP) and p75ICD-GFP virus. There was no significant difference between the two groups during conditioning. f Average freezing response of injected ChAT-cre p75in/in mice during fear extinction consolidation testing. Mutant mice that received p75ICD-GFP showed significantly less freezing during the Average CS period compared to Tau-GFP-injected mutants (two-way ANOVA, **p < 0.01; data are represented as mean±SEM). The number of animals analyzed is indicated in the graphs
Fig. 2. Arbour size of individual neurons is unaffected by the absence of p75NTR.
a Representative images of the prefrontal cortex (PFC; target) and medial septum (MS; injection site) and of cre-expressing control (ChAT-cre) and mutant (ChAT-cre p75in/in) mice injected with Tau-GFP. Sections were labeled using an anti-GFP antibody (green). Scale bar = 100 µm. b Average GFP-labeled axonal density per infected cell of mice injected with Tau-GFP AAV. No significant difference in axonal density/cBF neuron was observed between control and mutant mice. c Representative images of cBF axonal innervation of the PFC and infected cells in the MS of control and mutant mice injected with p75ICD-GFP. Sections were labeled using an anti-GFP antibody (green). d Average GFP-labeled axonal density per infected cell of animals injected with p75ICD-GFP AAV. No significant effect of the p75ICD-GFP injections were observed in animals of either genotype. The number of animals analyzed is indicated in the graphs
Fig. 3. p75NTR signalling affects synaptic connectivity of cBF neurons.
a Representative images of the prefrontal cortex (PFC) of cre-expressing controls (ChAT-cre), mutants (ChAT-cre p75in/in) and mutants injected with p75ICD-GFP. Postsynaptic cells in the PFC innervated by non-cholinergic basal forebrain neurons (green) were labeled against the GFP-expressing version of HSV129 virus (See Supplementary Figure S3A). Cells innervated by cBF neurons (red) were labeled against the tdTomato-expressing version of the HSV129 virus. Scale bar = 200 µm. b There was a significant increase in GFP labeling in the PFC of ChAT-cre p75in/in mutants compared to control and p75ICD-injected mutant mice (one-way ANOVA, **p < 0.01; the data are represented as mean ± SEM). c A significant increase in tdTomato labeling was also observed in the PFC of ChAT-cre p75in/in animals compared to controls, which reverted back to control levels upon injection of p75ICD (one-way ANOVA, *p < 0.05, **p < 0.01; data are represented as mean ± SEM)
Fig. 4. p75NTR affects behaviourally relevant neuronal activity.
a Representative images of the prefrontal cortex (PFC) of control and mutant mice. Activated cells are visualized with phosphorylated extracellular signal-regulated kinase (pERK). Scale bar = 400 µm. b A significant decrease in the number of pERK-positive cells was observed in the mPFC of naïve mutant animals compared to controls. c Animals were euthanized within one hour of Test 1 to analyze cells that were activated during the fear extinction consolidation test. d Representative images of the prefrontal cortex of extinction-tested control and mutant mice, immunostained for pERK. Scale bar = 400 µm. e A significant decrease in the number of pERK-positive cells was observed specifically within the IL of mutant animals (t test; *p < 0.05; data are represented as mean ± SEM; The number of animals analyzed is indicated in the graphs); Cg1 cingulate cortex, PrL prelimbic prefrontal cortex, IL infralimbic prefrontal cortex
References
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