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Beta2* Nicotinic Receptors Regulate Exploratory and Social Behavior Through Functionally Distinct Neuronal Populations

Alice AbbondanzaiD, Jan Elias, Chandani R. Verma, Amanda Rosanna Alves Barboza, Martin Capek, Trisha Jigar Dhabalia, Andrea Grigelova, Sylvie DumasiD, Veronique Bernard, Helena JanickovaiD

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Abstract

Beta2 subunit-containing nicotinic acetylcholine receptors (beta2*nAChRs) are highly expressed in the prefrontal cortex (PFC) and critically regulate behavioral and cognitive domains that are disrupted in neuropsychiatric disorders. Despite their therapeutic potential, the cell-type- and circuit-specific functions of beta2* nAChRs remain poorly defined, partially due to the lack of selective pharmacological tools. Here, we delineate the cellular expression patterns and behavioral roles of beta2* nAChRs expressed in a general vs. highly specific neuronal populations of the mouse PFC. Using fluorescence in situ hybridization (FISH), we mapped beta2 nicotinic subunit expression across cortical layers and major neuronal classes. We then employed CRISPR-mediated knockdown (KD) to reduce beta2* nAChRs expression selectively in two distinct PFC neuronal populations and, for comparison, in a striatal population. The KD of beta2* nAChRs in a mixed population of excitatory and inhibitory neurons in deep PFC layers resulted in a mild impairment of social and exploratory behaviors. In contrast, KD in a highly selective population of superficial-layer interneurons expressing the serotonin receptor 5HT3a induced a robust hypersocial phenotype, accompanied by changes in exploratory behavior. Notably, KD of beta2* nAChRs in a rare NPY-expressing striatal population slightly affected similar behavioral domains, but in the opposite direction to PFC manipulations. Together, these findings reveal opposing, cell-type- and region-specific contributions of beta2* nAChRs to behavioral regulations, highlighting the necessity of circuit-resolved targeting strategies to improve the precision and efficacy of therapeutic intervention for neuropsychiatric disorders.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (25 Sept 2026) · CC BY 4.0

Beta2 nicotinic receptor knockdown in Htr3a-Cre mouse interneurons gave strong hypersociability; prefrontal NPY-Cre neurons, weaker effects (reduced social preference); striatal NPY neurons, slight effects mostly opposite to those.

The paper says earlier genetic and pharmacological manipulations of prefrontal beta2* nicotinic receptors mostly targeted the whole PFC or broad subregions. Here, knockdown in defined neuronal populations and in a striatal population is compared in one behavioral battery.

Key results

  1. ShownChrnb2 mRNA was detected in all major PFC neuronal classes. Its prevalence depended on cell type more than on cortical layer, and Vip+ cells in layer VI were the exception.

    Fraction of Chrnb2+ cells: nearly all Pv+, about 80% of Vglut1+, about 70% of Npy+ and Htr3a+, about 50% of Vip+. Layer effect on percentage: F(3, 188)=0.14, p=0.936; cell-type effect: F(5, 188)=19.9, p<0.0001. · Analysis of beta2* nAChRs expression in specific…; Figure legends

  2. SuggestedThe NPY-Cre::Cas9-GFP line labelled a mixed, mostly excitatory cortical population. Gene editing was detected, but the drop in Chrnb2 mRNA was only a non-significant trend.

    About 70% of GFP+ cells were Vglut1+ and 30% Viaat+ (Sst 16%, Pv 13%, Htr3a 2.1%, Vip 1.4%). The T7 assay showed indels only in sgRNA-Chrnb2 samples. RNAscope Chrnb2+ cell count: 95% CI [-61; 15], p=0.167, one control and one mutant brain. · RNAscope; CRISPR KD of the beta2 nicotinic subunit in a…; Figure legends

  3. ShownKnockdown in the mixed PFC NPY-Cre population caused modest changes: reduced social preference in the 3-chamber test, a lower Y-maze alternation score, and more but shorter object interactions. Locomotion, anxiety-like and passive-coping tests were unchanged.

