Neurochemistry International

Background: ADHD is characterized by inattention and impulsivity. Stimulants such as amphetamine are the first line treatments for ADHD. Amphetamine (AMPH) works by increasing extracellular levels of dopamine and noradrenaline. Such stimulants are used to test predictive validity of animal tasks, where it has been reported that amphetamine improves rat 5-CSRTT accuracy when the session length is increased to 250 trials. This improvement in accuracy was observed later in the session as control performance declined. Proving that amphetamine can improve 5C-CPT task performance adds to the predictive validity of the task. No previous works has demonstrated the pro-attentional effects of amphetamine on sustained attention in rat 5C-CPT. We have previously shown that pharmacological manipulations of dopaminergic system improve waiting impulsivity in a rate dependent manner and the importance of analysing the data using Oldham method. The present study extends this work to investigate the effect of amphetamine on attentional processes. Objective: to investigate effects of amphetamine on attentional processes as measured by 5C-CPT task using correlational analysis and group performance. Methods: Trained female Lister hooded rats were tested with AMPH (0.1, 0.2 and 0.4 mg/kg) under reduced stimulus duration schedule (0.25,0.5,1,1.5 and 2s SD) over normal length session (120 trials). Correlational analysis (Oldham method) was used to understand the relationship between baseline performance measures and the change following administration of AMPH. Results: The highest dose of AMPH impaired attentional performance in the 5C-CPT under reduced SD challenge (0.5s,1s,1.5s and 2s). This impairment was due to increase in FAR. Previous studies in 5-CSRTT suggest that reducing the SD increases attentional demand in the form of reducing accuracy without affecting waiting impulsivity. However, the 5-CSRTT does not include non-target trials, therefore, it cannot be used to measure sustained attention (SI) and responsivity index. The 5C-CPT requires greater attentional resources than the 5-CSRTT.This study is the first to report that reducing the SD changes responsivity index (bias) rather than measures of sustained attention (sensitivity index) in the 5C-CPT. Control sensitivity index did not change until the SD was reduced to less than 0.5s and this change was due to a change in baseline responsivity index (bias). Performance under each reduced SD revealed that the highest dose of AMPH (0.4 mg/kg) showed a tendency toward reducing sensitivity index at 0.5, 1.0 and 1.5s SDs. Under 1.0s SD, the highest dose of AMPH changed sustained attention (SI, HR and FAR) and omission in a rate-dependent manner without changing responsivity index. Performance across the session revealed that the impairment in sustained attention was observed at the beginning of the session. In the third trial block, Oldham method revealed an inverse relationship between baseline accuracy and the change following administration of 0.2 mg/kg AMPH, indicative of a rate-dependent effect. Conclusions: This study successfully demonstrates rate-dependent effects of AMPH on measures of attentional performance in the 5C-CPT. This experiment also demonstrates that responsivity index is sensitive to reduced SD challenge. To our knowledge this is the first study to report the presence of a direct relationship between baseline sustained attention and magnitude of improvement following administration of 0.4 mg/kg AMPH and an inverse relationship between baseline accuracy and the change following administration of 0.2 mg/kg AMPH in 5C-CPT task. Future studies should test lower doses of amphetamine (0.05 and 0.1 mg/kg) over extended session length (250 trials) to reveal the pro-attentional effects of this drug on 5C-CPT performance measures.

Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE), which leads to developmental defects during embryogenesis. Angiogenesis is one facet of development that is heavily dysregulated during PAE and where such impairment is poorly understood. Here we found that expression levels of focal adhesion molecules Tensin 1 (Tns1) and Tensin 2 (Tns2) were significantly reduced by alcohol exposure in brain microvascular endothelial cells (BMVECs). We also found that miR-150-5p, which we previously found was increased by PAE, regulates the expression of Tns1 and Tns2. Dual-luciferase assays demonstrated direct binding of miR-150-5p to the 3UTRs of Tns1 and Tns2. Additionally, we utilized sprouting angiogenesis bead assays to evaluate endothelial cell sprouting in response to ethanol (EtOH) treatment and following Tns1 and/or Tns2 overexpression as well as miR-150-5p modulation. Tns1 and Tns2 rescued both EtOH- and miR-150-5p-mediated deficits in sprouting angiogenesis. Overall, we found that miR-150-5p negatively regulates Tns1 and Tns2 in BMVECs and that augmenting Tns1 and Tns2 expression may reverse the negative effects of EtOH, and subsequent miR-150-5p elevation, thus improving endothelial cell sprouting during cerebrovascular development.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Alcohol use disorder is associated with persistent neuroadaptations and cognitive impairments, particularly during withdrawal, yet the temporal dynamics and interactions between these processes remain poorly understood. In the present study, we investigated time-dependent alterations in the oxytocin receptor (OTR), -opioid receptor (MOPr), metabotropic glutamate receptor 5 (mGlu5R), and translocator protein (TSPO) following chronic ethanol exposure and withdrawal, and their relationship to behavioral impairments. Male C57BL/6J mice were exposed to a 10-day escalating-dose ethanol liquid diet paradigm followed by 1, 4, or 7 days of withdrawal. Motor coordination and recognition memory were assessed using rotarod and novel object recognition tests, and receptor binding was quantified using autoradiography across multiple brain regions. Chronic ethanol exposure induced motor deficits that persisted during early withdrawal, whereas recognition memory impairments emerged after 7 days of withdrawal. Ethanol exposure and withdrawal produced region- and time-dependent alterations across all systems examined, with increased OTR binding in septal regions and transient reductions in basomedial amygdala OTR binding during intermediate withdrawal that normalized following prolonged withdrawal, transient increases in MOPr binding in striatal regions at 4 days of withdrawal, increased mGlu5R binding in the nucleus accumbens core, basolateral amygdala, and hippocampus, and early transient increases in TSPO binding indicative of neuroimmune activation. Notably, mGlu5R binding in the caudate-putamen and amygdala was associated with recognition memory deficits in a withdrawal stage-dependent manner. Bioinformatic analysis of human postmortem hippocampal transcriptomic datasets from individuals with alcohol use disorder revealed no consistent changes in GRM5 expression, suggesting that mGlu5R dysregulation may be driven by post-transcriptional mechanisms. These findings demonstrate that ethanol withdrawal is characterized by temporally distinct neuroadaptations across multiple systems and identify mGlu5R as a key correlate of withdrawal-associated memory impairment, highlighting its potential as a translational therapeutic target.

Cannabis is one of the most widely consumed substances in the world. Consumers seek out cannabis cultivars with varying levels of phytocannabinoids, primarily delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). The effects of THC, CBD or the combination of THC:CBD have distinct outcomes on cognitive processes, cellular functions, and phytocannabinoid pharmacokinetics. The majority of research on cannabis effects on the brain has focussed on neurons, and few studies have investigated the impact of different cannabis cultivars on glia. In particular, the impact of varying levels of THC:CBD on oligodendrocyte lineage cells, which play numerous support roles in the brain essential to proper neuronal communication, is relatively unknown. This study set out to examine the acute impact of different cultivars of vaporized cannabis on oligodendrocyte lineage cells in the forceps minor of adult male and female mice. Mice were exposed to vapor from cannabis flower high in THC, high in CBD or balanced in THC:CBD over 15 minutes, and brains were fixed 30 minutes post-cannabis onset. Using immunofluorescence microscopy, we observed significant changes to oligodendrocyte progenitor cell (OPC) morphology in mice exposed to balanced cannabis, and, using correlative light and electron microscopy, we observed alterations to OPC mitochondria. The alterations observed (i.e., enlarged soma and nucleus volume, reduced density and increased area of mitochondria in the soma) in OPCs due to balanced cannabis are reminiscent of the very early changes seen during OPC differentiation. This study highlights the differing effects of cannabis cultivars on OPCs and the rapidity of the OPC response to inhaled phytocannabinoids.

