Perinatal Exposure to Metal Mixtures Disrupts Neuronal Function and Behavior
Naveen ChandraiD, Benyamin KarimiiD, Ankita BhobeiD, Susmitha R. Pagadala, Shekhar PandeyiD, Sylvia S. SancheziD, Bonnie H. Yeung-LukiD, Emily Rivera, Mark Jeffrey KohriD, L. Elizabeth RiessiD, Keeve Edward NachmaniD, Shyam S. BiswaliD, Fenna C.M. SilléiD, Austin R. GravesiD
Abstract
BackgroundEnvironmental exposure to heavy metals such as lead (Pb), arsenic (As), hexavalent chromium [Cr(VI)], and cadmium (Cd), (PACC), is linked to neurodevelopmental disorders. These metals often co-occur in contaminated environments, but their combined effects on brain development remain poorly understood. ObjectiveTo test the hypothesis that perinatal exposure to a mixture of environmentally relevant levels of Pb, As, Cd, and Cr(VI), causes developmental defects in cognition, behavior, and neuronal function. MethodsFemale C57BL/6J mice were exposed to either a single metal or the PACC mixture in drinking water. Exposure began two weeks preconception and continued until weaning at postnatal day 21. Juvenile mice were tested at 4-5 weeks of age in open field (locomotion), novel object recognition (short-term memory), Y-maze (working memory), and elevated plus maze (anxiety-like behavior). A subset of animals underwent Whole-cell patch-clamp recordings in the medial prefrontal cortex (mPFC) and hippocampal CA1 neurons. ResultsPerinatal exposure to PACC metal mixture increased anxiety-like behavior and impaired short-term memory but not locomotion or working memory. Pyramidal neurons in mPFC and hippocampal CA1 displayed increased intrinsic excitability, mPFC neurons also showed elevated amplitude in spontaneous excitatory postsynaptic currents. DiscussionOur findings suggest that perinatal exposure to the PACC metal mixture impairs cognition, increases anxiety-like behavior, and alters neuronal function in specific brain regions of juvenile mice, leading to disruption in neuronal function and behavior later in life. Further studies are needed to provide mechanistic insight into how perinatal heavy metal exposure affects neuronal development. HighlightsO_LIPerinatal exposure to a metal mixture including lead (Pb), arsenic (As), hexavalent chromium (Cr(VI)), and cadmium (Cd), collectively termed PACC metal mixture--impairs cognition and increases anxiety in mice. C_LIO_LINeuronal excitability and synaptic transmission are altered in medial prefrontal cortex after PACC metal mixture exposure. C_LIO_LIPACC mixture exposure decreases short-term memory in both males and females, and increases anxiety in males C_LIO_LIPrincipal component and clustering analyses reveal that PACC mixture exposure and control mice form distinct, nonoverlapping populations in physiological-behavioral space. C_LIO_LIEnvironmentally relevant PACC metal mixtures exert stronger effects than individual metals alone. C_LI
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (11 Sept 2025) · CC BY 4.0
Perinatal exposure of mice to a Pb, As, Cd and Cr(VI) mixture at EPA levels impaired recognition memory, raised anxiety-like behavior, and altered mPFC neuron function.
Heavy metals co-occur at contaminated sites, yet most toxicology work tested Pb, As, Cd or Cr(VI) alone. This study exposes mice to all four at EPA-defined levels and pairs behavior with patch-clamp recordings in mPFC and CA1.
Key results
ShownThe metal mixture reduced novel-object preference (short-term recognition memory) in both sexes. Some single metals (As, Cd, Cr) also reduced it, but only in particular subsets.
Mix vs CTRL: males p=0.0099, females p<0.0001, pooled sexes p<0.0001. Pooled As p=0.0264 and Cd p=0.007 vs pooled control; female As p=0.0476, Cr p=0.0311. n≥7 per group. · PACC Metal Mixture Exposure Impaired Recognition…; Animal Care and Metals Exposure Models
ShownThe mixture lowered open-arm time on the elevated plus maze in females and in pooled sexes, indicating more anxiety-like behavior. Males showed only a non-significant trend. Pb (females) and As (pooled) also lowered open-arm time.