    Social preference genotype x stimulus F(1, 94)=7.7, p=0.007; Y-maze alternation 95% CI [-12.2; -3.9], p=0.0003; NOR interactions genotype effect F(1, 88)=6.4, p=0.013. n(ctrl)=21-23, n(mutant)=24-27, two pooled cohorts. · KD of beta2* nAChRs in the mixed population of…; Figure legends

  4. ShownKnockdown in the sparse, superficial-layer Htr3a-Cre interneurons produced a stronger and opposite social phenotype (hypersociability), plus more object interactions in NOR. The Y-maze score was unchanged.

    Relative interaction time genotype x stimulus F(1, 58)=24, p<0.0001; time with social stimulus 95% CI [-59; -12], p=0.0018; NOR interactions genotype effect F(1, 58)=11, p=0.0013; Y-maze p=0.11. n(ctrl)=13-15, n(mutant)=17-18. · KD of beta2* nAChRs in a specific population of…; Figure legends

  5. SuggestedKnockdown in striatal NPY interneurons affected overlapping domains, mostly opposite to the PFC NPY-Cre knockdown. It slightly raised sociability, lowered object interactions in NOR, and showed an anxiolytic-like effect in the elevated plus maze, which was not seen in either PFC knockdown.

    Social preference genotype x stimulus F(1, 40)=5, p=0.0303; NOR interactions F(1, 38)=5.6, p=0.022; EPM genotype x arm F(1, 40)=8.5, p=0.0056. n(ctrl)=10-11, n(mutant)=10-12. · KD of beta2* nAChRs in the NPY-expressing…; Figure legends

Methods, data and reproducibility

Double-probe FISH mapped Chrnb2 expression across PFC layers and cell classes. Cre-dependent Cas9 mice received AAV-delivered sgRNA against Chrnb2 or a scrambled control in PFC or dorsal striatum, followed by a behavioral test battery analysed with t-tests and two-way ANOVA.

  • FISH of Chrnb2 and cell-type markers in PFC sections, bregma +2.8 to +1.6 · mouse
  • PFC NPY-Cre::Cas9-GFP behavioral cohorts (sgRNA-scr vs sgRNA-Chrnb2), two pooled cohorts, male mice · mouse · n = n(ctrl)=21-23, n(mutant)=24-27
  • PFC Htr3a-Cre::Cas9-GFP behavioral cohorts, two pooled cohorts, male mice · mouse · n = n(ctrl)=13-15, n(mutant)=17-18
  • Dorsal striatum NPY-Cre::Cas9-GFP behavioral cohort, male mice · mouse · n = n(ctrl)=10-11, n(mutant)=10-12
  • Knockdown validation: T7 assay (3 mutant, 2 scrambled, 1 non-injected) and RNAscope (1 control, 1 mutant brain) · mouse · n = 6 animals (T7); 2 brains (RNAscope)
Methods & tools
GraphPad Prism 10, QuPath, NDP.view2, AAV5 vectors, Addgene plasmid #87916, T7 endonuclease assay, RNAscope, FISH, CRISPR/Cas9 (Cre-inducible Cas9-GFP mice)
Reagents & environment
GraphPad Prism version 10 for statistics; mouse lines listed in Supplementary Table S1.
Reproducibility gaps
Data are available from the corresponding author upon request; no code or public repository is given.
Limitations (5)
  • Only male mice were used, which limits generalizability to females. · stated by the authors
  • mRNA detection does not guarantee functional receptors. The NPY-Cre line labels a mixed population from developmental promoter activity, so the knockdown there is treated as nonspecific. · stated by the authors
  • Differences in housing, baseline stress or individual social stimuli may cause variability or poor reproducibility. Results are shown as significant only when consistent across two cohorts. · stated by the authors
  • Knockdown efficiency is weakly established: RNAscope used one control and one mutant brain and showed only a non-significant trend, with no protein or functional measure of receptor loss. · noted in this summary
  • Many behavioral tests were run per cohort. Several effects have modest p-values (0.007 to 0.036) and the text does not describe correction across tests; the striatal groups are small (10-12). · noted in this summary

Read this if you study cell-type-specific cholinergic or nicotinic control of social and exploratory behavior and want a CRISPR knockdown comparison across prefrontal and striatal populations.

Citations
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