Chemical effects on neural network activity: Comparison of acute versus network formation exposure in microelectrode array assays.

New approach methodologies (NAMs) can address information gaps on potential neurotoxicity or developmental neurotoxicity hazard for data-poor chemicals. Two assays have been previously developed using microelectrode arrays (MEA), a technology which measures neural activity. The MEA acute network function assay (AcN) uses dissociated rat cortical cells cultured at postnatal day 0 and evaluates network activity during a 40-minute chemical exposure on day in vitro (DIV)13 or 15. In contrast, the MEA network formation assay (NFA) uses a developmental exposure paradigm spanning DIV0 through DIV12. Measures of network activity over time at DIV5, 7, 9, and 12 in the NFA are reduced to an estimated area under the curve to facilitate concentration-response evaluation. Here, we evaluated the hypothesis that chemicals with effects in the AcN also perturb the NFA by examining quantitative and qualitative concordance between assays. Out of 243 chemicals screened in both assays, we observed 70.3% concordance between the AcN and NFA after eliminating activity inferred to be cytotoxic (selective activity), with the majority of discordance explained by chemicals that altered selective activity in the AcN but not NFA. The NFA detected more active chemicals when evaluating activity associated with cytotoxicity. Median potency values were lower in the NFA compared to the AcN, but within-chemical potency values were not uniformly lower in the NFA than the AcN. Lastly, the AcN and NFA captured unique bioactivity fingerprints; the AcN was more informative for identifying chemicals with a shared mode of action, while the NFA provided information relevant to developmental exposure. Taken together, this analysis provides a rationale for using both approaches for chemical evaluation with consideration of the context of use, such as screening/ prioritization, hazard identification, or to address questions regarding biological mechanism or function.

A Comparison of In Vitro Points of Departure with Human Blood Levels for Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are widely used, and their fluorinated state contributes to unique uses and stability but also long half-lives in the environment and humans. PFAS have been shown to be toxic, leading to immunosuppression, cancer, and other adverse health outcomes. Only a small fraction of the PFAS in commerce have been evaluated for toxicity using in vivo tests, which leads to a need to prioritize which compounds to examine further. Here, we demonstrate a prioritization approach that combines human biomonitoring data (blood concentrations) with bioactivity data (concentrations at which bioactivity is observed in vitro) for 31 PFAS. The in vitro data are taken from a battery of cell-based assays, mostly run on human cells. The result is a Bioactive Concentration to Blood Concentration Ratio (BCBCR), similar to a margin of exposure (MoE). Chemicals with low BCBCR values could then be prioritized for further risk assessment. Using this method, two of the PFAS, PFOA (Perfluorooctanoic Acid) and PFOS (Perfluorooctane Sulfonic Acid), have BCBCR values < 1 for some populations. An additional 9 PFAS have BCBCR values < 100 for some populations. This study shows a promising approach to screening level risk assessments of compounds such as PFAS that are long-lived in humans and other species.

Evaluation of Per- and Polyfluoroalkyl Substances (PFAS) In Vitro Toxicity Testing for Developmental Neurotoxicity.

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

An expert-driven literature review of "negative" chemicals for developmental neurotoxicity (DNT) in vitro assay evaluation.

To date, approximately 200 chemicals have been tested in US Environmental Protection Agency (EPA) or Organization for Economic Co-operation and Development (OECD) developmental neurotoxicity (DNT) guideline studies, leaving thousands of chemicals without traditional animal information on DNT hazard potential. To address this data gap, a battery of in vitro DNT new approach methodologies (NAMs) has been proposed. Evaluation of the performance of this battery will increase the confidence in its use to determine DNT chemical hazards. One approach to evaluate DNT NAM performance is to use a set of chemicals to evaluate sensitivity and specificity. Since a list of chemicals with potential evidence of in vivo DNT has been established, this study aims to develop a curated list of "negative" chemicals for inclusion in a "DNT NAM evaluation set". A workflow, including a literature search followed by an expert-driven literature review, was used to systematically screen 39 chemicals for lack of DNT effect. Expert panel members evaluated the scientific robustness of relevant studies to inform chemical categorizations. Following review, the panel discussed each chemical and made categorical determinations of "Favorable", "Not Favorable", or "Indeterminate" reflecting acceptance, lack of suitability, or uncertainty given specific limitations and considerations, respectively. The panel determined that 10, 22, and 7 chemicals met the criteria for "Favorable", "Not Favorable", and "Indeterminate", for use as negatives in a DNT NAM evaluation set. Ultimately, this approach not only supports DNT NAM performance evaluation but also highlights challenges in identifying large numbers of negative DNT chemicals.

