Journal: bioRxiv

Article Title: Perinatal Exposure to Metal Mixtures Disrupts Neuronal Function and Behavior

doi: 10.1101/2025.09.05.673216

Figure Lengend Snippet: (A) Timeline and experimental workflow. Female C57BL/6J mice were exposed to either individual metals (Pb, As, Cd, or Cr(VI)), a combined mixture (PACC; labeled as “Mix”), or control (CTRL) through drinking water starting 2 weeks before mating. Exposure continued throughout gestation (3 weeks) and lactation until postnatal day 21 (weaning). Offspring were tested at 4–5 weeks of age using a behavioral battery including Open Field (locomotor activity), Elevated Plus Maze (EPM; anxiety-like behavior), Novel Object Recognition (NOR; short-term memory), and Y-Maze (spatial working memory). After behavioral testing, tissues were collected for electrophysiology, cardiac assessment, and histological or molecular analysis. (B) Exposure groups included unexposed controls (CTRL), individual metal exposures (Pb, As, Cd, Cr), and the PACC metal mixture group (Mix). All metals were administered at maximum contaminant levels (MCLs) for As, Cd, and Cr(VI) , or the historical U.S. EPA action level (AL – 15 ppb, as it lacks an MCL) for Pb # via drinking water . Offspring from each group were evaluated in the same behavioral and physiological pipeline. All behavioral and electrophysiological experiments were performed blind to exposure conditions. # NOTE: Since October 2024, the U.S. EPA has suggested a new “trigger level” for lead of 10 ppb as part of updated regulations .

Article Snippet: Among environmental neurotoxicants, lead (Pb), arsenic (As), cadmium (Cd), and hexavalent chromium [Cr(VI)], collectively referred to as the PACC metal mixture—rank among the top hazardous substances on the Agency for Toxic Substances and Disease Registry (ATSDR)’s Priority List, occupying the 1 st , 2 nd , 7 th , and 17 th positions, respectively .

Techniques: Labeling, Control, Battery, Activity Assay

Journal: bioRxiv

Article Title: Perinatal Exposure to Metal Mixtures Disrupts Neuronal Function and Behavior

doi: 10.1101/2025.09.05.673216

Figure Lengend Snippet: ( A–C ) Novel Object Recognition (NOR) test assessing short-term recognition memory in ( A ) male, ( B ) female, and ( C ) all mice combined. PACC metal mixture-exposed mice (labeled “MIX”) showed significantly reduced novel object preference compared to untreated controls (“CTRL”), indicating impaired short-term memory. In some comparisons, individual metals such as Pb and Cd also reduced performance, but PACC metal mixture had a more consistent and stronger effect across sex. ( D–F) Elevated Plus Maze (EPM) test measuring anxiety-like behavior in ( D ) male, ( E ) female, and ( F ) all mice combined. PACC metal mixture exposure significantly decreased open arm time, indicating heightened anxiety. While some individual metals such as Pb also reduced open arm time, the effect of PACC metal mixture was generally more pronounced. Open-arm exploration time in PACC metal mixture-treated mice was also significantly lower than individual metals, again supporting the notion of combined toxicity exceeding that of individual metals. (G–I) Y-Maze spontaneous alternation, assessing spatial working memory, in ( G ) male, ( H ) female, and ( I ) all mice combined. No significant differences were observed across groups, indicating that PACC metal mixture and individual metals did not impair spatial working memory under the tested conditions. (J–L) Open Field Test measuring general locomotor activity in ( J ) male, ( K ) female, and ( L ) all mice. Total distance traveled did not differ significantly between control, PACC metal mixture, or any of the individual metal groups, suggesting that the observed behavioral deficits in NOR and EPM were not due to changes in gross motor function or arousal. Data are presented as mean ± SEM, with each dot representing one mouse. One-way ANOVA with multiple comparisons was used for statistical analysis. Blue significance bars denote comparisons between the untreated control group and each exposure group (PACC metal mixture or individual metals). Black significance bars denote comparisons between the PACC metal mixture group and each individual metal group. Exact p -values are shown above each line. The numbers at the bottom of each bar graph denote the number of animals per group (n≥7 combined from 4 repeat experiments, ≥4males and ≥3 females/group).

