Low-level Adolescent Lead Exposure Effect on Alcohol-drinking and Punishment Resistance in Mice

Lead has been utilized for thousands of years for various uses, ranging from increasing the durability of paint to allowing gasoline to reduce exhaust valve wear and strain in cars. Although this versatility comes at a price, modern studies have shown that any level of lead exposure can have devastating effects on human health. Lead has been shown to circulate through the brain, liver, kidneys, and bones, and is stored in the teeth and bones (WHO, 2024).  Environments that may be contaminated with lead include older infrastructure, especially in impoverished areas, contaminated soils and dust particles, and contaminated water sources (Hauptman et al., 2023; WHO, 2024). Over time, lead accumulates in the teeth and bones and can serve as a point of transfer from a pregnant mother to the fetus (WHO, 2024). Children are at the biggest risk of long-term effects of lead exposure, as their central nervous systems are still developing and are at risk of permanent cognitive damage. A multitude of studies have determined that there is an association between lead exposure and negative effects on human health, including kidney, dental, and gingival diseases, and behavioral disorders like ADHD (Olufemi, Mji, & Mukhola, 2022). 

Currently, CDC uses 3.5 micrograms per deciliter (µg/dL) as a blood lead level (BLL) reference point (CDC, 2025). This value is above the 97.5th percentile of children's BLLs, so children with a BLL above 3.5 µg/dL have the top 2.5% BLL (CDC, 2025). For this proposed experiment, a lower level of lead is used to simulate the typical amount of lead that is seen in children who come from areas of high lead exposure.         

Prior research highlights the harmful effects of lead and other heavy metals on human development, with adolescent exposure linked to neurotoxic impacts, as well as behavioral and cognitive disorders (Albores-Garcia et al., 2022). Such cognitive dysfunction has also been associated with substance abuse, suggesting adolescent lead exposure may increase vulnerability to addiction (Albores-Garcia et al., 2022). Since lead exposure has been associated with behavioral and cognitive disorders, the goal of this study is to determine if lead exposure could also be linked to substance use disorders (SUD). These disorders share the fact that they primarily affect the brain, and some effects of SUDs have similar deficits of self-awareness and behavioral control as other neuropsychiatric disorders (Ramey & Regier, 2018). 

Animal Model/Husbandry 

We will be using C57BL/6J mice, which is a specific strain of mice that is utilized to eliminate genetic variability. This strain is easily trainable and known to respond for EtOH. The mice will be randomly assigned to two groups, experimental and control, with equal numbers of males and females in each group. They will be housed socially, with two mice per home cage based on their experimental condition and their sex. The mice will be housed in standard shoebox cages, with climate control and a 12h:12h reverse light:dark cycle. Once the study has concluded, the mice will be euthanized using a CO2 chamber and disposed of properly following Environmental and Occupational Health and Safety guidelines. 

Power Analysis 

In order to ensure our experiment is properly powered, an ANOVA: Repeated measures, within-between interactions power analysis was run. There were four groups included: lead-exposed females, lead-exposed males, non-lead exposed females, and non-lead exposed males. An effect size of 0.75 was used, along with 3 measurements (number of groups - 1). The analysis provided a sample size of 12 for each group, where the total sample size is 48 mice. 

Adolescent Mice Lead Exposure

For mice, adolescence is considered to be from postnatal day (PND) 21 to PND 42. This is about three weeks of development where lead exposure will occur. We are using an established lead exposure protocol outlined in identified published studies (Garcy & Boehm, 2023). After weaning on PND 21, the mice will be moved to a separate vivarium away from the breeding colony and randomly selected to be in the Pb-exposed group or the non-exposed control group. A fresh 30 ppm solution of Pb drinking solution will be made by dissolving 0.0305 g of Pb-IV acetate in 1 Liter of deionized (DI) water and 4 mL glacial acetic acid each week. The control solution will also be made fresh by adding 4 mL of glacial acetic acid to 1 Liter of DI water. Each solution will be transferred into screw-top plastic bottles and placed on the cage tops. To follow the Environmental and Occupational Health and Safety guidelines, cages with the Pb solution will be labeled as biohazardous proper disposal methods will be used. The mice will have unlimited access to their respective solution for 3 weeks, until PND 42, where they will be switched to regular tap water for the duration of the study. After this, the mice will undergo weekly cage changes and regular maintenance until they are ready for testing at PND 60.  