Individuals carrying a G4C2 repeat expansion in C9orf72 can develop amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), at various and unpredictable ages. The contribution of the expansion to this extreme clinical variability remained a matter of debate. In this work, we validated the use of optical genome mapping to efficiently size this expansion from blood and applied this method to a cohort of 54 repeat expansion carriers including patients with FTD (n = 23), ALS (n = 22) and presymptomatic individuals (n = 9). Despite the high variability in expansion profiles, distinct groups can be inferred. Although consensus repeat profiles were similar between FTD and ALS patients, relationships between repeat number, indexes of repeat instability, and the age at sampling differed between clinical conditions. An age at sampling-dependent increase of repeat instability in blood was observed in presymptomatic carriers and FTD patients (p = 0.01 and p = 0.04 respectively), in contrast to patients with ALS (p = 0.9). The proportion of shorter repeats ([500-1500 repeats]) increased with the age at sampling in ALS patients but decreased in presymptomatic and FTD cohorts, while the proportion of larger repeats (>2000 G4C2), in particular those exceeding 4000 repeats, increased along with the age at sampling in presymptomatic carriers and FTD cases (p <1.10-e4 and p = 0.006 respectively) conversely to patients with ALS (p = 0.98). In conclusion, this work strongly supports different dynamics of repeat instability of the C9orf72 pathogenic expansion in blood linked to the clinical evolution. These findings provide new insights into the relationship between expansion profiles and clinical variability.

Nerve Growth Factor (NGF) supports neuronal survival, plasticity, and growth by binding to p75NTR and TrkA receptors. There is a strong link between NGF and the cholinergic hippocampus, and changes in NGF pathways are observed in Alzheimer's disease (AD). Research shows NGF's neuroprotective and regenerative effects on cholinergic neurons, with positive results in AD models and clinical studies. However, NGF therapy faces challenges: limited blood-brain barrier crossing and dose-dependent pain. To address pain, a mutated NGF called CHF6467 was developed. Thanks to the R100E mutation, it prevents binding to p75NTR while maintaining TrkA affinity, which helps reduce pain side effects. Understanding CHF6467's mechanism remains a challenge. In this context, untargeted metabolomics is effective for uncovering unforeseen responses, making it suitable for assessing CHF6467's overall effects. Building on our previous research and recognizing the importance of amyloid-{beta} peptide as a critical pathological marker of AD, we used NMR-metabolomics to study the effect of CHF6467 on SH-SY5Y cells. The study examined metabolomic profiles of cells treated with CHF6467, with and without A{beta}(1-42), using NMR. Results show CHF6467 influences the metabolic changes caused by A{beta}(1-42), inducing biochemical effects consistent with the promotion of phospholipid precursors, reduced glutamate excitotoxicity, and restored energy metabolism.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Background: Determining the cause of death in electrocution is often challenging at autopsy, particularly when characteristic external electrical marks are absent. Reliable internal markers are scarce, and the neuropathological changes associated with electrocution remain underexplored. Objective: To identify and characterize histopathological alterations in the brain in cases of fatal electrocution and evaluate their diagnostic utility. Methods: A prospective observational study was conducted on 12 autopsy cases, including 7 confirmed electrocution deaths and 5 non-electrocution deaths (control). Brain tissue samples from multiple regions were processed using H&E and Golgi Cox staining. Histopathological parameters, including neuronal morphology, vascular changes, haemorrhage, and dendritic architecture, were evaluated. Results: Electrocution cases demonstrated neuronal destruction, axonal ballooning, focal hemorrhages, and dendritic disruption. Cerebellar involvement with Purkinje cell damage. Conclusion: Comprehensive neurohistological technique may helpful in evaluation neuronal damage in electrocution death. Neuropathological findings, particularly neuronal and dendritic injury, may serve as supportive evidence in determining cause of death when external signs are absent. Keywords: Electrocution, Neuropathology, Golgi Cox stain, Forensic histopathology, death.