Mix: females p=0.007, pooled p=0.0005. Pb females p=0.0015; As pooled p=0.007. Male mixture effect not significant in the Results text. · Anxiety-Like Behavior is Increased in Offspring…; Figure legends
SuggestedIn mPFC pyramidal neurons the mixture depolarized resting potential and enlarged sEPSC amplitude, with no change in sEPSC frequency or decay. CA1 neurons showed no significant change, and spike output was not significantly raised at current steps.
mPFC resting membrane potential p=0.0174; sEPSC amplitude p=0.0043; input resistance, frequency and tau unchanged. CA1 resting potential, input resistance, spike counts n.s. At least 8 mice per group, 4 males and 4 females. · Perinatal PACC Metal Mixture Exposure Enhanced…; Figure legends; Electrophysiology
ShownLocomotion (open field) and spatial working memory (Y-maze) were not significantly changed by the mixture or any single metal. Normalized heart weight-to-tibia length also showed no mixture effect, arguing against gross motor or cardiac confounds.
No significant group differences in total distance traveled or spontaneous alternation across six groups. Heart ratio unchanged for Mix vs CTRL; female Cr hearts larger than female Mix, neither differing from control. · Perinatal Metal Exposure Does Not Impair…; Perinatal Metal Exposure Does Not Alter…; Figure legends; Maternal and Offspring Health Metrics Were…
SuggestedA PCA of behavior plus mPFC physiology separated mixture-exposed from control mice. K-means (k=2) reproduced the grouping with one error, and a permutation test supported it.
PC1 and PC2 explained 64% of variance. K-means misclassified a single cell. Observed clustering had the second smallest summed centroid distance among 43,758 possible 10-vs-8 groupings; n=18 (10 PACC, 8 control). · Statistical Analysis; PCA and Clustering Reveal Distinct…; Figure legends
Methods, data and reproducibility
Female C57BL/6J mice received Pb, As, Cd and Cr(VI) singly or combined in drinking water from two weeks preconception to weaning. Juvenile offspring then underwent open field, NOR, Y-maze and EPM tests, followed by slice patch-clamp, with PCA and k-means integration.
- F1 offspring behavior across six groups (Control, Pb, As, Cd, Cr(VI), PACC mixture); doses As 10 ppb, Cd 5 ppb, Cr(VI) 100 ppb, Pb 15 ppb · Mouse (C57BL/6J) · n = n≥7 per group, ≥4 males and ≥3 females, from 4 repeat experiments
- Breeding pairs set up across four waves; pairs that became pregnant: 5 control, 9 mixture, 2 Pb, 3 As, 8 Cd, 5 Cr(VI) · Mouse (C57BL/6J) · n = 120 breeding pairs (20 pairs per group)
- Whole-cell patch-clamp of mPFC and dorsal CA1 pyramidal neurons, control vs PACC mixture only · Mouse (C57BL/6J) · n = At least 8 mice per group (4 males, 4 females)
- Multivariate dataset: NOR, EPM and mPFC electrophysiology features for PCA and k-means · Mouse (C57BL/6J) · n = n=18 (10 PACC, 8 control)
- Methods & tools
- Novel object recognition, Open field test, Y-maze spontaneous alternation, Elevated plus maze, Whole-cell patch-clamp, ANY-maze, pClamp / Clampfit 11.2, GraphPad Prism 10, Mixed-effects models with Tukey or Sidak post-hoc tests, PCA and K-means clustering (k=2), Grubbs' test, Hertzberg four-step dose additivity approach
- Reagents & environment
- GraphPad Prism 10 for statistics; Clampfit 11.2 for electrophysiology; ANY-maze for behavior tracking.
- Reproducibility gaps
- No code or data repository URL is given. Detailed procedures, solution tables and additivity analyses are in supplementary materials.
Limitations (5)
- Each metal was tested at a single dose, so interaction (additive, synergistic or antagonistic) cannot be formally assessed; the abbreviated additivity analysis suggested less than additive effects or antagonism. · stated by the authors
- Cardiac size was the only cardiac measure; more sensitive functional assays (echocardiography, ECG, serum biomarkers) would be needed to exclude subtle cardiac effects. · stated by the authors
- The authors leave open whether the heightened excitatory drive is a downstream reaction to earlier damage or an initial maladaptive change that drives pathology; molecular mechanisms were not tested. · stated by the authors
- Electrophysiology covered only control and mixture groups, so the link between mPFC changes and behavior is correlational. The text calls intrinsic excitability increased, yet spike counts were not significant at the steps tested. · noted in this summary
- Pregnancy rates were low in some groups (2 of 20 pairs for Pb, 3 for As). Mice are plotted as individual units, and litter clustering is not described, so single-metal comparisons may be underpowered or confounded by litter. · noted in this summary
Who should read:Read this if you study developmental neurotoxicology of environmental metal mixtures and want behavior paired with mPFC and CA1 patch-clamp data.
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