Influence of Developmental Lead Exposure on Impulsive Behaviors in Mice Using a Fixed-Ratio Waiting for Reward (FR wait) Paradigm

Lead has been found to be associated with impulsive behavior in children and adolescents (Reyes 2015).
The goal of this project was to investigate whether early-life lead exposure impacts decision-making and impulsivity in mice.
A fixed-ratio waiting-for-reward (FR wait) paradigm was used, modeled off of protocols used by Brockel & Cory-Slechta (1998) who experimented with rats.
We hypothesized that exposure during early life can impair thinking by increasing impulsivity. This would be shown by lead-exposed mice having an increase in earned rewards, with a decrease in free rewards.

Methods

Subjects: C57BL/6J male and female mice were generated from breeding pairs purchased from The Jackson Laboratory, Bar Harbor, ME.

Lead Exposure: The experimental group of female and male mice were exposed to 30 ppm of lead acetate (dissolved by acetic acid) in their drinking water from postnatal day (PND) 21 to 42. The control group was given acetic acid in their drinking water.

Behavioral Testing: Mice were trained in operant conditioning chambers equipped with a nose-poke hole. Once mice reached adulthood on postnatal day 60 (PND 60), they were placed on food restriction for 7-10 days to enhance motivation for the reward pellets. Following this period of restriction, training began. Mice completed approximately 30 total sessions, conducted five days per week, with each session lasting around 20 minutes. During early training, mice learned to nose-poke to earn a reward. The number of required responses to earn one reward gradually increased until mice reached the fixed ratio response requirement of 25 (FR25). Once the FR25 is achieved, a waiting period was implemented where mice could earn a ‘free’ pellet by not responding for a specific set time (7s, 10s, 13s, etc.). If they responded too early, the task restarted. Both control and lead-exposed groups followed the same procedure to compare impulsive behavior and waiting ability.

Once fully trained, the mice were tested on FR25 consistently with increasing wait times (16, 22, 28, 34, 40, 46). This was done to determine at what wait time the mice would switch from waiting for the free pellets to nose poking to earn their rewards more efficiently.

Statistical Plan: For the training results, a three-way ANOVA with wait time, treatment, and nose poke type as the independent variables and number of nose pokes as the dependent variable was done fot both females and males. For the free and earned average rewards, a 3-way ANOVA was conducted with sex, treatment and wait time as the indpendent variables and number of rewards as the dependent variable. For the ratio of free/earned rewards, a 2-way ANOVA was conducted for each wait time with sex and treatment as the independent varaiblles and the ratio of free/earned rewards as the dependent variable. All analyses were done in GraphPad Prism 11.0.0.

Results/Findings

Training Results

A graph representing the number of active vs. inactive nose pokes by female control and lead mice during the training phase (FR1, FR5, and FR10). There was no significant treatment difference, with both groups increasing in active and decreasing in inactive nose pokes as training progressed.

Figure 1A. Female

A graph showing the number of active vs. inactive nose pokes in both control and lead-treated male mice during the training period (FR1, FR5, and FR10). There was no significant difference between treatments, with both groups increasing in active nose pokes and decreasing in inactive nose pokes as training progressed.

Figure 1B. Male

Figure 1A-B. The training phase of FR 1, FR 5, and FR 10, illustrating how the mice learned the paradigm because they successfully responded more on the active side. This was analyzed using a three-way ANOVA, with ** = p-value <0.0001

Free and Earned Average Rewards

A graph showing the average free rewards from wait times 16 to 46. The left side shows female mice and the right side shows male mice. Each wait time for each sex has a bar on the left representing the acetic acid group and a bar on the right representing the lead-treated group. Over time, all mice decreased their average free rewards. There was a significant treatment difference in females at wait time 40, with the lead-treated mice earning more free rewards compared to the acetic acid controls.