BACKGROUND: Cognitive resilience to Alzheimer's disease (AD) can be modelled in amyloid-expressing rodents through early handling (EH). This study investigated how EH and sex affect cognition and pathology over time. METHODS: We assessed cognition (Open field, Novel object recognition test, Barnes maze) in 6- and 12-month-old APP/PS1 and WT mice (N=180, EH/Ctrl X M/F), performed biochemical and histological analyses of several brain areas to assess effects on amyloid-beta, glial cells, and other markers of pathology, and investigated how behavior correlates with pathology. RESULTS: EH improved non-spatial memory at 6 months, consistent with a reduction of hippocampal microglia density, and normalized locomotor activity at 12 months (both p<0.05). Females demonstrated similar (6 & 12 months) or better (12 months) cognition, despite greater pathology across timepoints. Correlations implicated microglial recruitment in EH-related effects, supported by morphological analysis of prefrontal regions (p<0.05). CONCLUSION: Our findings highlight the critical role of early postnatal experiences and sex in shaping resilience in the context of AD pathology.

Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

It is estimated that approximately fifty percent of the population will encounter at least one traumatic event during their lifetime; however, only six percent develop Posttraumatic Stress Disorder (PTSD), a trauma-related disorder characterized by persistent alterations in affect and cognition, including an exaggerated fear response. It is well established that females (9.7%) are more than twice as likely as males (3.6%) to be diagnosed with PTSD. Nevertheless, a significant challenge remains in understanding how the severity of traumas experienced in childhood versus adulthood influences PTSD development across sexes. To address this challenge effectively, the adoption of preclinical animal models that accurately reflect these key characteristics is crucial. The Stress Enhanced Fear Learning (SEFL) paradigm in rodents encapsulates some of these complexities associated with PTSD, such as persistent fear sensitization following exposure to acute trauma. This study investigates how stress severity and developmental timing affect the long-term maintenance of SEFL in male and female rats. Juvenile and adult rats were initially subjected to no stress (control, context only), low stress (1 footshock), moderate stress (3 footshocks), or high stress (15 footshocks), followed by an extinction session either one day (recent) or sixty days (remote) later. Subsequently, to evaluate SEFL in a new context, all subjects received a single footshock and were tested the following day. Shortly after stress induction, all groups displayed SEFL. Although the effects persisted for sixty days in adult males and juvenile females, even at the lowest stress level, they were significantly reduced in juvenile males and adult females. These findings offer novel insights into sex-related differences in the persistence of SEFL and introduce new considerations regarding how the timing of developmental stressors influences future threat encoding and fear responses.

Autism spectrum disorder (ASD) is a common neurodevelopmental disease with no currently effective treatments. In this study, Xenon (Xe), a noble gas used clinically as an anesthetic and a neuroprotectant, has been investigated for its potential therapeutic effects in ASD. Two genetic ASD mouse models (Mef2c mutant and Shank3-/- mice) were exposed to a low concentration of xenon (25%) mixed with oxygen (30%) and balanced with nitrogen for a single exposure of 30 min or for 10 min per day for 10 days. These Xe exposure paradigms rescued ASD-like behaviors in both male and female mice. The behavioral improvements persisted for up to 10 days following termination of the 10-day exposure paradigm. Mechanistically, Xe reduced glutamic acid decarboxylase expression in the inhibitory neurons (verified with RNAseq) of the paraventricular thalamic nucleus (PVT) and decreased GABA release during social interaction. Targeted infusion of the GABAA receptor agonist muscimol into the PVT abolished the Xe-mediated behavioral improvements. Reduced GABAergic transmission following Xe exposure increased PVT glutamatergic neuronal activity. Chemogenetic inhibition of these neurons eliminated the therapeutic effects of Xe, whereas chemogenetic activation mimicked the Xe-induced behavioral improvements. Furthermore, opto-inhibition of PVT glutamatergic projections to the central amygdala (CeA) blocked Xe-mediated behavioral improvement. Opto-activation of the PVT-CeA glutamatergic circuit reversed ASD-like behaviors. These findings demonstrate that Xe ameliorates ASD-like phenotypes in mouse models by modulating the PVT Glu-CeA circuit, supporting Xe as a novel potential therapy for ASD.

