# Uncovering Hidden Phenotypes in NEX‐Cre Mice: Behavioral and Cellular Alterations Demand Re‐Evaluation of a Widely Used Transgenic Line

## TL;DR

This study finds unexpected behavioral and brain changes in NEX-Cre mice, suggesting these commonly used mice need re-evaluation for accurate neuroscience research.

## Contribution

The study reveals previously unnoticed behavioral and cellular changes in NEX-Cre mice using behavioral and histological analyses.

## Key findings

- NEX-Cre mice show reduced anxiety-like behavior, altered reward behavior, and increased locomotion.
- SVM analysis detected subtle genotype-specific behavioral traits in NEX-Cre mice compared to C57BL/6J controls.
- Dendritic spine density changes were observed in key brain regions like the hippocampus and prefrontal cortex.

## Abstract

Transgenic mouse strains are essential tools in neuroscience, enabling targeted genetic manipulations to investigate brain function and neurological diseases. The NEX‐Cre mouse line, which targets glutamatergic principal neurons in the neocortex and hippocampus by expressing Cre‐recombinase under the NEX (NeuroD6) promoter, has been widely used for conditional gene manipulation. Contrary to previous reports suggesting no behavioral and histological abnormalities in NEX‐Cre mice, our study reveals distinct behavioral and cellular phenotypes. Behavioral analyses indicate reduced anxiety‐like behavior, altered reward‐related behavior, and increased locomotor activity in NEX (Cre/Cre) mice. Additionally, Support Vector Machine (SVM) analysis uncovered subtle strain‐specific and genotype‐specific behavioral traits across all NEX‐Cre genotypes relative to the commonly used C57BL/6J mouse strain. While overt behavioral abnormalities were most prominent in NEX (Cre/Cre) mice, SVM‐based analysis revealed subtle genotype‐ and strain‐specific behavioral signatures across NEX‐Cre genotypes. This underlines the importance of using littermate controls rather than independently maintained or purchased C57BL/6J animals when interpreting genotype‐related effects. Histological analyses of Golgi‐Cox‐stained brain slices revealed alterations in dendritic spine density across key brain regions, including the caudate putamen, hippocampal CA1, nucleus accumbens core region, lateral septum, and medial prefrontal cortex. These findings highlight significant inter‐ and intra‐strain variability, emphasizing the importance of careful characterization of transgenic models and the need for appropriate control groups and experimental designs to ensure the reliability and validity of studies utilizing Cre‐Driver lines.

Cre‐driver mouse lines are widely used for targeted gene manipulation, yet their baseline phenotypes are often assumed to be neutral. Here, we reveal hidden behavioral and synaptic alterations in the commonly used NEX‐Cre line, including changes in locomotion, anxiety‐like behavior, reward‐related behavior, and region‐specific dendritic spine density. Machine‐learning analysis further detects subtle genotype‐dependent behavioral signatures across NEX‐Cre genotypes compared with C57BL/6J controls. These findings highlight the need to evaluate Cre‐driver baselines and to prioritize littermate‐controlled experimental designs.