Figure 3A. Free rewards

A graph showing the average earned rewards for all mice from wait times 16 through 46. The female mice are represented on the left half of the graph while the males are on the right half. Each wait time for each sex has two bars, with the bar on the left representing the acetic acid controls and the right bar representing the lead-treated mice. All groups increased their earned rewards over time. In females, treatment was significantly different at wait time 28, with the controls earning more compared to the lead-treated mice. In males, there was a treatment difference at wait time 16, with lead-treated mice earning more compared to controls.

Figure 3B. Earned rewards

Figure 3A-B. The average amount of rewards given to subjects per fixed ratio wait time and the type of sucrose reward received. Treatment is also shown along with gender. (A) depicts the average free rewards dispensed in every session, (B) shows the average earned rewards dispensed per session. Both were analyzed using a three-way ANOVA, * = p < 0.05, ** = p <0.01

Average Ratio of Free/Earned Rewards

This is a picture of six graphs showing the average ratio of free-to-earned rewards. Each graph represents a wait time from wait time 16 to wait time 46, with the wait times being shown top-to-bottom, left-to-right. As the wait time increased, the ratio of free/earned rewards decreased, as each subsequent graph has a smaller y-axis scale. The female mice were on the left half of each graph and the males were on the right. The left bar for each sex represents the acetic acid controls, while the right bar represents the lead-treated mice. Treatment was significantly different at wait time 40 in females, where the lead-treated mice had a significantly higher ratio compared to controls.

Figure 4. The average ratio of free/earned rewards at wait times 16 through 48, calculated by dividing the average free rewards by the average earned rewards for each mouse. Graphs are split by sex on the x-axis, while blue bars represent control mice treated with acetic acid and orange bars represent lead treated mice Each wait time was analyzed using a two-way ANOVA.* = p < 0.05, ** = p < 0.01

Conclusions

Mice can learn how to nose poke up to FR 10 regardless of exposure to lead.
For females at wait time 40, lead treatment increased the amount of free rewards, not supporting our hypothesis. Lead treatment did have an interaction with wait time, causing lead-treated mice to earn less free rewards compared to controls at lower wait times but more at larger wait times.
Lead treatment decreased the amount of earned rewards at wait time 28 in females, not supporting our hypothesis, but supported it at wait time 16 in males by increasing the number of earned rewards. There was also an interaction between wait time and lead treatment which overall resulted in lead-treated mice earning a greater number of rewards compared to controls at larger wait times compared to shorter wait times.
Lead treatment was significant at wait time 40, where it increased the ratio of free/earned rewards, going against our hypothesis.
Overall, our hypothesis was not supported. While lead treatment does seem to lead to an increase in earned rewards, it also leads to an increase in free rewards, especially at longer wait times. This indicates that lead may cause mice to be more responsive or focused on the paradigm at higher wait times compared to controls.

The following is an image of our poster, presented at the 2026 Undergraduate Research Forum.

Acknowledgements

Research supported by the National Institute on Drug Abuse and the Office of Research for Undergraduates at Miami University.

References

NACE Career Readiness Competencies

Critical Thinking

The research team improved their critical thinking through this experience by the problems they had to solve as we ran their paradigm. As it was a new paradigm that had never been conducted by the lab before, they ran into several problems particularly at the beginning of the experiment with getting equipment to work properly. They had to work together amongst themselves and with other members of the lab to find creative solutions to get everything to work together properly.

Teamwork

There was a lot of opportunity for teamwork skills to be improved during this project, as the research was conducted in equal part by the four undergraduate researchers. They had to work together in every step of the project, from planning out what they wanted to do for their experiment, to creating and adhering to a schedule for training and testing to mice, to finally creating their research poster and presenting at the URF. They learned to listen to everyone's ideas to choose a project everyone was interested in, to follow the schedule they created together and to find a solution when conflicts arose.

Technology

While they were testing their mice and analyzing their data, the undergraduate researchers had to learn to use new technology and software to collect data and to analyze it. They had to adapt to using the new technology quickly and had to successfully utilize it to achieve their final goal of presenting at the URF.

Research Compliance Protocols

The experiment conducted for this study was reviewed and approved by the Miami University Institutional Animal Care and Use Committee (IACUC).

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