Article Snippet: Among environmental neurotoxicants, lead (Pb), arsenic (As), cadmium (Cd), and hexavalent chromium [Cr(VI)], collectively referred to as the PACC metal mixture—rank among the top hazardous substances on the Agency for Toxic Substances and Disease Registry (ATSDR)’s Priority List, occupying the 1 st , 2 nd , 7 th , and 17 th positions, respectively .

Techniques: Labeling, Activity Assay, Control

Journal: bioRxiv

Article Title: Perinatal Exposure to Metal Mixtures Disrupts Neuronal Function and Behavior

doi: 10.1101/2025.09.05.673216

Figure Lengend Snippet: ( A-D ) Dorsal Hippocampus CA1, ( A ) Representative traces for dorsal CA1. ( B ) Resting membrane potential (RMP) did not differ between control (“CTRL”) and PACC metal mixture-exposed groups (“Mix”). ( C ) Input resistance (Rin) was also unchanged. ( D ) Spike counts during stepwise depolarizing current injection (–50 to +200 pA) showed a non-significant trend toward increased excitability in the PACC group. Top left: The traces show representative action potential firing patterns recorded from CA1 pyramidal neurons in CTRL (black) and PACC metal mixture-exposed (red) mice during depolarizing current steps ( E ) Representative traces for Medial Prefrontal Cortex (mPFC), ( F ) PACC metal mixture exposure caused a significant depolarization of RMP ( p =0.0174), indicating increased baseline excitability. ( G ) Input resistance remained unchanged between groups. ( H ) Spike output in mPFC was not significantly increased in PACC metal mixture-exposed neurons at multiple current steps, although a non-significant trend toward increased excitability in the PACC metal mixture group was observed. ( I ) Representative traces for Spontaneous Excitatory Postsynaptic Currents (sEPSCs) in mPFC. ( J ) PACC metal mixture exposure significantly increased sEPSC amplitude ( p =0.0043), suggesting enhanced postsynaptic response to excitatory inputs. ( K ) No significant difference in decay kinetics (tau). ( L ) Frequency of sEPSC events was not significantly altered, indicating that presynaptic release probability or event rate was unaffected. Total number of mice: 8, 4 males and 4 females/group. Data are expressed as mean ± SEM. Statistical significance determined using unpaired t-tests for single-point comparisons and two-way repeated-measures ANOVA with Sidak’s multiple comparisons test for spike-frequency analysis.

Article Snippet: Among environmental neurotoxicants, lead (Pb), arsenic (As), cadmium (Cd), and hexavalent chromium [Cr(VI)], collectively referred to as the PACC metal mixture—rank among the top hazardous substances on the Agency for Toxic Substances and Disease Registry (ATSDR)’s Priority List, occupying the 1 st , 2 nd , 7 th , and 17 th positions, respectively .

Techniques: Membrane, Control, Injection

Journal: bioRxiv

Article Title: Perinatal Exposure to Metal Mixtures Disrupts Neuronal Function and Behavior

doi: 10.1101/2025.09.05.673216

Figure Lengend Snippet: (A) Table of each Principal Component (PC), its Eigenvalue, variance explained by each individual PC, and the cumulative variance explained by including each successive PC. (B) Plot of all data (n=18 cells) for first two PCs, colored by exposure group (red is PACC metal mixture, n=10; black is untreated control, n=8). (C) Unsupervised K-means clustering into 2 groups (k=2) divides experimentally observed data into two very similar clusters as separated by exposure condition, with only a single cell misassigned (marked by dagger). (D) Plotting each cell as a function of distance from its cluster center (either C1 or C2, respectively) revealed significant separation between clusters ( p <0.05 1-way ANOVA). (E) Histogram shows distribution of all possible distances to all possible cluster centers. Experimentally observed clustering (red) is the second lowest summed distance (i.e., second tightest clustering) of all possible permutations, supporting the extreme unlikelihood that observed clustering occurred by chance.

Article Snippet: Among environmental neurotoxicants, lead (Pb), arsenic (As), cadmium (Cd), and hexavalent chromium [Cr(VI)], collectively referred to as the PACC metal mixture—rank among the top hazardous substances on the Agency for Toxic Substances and Disease Registry (ATSDR)’s Priority List, occupying the 1 st , 2 nd , 7 th , and 17 th positions, respectively .