Circadian clock genes in the striatum modulate alcohol drinking behavior in female mice, but the underlying synaptic mechanisms remain unknown. Medium spiny neurons (MSNs) of the striatum express D1- or D2-type dopamine receptors, which are differentially modulated by alcohol. Here we used whole-cell patch-clamp recordings to examine how deletion of the clock gene Bmal1 affects spontaneous excitatory postsynaptic currents (sEPSCs) in electrophysiologically identified D1 and D2 neurons of the female mouse dorsomedial striatum (DMS). Prior intermittent access to alcohol increased sEPSC amplitude in putative D1 neurons of wildtype mice without affecting sEPSC frequency, and this increase was accompanied by greater dendritic branching and complexity in biocytin-filled D1 MSNs. Bmal1 knockout had the opposite effect in D1 neurons, reducing sEPSC amplitude in both alcohol-exposed and alcohol-naive animals. In D2 neurons, alcohol produced only a non-significant trend toward increased sEPSC amplitude in wildtype mice, but a significant increase in Bmal1 knockout mice. Thus, local disruption of Bmal1 appears to block the alcohol-induced enhancement of sEPSCs in D1 neurons while unmasking a similar enhancement in D2 neurons. These findings may help explain how Bmal1 deletion in the DMS reduces alcohol consumption in female mice.

Background and Purpose: Neonatal hypoxia-ischemia (NHI) is a leading cause of mortality and long-term neurodevelopmental disability. Therapeutic hypothermia (TH) is currently the only established treatment for NHI; however, its efficacy remains limited, underscoring the need for adjunctive neuroprotective strategies. Lactate is now regarded as a major metabolic substrate of the brain and has previously been shown to exert neuroprotective effects following experimental NHI. We investigated whether lactate enhances hypothermia-induced neuroprotection and evaluated the optimal timing of administration and potential underlying mechanisms. Methods: Postnatal day 7 Wistar rats were subjected to NHI using the Rice-Vannucci model and treated with TH alone (2h at 32{degrees}C), sodium L-lactate alone, or combined L-lactate-TH. Animals underwent longitudinal magnetic resonance imaging (MRI) and behavioral assessments, complemented by histological, immunohistochemical, and Western blot analyses, including assessment of protein lactylation. The effect of sodium L-lactate administration timing relative to TH was also evaluated. Results: Lactate significantly enhanced the neuroprotective effects of TH, particularly when administered before hypothermia, resulting in greater lesion recovery, reduced neuroinflammation and apoptotic cell death, preserved neuronal integrity, and improved long-term motor and cognitive outcomes. Combined treatment preserved neuronal lactate transporter, prevented NHI-induced astrocytic glutamate transporter upregulation, modulated lactylation markers, supporting coordinated metabolic and epigenetic adaptations following NHI. Conclusions: Early lactate administration enhances the neuroprotective efficacy of TH following NHI. These findings identify lactate as a promising adjunct to TH and support its translational potential as a metabolic strategy to improve outcomes following NHI brain injury.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