Ethanol Training/sucrose fading

The following ethanol training and footshock paradigm is based on the protocol described in Sneddon et al., (2022). Maturation of mice is considered to be concluded on PND 60. This is when we will begin ethanol (EtOH) response training. Both conditions will undergo EtOH response training. The mice will be restricted to 85% of their free feeding weight in order to encourage engagement in the response training. After the conclusion of training for the day, mice will be fed the appropriate amount of standard pellets, adjusted to 85% of their weight. EtOH training, as well on the footshock paradigm, will occur from approximately from 10 a.m. to 1 p.m. Monday through Friday. The team will be responsible for feeding the mice during the weekend, at the same time as training occurs during the week. 

Mice will be trained to respond for EtOH using operant conditioning. We will begin with using positive reinforcement to encourage the mice to nose poke for a pellet of food, then introduce the respective drinking solutions. Since EtOH does not have the most pleasant taste, sucrose fading is used to adjust the mice. Sucrose fading begins with a 10% sucrose solution, then moves onto a 10% sucrose and 10% EtOH solution, then 5% sucrose and 10% EtOH, and then finally, a solution containing only 10 % EtOH. All solutions will be made with reverse osmosis (RO) water and made fresh daily. EtOH responding is meant to model addiction and will serve as our baseline for next steps. 

Footshock paradigm

Once EtOH responding has stabilized within both groups of mice, they will be introduced to the footshock paradigm. This part of the experiment will quantify how long the mice will respond for EtOH despite a direct, negative consequence (footshock). The same operant conditioning chamber used for ethanol training will also be used for the footshock paradigm. The footshock session will last for 30 minutes, regardless if the mouse is responding or not. The footshock punishment will be delivered using an aversive stimulator/scrambler (ENV-414S).

The footshock punishment will begin at 0.1 mA, and increase by an interval of 0.05 mA. Each session will consist of a singular amplitude of footshock. Once the mouse is placed in the operant chamber, the session will begin. The shock will be delivered on the second nose-poke, lasting for 0.5 seconds. Access to the EtOH drinking solution will be given after the nose-poke. The amount of EtOH consumed will be recorded by the lickometer. The same procedure will be followed for each of the following amplitudes: 0.15 mA, 0.2 mA, 0.25 mA, 0.3 mA, 0.35 mA, 0.4 mA. It is expected that mice will stop responding for EtOH around 0.4 mA. They will not be forced to respond after their individual threshold has been met. After the 30-minute session concludes, mice will be removed from the operant chamber and placed in their respective home cage. The chamber will be disinfected between individual mice. 

Repeated Measures ANOVA Within-between Interaction

For the proposed experiment, a two-way repeated-measures analysis of variance (ANOVA) with a within-between interaction will be used. The purpose of this analysis is to determine if there is an interaction between the two factors on the dependent variable. In this experiment, lead exposure, the shock amplitude, and the number of nose pokes will be analyzed in relation to the amount of alcohol consumed. Additionally, sex differences will be analyzed to determine if there is a significant difference between sexes and to what degree. All data will be expressed as the mean ± standard error of the mean (SEM), and GraphPad Prism will be used to analyze data and construct graphs.  

The amplitude of the last foot shock and the number of nose-pokes will be documented and then averaged for each group, respectively. Then two ANOVA tests will be conducted with the between-subjects factor being lead exposure, either amplitude of foot shock or nose-pokes as the within-subjects factor, and alcohol consumption as the dependent variable. 

Analysis of sex differences will be similar with an ANOVA test, with shock amplitude as the within-subjects factor. Along with post hoc tests that may determine if the previous ANOVA tests contain significant sex differences. 

● Lead-exposed male and female mice were able to learn the nose poke task and responded consistently for EtOH across training and baseline sessions. 

● There was no significant difference in the amplitude of shock needed for cessation of drinking behaviors to occur across all groups.

● Pb females jumped at lower foot shock amplitudes, suggesting they are more sensitive to foot shock.

● Control females compensated for fewer responses during foot shocks with more sips, while Pb females did not. 

● Pb exposure may impact motivation to drink under punishment in female mice.

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