Techniques: Control

Journal: bioRxiv

Article Title: An astrocytic AMPK clock drives circadian behaviour

doi: 10.1101/2025.08.14.670385

Figure Lengend Snippet: a, Representative immunofluorescence images showing pACCα in the SCN at ZT4 following overnight fasting. DAPI staining (left) delineates SCN structure; middle and right panels show pACCα signal, with inset highlighting the ventrolateral SCN domain. b, Western blot analysis of AMPK signalling components in hypothalamic lysates from control and α1cKO mice at ZT8. c, Locomotor activity rhythms of control and α1cKO mice under 12:12 hours LD and DD conditions (upper panels). Lomb-Scargle periodogram analysis of rhythmic period under LD and DD (lower panels) (n = 5-13). d, Representative double-plotted actograms from control and α1cKO mice under LD and DD. e, Levels of clock proteins (PER2, CRY1) and neuropeptides (VIP, AVP) in hypothalamic tissue from control and α1cKO mice at ZT8. Western blot quantification (left) (n = 3-5) and representative immunoblots (right). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, **** P < 0.0001 (unpaired two-tailed t-test or two-way ANOVA).

Article Snippet: Membranes were incubated with primary antibodies against: PER2 (1:1000, Invitrogen PA5-89045), phospho-PER2 (Ser662) (1:1000, Invitrogen PA538901); CRY1 (1:1000, Invitrogen PA5-89349), UCP1 (1:1000, Abcam ab10983), phospho-AMPK (1:1000, Cell Signaling #2535S), total AMPK (1:1000, Cell Signaling #2532), AMPKγ1 (1:1000, Thermo Fisher PA5-27471), VIP (1:500, Invitrogen PA5-78224), AVP (1:1000, Santa Cruz Biotechnology sc-390723), phospho-ACC (pACC) (1:1000, Cell Signaling #3661S), and total ACC (1:1000, Cell Signaling #3662S).

Techniques: Immunofluorescence, Staining, Western Blot, Control, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: An astrocytic AMPK clock drives circadian behaviour

doi: 10.1101/2025.08.14.670385

Figure Lengend Snippet: a, Heatmap of rhythmic hypothalamic phosphoproteins after 24-h fasting (left) or ad libitum feeding (right) across four circadian time points (n = 3-4 per group). b, Polar plot showing phase distribution of the 347 rhythmic phosphoproteins detected under fasting, highlighting “rush hours” of phosphorylation at ZT0 and ZT13. c, Kinase-substrate enrichment analysis showing the top predicted upstream kinases for daytime (yellow) and night-time (grey) phosphorylation profiles; red text indicates AMPK subunits. d, KEGG pathway enrichment analysis of differentially phosphorylated proteins at ZT0 (daytime, yellow) and ZT13 (night-time, grey). Bubble size represents -log₁₀ (p value), position indicates fold enrichment, and colour denotes peak phase. e, Quantification (left) and representative immunoblots (right) of pAMPK and pACCα in hypothalamic extracts from wild-type and IP₃R2KO mice under ad libitum feeding at ZT0 and ZT12 (n = 4-6 per group). f, Schematic model of night-time Ca²⁺-dependent AMPK activation in astrocytes. During the night, IP₃R2-mediated Ca²⁺ release activates CaMKK2, leading to AMPK phosphorylation. During the day, reduced Ca²⁺ flux limits AMPK activation. Data are mean ± s.e.m. * P < 0.05, **** P < 0.0001 (unpaired two-tailed t-test).

Article Snippet: Membranes were incubated with primary antibodies against: PER2 (1:1000, Invitrogen PA5-89045), phospho-PER2 (Ser662) (1:1000, Invitrogen PA538901); CRY1 (1:1000, Invitrogen PA5-89349), UCP1 (1:1000, Abcam ab10983), phospho-AMPK (1:1000, Cell Signaling #2535S), total AMPK (1:1000, Cell Signaling #2532), AMPKγ1 (1:1000, Thermo Fisher PA5-27471), VIP (1:500, Invitrogen PA5-78224), AVP (1:1000, Santa Cruz Biotechnology sc-390723), phospho-ACC (pACC) (1:1000, Cell Signaling #3661S), and total ACC (1:1000, Cell Signaling #3662S).

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Two Tailed Test