Understanding how rodents  navigate complex environments and interpret social cues is central to the study of cognitive neuroscience. Traditional behavioral assays, such as the Operant chamber and 3-chamber Sociability test often examine spatial or social cognition in isolation, limiting insights into how these processes interact in real-world contexts. To address this gap, the Vertical Maze (VM)—a novel, three-dimensional behavioral apparatus was designed to simultaneously assess spatial and social decision-making in rodents.

The VM expands on conventional three-chambered social mazes by adding a vertical dimension, allowing researchers to present social stimuli (i.e., conspecifics) at varying heights and distances. This setup mimics the layered spatial environments rodents naturally encounter and engages multiple sensory modalities including olfaction, vision, and audition. Through both spontaneous exploration and operant conditioning paradigms, the VM enables precise measurements of how animals integrate spatial layout and social information to guide behavior.

By combining multi-level spatial challenges with dynamic social contexts, the VM offers a powerful platform for investigating the neural and behavioral mechanisms underlying complex cognition. It holds particular promise for studying conditions where the integration of spatial and social cues may be disrupted, such as in autism spectrum disorders, schizophrenia, or age-related cognitive decline.

1. Subjects

• Use both male and female rats or mice

• Divide the subjects into two groups:

  • Experimental subjects (explore the maze).

  • Stimulus subjects (placed in lower chambers as social cues).

2. Apparatus: Vertical Maze (VM) Design

• Each column has a compartment at the base that can hold a conspecific subject (stimulus animal).

• The top arena allows free movement and visual access to lower compartments.

• Include cameras above the arena for behavior tracking.

3. Habituation

• Acclimate experimental subjects with handling and in the VM environment over 2–3 sessions.

• No stimulus subjects are present during this phase.

Behavioral Paradigms:

Two main types of tasks were examined using the 3D Vertical Maze:

1. Spontaneous Exploration Tests:

   • Rats were introduced to the VM and allowed to explore freely.

   • Researchers recorded the time spent by rats near different social stimuli (e.g., familiar vs. novel rats; close vs. distant columns).

   • These tests assessed innate preferences based on social novelty and spatial distance

2. Operant Conditioning Tasks:

• • Rats were trained to respond to the presence or location of specific social cues to receive a reward.

  • These tasks evaluated how well rats could learn and recall spatial-social associations.

The 3D Vertical Maze: A new model system for studying the interactions between social and spatial cognition

The 3D Vertical Maze (VM) was utilized to investigate how rats integrate spatial and social information. Adult Long-Evans rats were used as both experimental subjects and social stimuli.

Two types of behavioral assessments were conducted: spontaneous exploration and operant conditioning. In spontaneous exploration trials, experimental rats were allowed to move freely across the upper maze while conspecifics of varying familiarity (familiar vs. unfamiliar) were positioned in different vertical columns. The objective was to measure time spent in proximity to each social stimulus to assess preferences based on both social novelty and spatial distance. In operant conditioning tasks (when used), rats were trained to associate specific zones or stimuli with rewards, testing their ability to learn and apply spatial-social associations. All behavior was recorded via overhead cameras and analyzed using tracking software to quantify metrics such as time spent near stimulus zones and exploration patterns. Data were statistically analyzed to determine the significance of observed behavioral preferences and learning effects.

Key Findings

• Rats Integrate Social and Spatial Information

  • Rats were able to discriminate between conspecifics (stimulus rats) based on both social identity (familiar vs. unfamiliar) and spatial positioning (vertical distance).

  • This indicates that rats naturally combine social and spatial cues during exploration.

• Spontaneous Social Preference is Spatially Sensitive

  • Rats preferred unfamiliar conspecifics, consistent with known social novelty-seeking behavior.

  • Importantly, this preference varied depending on the vertical location of the conspecifics, showing that spatial distance modulates social interest.

• Use of Multisensory Cues

  • The rats utilized visual, olfactory, and auditory cues to detect and assess the social stimuli, even when there was no direct access.

  • This supports the validity of the maze for studying naturalistic multisensory processing.

• Three-Dimensional Spatial Processing

  • Rats were capable of navigating and making decisions in a vertically structured environment, demonstrating sophisticated spatial cognition beyond two-dimensional settings.

• Potential for Operant Learning 

  • In some conditions, rats were shown to learn associations between specific spatial-social configurations and outcomes (e.g., rewards), indicating that the maze can support conditioning paradigms.

1. Wise TB, Templer VL, Burwell RD. The 3D Vertical Maze: A new model system for studying the interactions between social and spatial cognition. J Neurosci Methods. 2025 Jun;418:110414. doi: 10.1016/j.jneumeth.2025.110414. Epub 2025 Feb 28. PMID: 40024460.