BACKGROUND: Pesticides are extensively used in agriculture, raising concerns about their potential impact on human health through dietary and environmental exposure. OBJECTIVES: This study evaluated the in vitro cytotoxicity of ten commonly used pesticides and their mixtures (lambda-cyhalothrin, cypermethrin, deltamethrin, tebuconazole, glyphosate, acetamiprid, cyprodinil, piperonyl butoxide, fluopyram, and imazalil) on human intestinal Caco-2 cells. METHODS: Cytotoxicity was assessed using the MTT assay, as a measure of metabolic activity, and the trypan blue exclusion test, as an indicator of cell membrane integrity. FINDINGS: Results showed that high concentrations (100 mg/L) of all pesticides significantly reduced cell viability and vitality. Notably, glyphosate and tebuconazole exhibited significant toxicity even at lower concentrations, respectively 0.1 mg/L and 10 mg/L. Combination treatments (Top 3 and Top 8 pesticide mixtures) retained the cytotoxic effects observed for individual compounds, showing additive (non-synergistic) effects. CONCLUSIONS: Overall, these findings indicate that certain pesticides-based herbicides can exert cytotoxic effects on intestinal cells even at relatively low concentrations and highlight the importance of using the component-based approach in mixture risk assessment for humans. This study was performed as part of the EU SPRINT (Sustainable Plant Protection Transition: A Global Health Approach) project.

Metabotropic glutamate receptors (mGluR) are G-protein coupled receptors that regulate neuroplasticity and have been shown to play a role in auditory processing. However, not much is known about the expression of mGluRs during cochlear nucleus development in the context of Fragile X syndrome (FXS). FXS is the most common heritable form of intellectual disability and autism and is associated with auditory processing deficits including hypersensitivity. Audiogenic seizure is a notable feature of the Fmr1 knock-out (KO) mouse model for FXS, in which mGluR5 has been shown to be necessary. In this study, we investigated the expression of eight metabotropic glutamate receptors (mGluRs) in the cochlear nucleus at both young and adult ages and in wildtype and Fmr1 KO mice. While it is established that mGluR1 is the predominantly expressed mGluR in adult cochlear nucleus, we report the following new finding that mGluR5 is the more abundant receptor during early development. The expression of Group II and III mGluRs (2,3,4,6,7,8) was relatively stable during development. We show that mGluR1 and mGluR5 are co-expressed across multiple neuron types. Fmr1 genotype did not have statistically significant impact on the expression pattern of mGluRs in cochlear nucleus. Although hearing onset occurs during this mGluR5 to 1 transition period, mGluR expression patterns were not impacted by cochlear activity. We conclude that there is an mGluR5 is to mGluR1 transition that occurs within cochlear nucleus neurons during the critical period closure that is intrinsic to neuronal development.

We have examined how ibogaine may enhance synaptic plasticity and neural repair. Extracellular efflux analysis showed that ibogaine (3, 10 or 30 M) produced a rapid (<5 minutes) and concentration-dependent suppression of mitochondrial function and glycolysis in mouse cortical neurons. Cell-based imaging demonstrated this metabolic inhibition was preceded by an elevation of the mitochondrial membrane potential and followed by increased mitochondrial reactive oxygen species levels. Like ibogaine, which blocks potassium channels, mitochondrial Kv1.3 channel (mitoKv1.3) inhibitors produce concordant changes over a similar timeframe. Following treatment with ibogaine (3, 10 or 30 M) for 4 hours, a marked concentration-dependent rebound of energy production occurred 20 hours later suggestive of metabolic preconditioning. Indeed, proteomic profiling showed that oral ibogaine (100 mg/kg) administration to female mice produced changes in spinal cord protein abundance indicative of increasing metabolic stress from 0.25-24 hours that resolved by 72 hours. This was accompanied by activation, inhibition and reactivation of synaptic plasticity and neural repair signaling pathways at 0.25, 4-24 and 72 hours, respectively. Hence, by inducing transient metabolic stress, ibogaine may re-open a neurodevelopmental window characterized by enhanced synaptic plasticity and neural repair. Ibogaine binds with high affinity to the sigma-2 receptor. Like mitoKv1-3 inhibitors and sigma-2 receptor ligands, ibogaine transiently elevated AMP-activated protein kinase (0.25 hours) and temporally suppressed mechanistic target of rapamycin and lipid synthesis pathways (0.25-24 hours) implicated in enhanced synaptic plasticity and neural repair. These findings suggest ibogaine rewires and repairs neural circuits by a metabolic preconditioning mechanism, perhaps involving mitoKv1.3 and the sigma-2 receptor.