Integrating Data From In Vitro New Approach Methodologies for Developmental Neurotoxicity.

In vivo developmental neurotoxicity (DNT) testing is resource intensive and lacks information on cellular processes affected by chemicals. To address this, DNT new approach methodologies (NAMs) are being evaluated, including: the microelectrode array neuronal network formation assay; and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work addresses 3 hypotheses: (1) a broad screening battery provides a sensitive marker of DNT bioactivity; (2) selective bioactivity (occurring at noncytotoxic concentrations) may indicate functional processes disrupted; and, (3) a subset of endpoints may optimally classify chemicals with in vivo evidence for DNT. The dataset was comprised of 92 chemicals screened in all 57 assay endpoints sourced from publicly available data, including a set of DNT NAM evaluation chemicals with putative positives (53) and negatives (13). The DNT NAM battery provides a sensitive marker of DNT bioactivity, particularly in cytotoxicity and network connectivity parameters. Hierarchical clustering suggested potency (including cytotoxicity) was important for classifying positive chemicals with high sensitivity (93%) but failed to distinguish patterns of disrupted functional processes. In contrast, clustering of selective values revealed informative patterns of differential activity but demonstrated lower sensitivity (74%). The false negatives were associated with several limitations, such as the maximal concentration tested or gaps in the biology captured by the current battery. This work demonstrates that this multi-dimensional assay suite provides a sensitive biomarker for DNT bioactivity, with selective activity providing possible insight into specific functional processes affected by chemical exposure and a basis for further research.

Perineuronal net degradation rescues CA2 plasticity in a mouse model of Rett syndrome.

Perineuronal nets (PNNs), a specialized form of extracellular matrix, are abnormal in the brains of people with Rett syndrome (RTT). We previously reported that PNNs function to restrict synaptic plasticity in hippocampal area CA2, which is unusually resistant to long-term potentiation (LTP) and has been linked to social learning in mice. Here we report that PNNs appear elevated in area CA2 of the hippocampus of an individual with RTT and that PNNs develop precociously and remain elevated in area CA2 of a mouse model of RTT (Mecp2-null). Further, we provide evidence that LTP could be induced at CA2 synapses prior to PNN maturation (postnatal day 8-11) in wild-type mice and that this window of plasticity was prematurely restricted at CA2 synapses in Mecp2-null mice. Degrading PNNs in Mecp2-null hippocampus was sufficient to rescue the premature disruption of CA2 plasticity. We identified several molecular targets that were altered in the developing Mecp2-null hippocampus that may explain aberrant PNNs and CA2 plasticity, and we discovered that CA2 PNNs are negatively regulated by neuronal activity. Collectively, our findings demonstrate that CA2 PNN development is regulated by Mecp2 and identify a window of hippocampal plasticity that is disrupted in a mouse model of RTT.

Distinct and dementia-related synaptopathy in the hippocampus after military blast exposures.

Explosive shockwaves, and other types of blast exposures, are linked to injuries commonly associated with military service and to an increased risk for the onset of dementia. Neurological complications following a blast injury, including depression, anxiety, and memory problems, often persist even when brain damage is undetectable. Here, hippocampal explants were exposed to the explosive 1,3,5-trinitro-1,3,5-triazinane (RDX) to identify indicators of blast-induced changes within important neuronal circuitries. Highly controlled detonations of small, 1.7-gram RDX spherical charges reduced synaptic markers known to be downregulated in cognitive disorders, but without causing overt neuronal loss or astroglial responses. In the absence of neuromorphological alterations, levels of synaptophysin, GluA1, and synapsin IIb were significantly diminished within 24 hr, and these synaptic components exhibited progressive reductions following blast exposure as compared to their stable maintenance in control explants. In contrast, labeling of the synapsin IIa isoform remained unaltered, while neuropilar staining of other markers decreased, including synapsin IIb and neural cell adhesion molecule (NCAM) isoforms, along with evidence of NCAM proteolytic breakdown. NCAM180 displayed a distinct decline after the RDX blasts, whereas NCAM140 and NCAM120 exhibited smaller or no deterioration, respectively. Interestingly, the extent of synaptic marker reduction correlated with AT8-positive tau levels, with tau pathology stochastically found in CA1 neurons and their dendrites. The decline in synaptic components was also reflected in the size of evoked postsynaptic currents recorded from CA1 pyramidals, which exhibited a severe and selective reduction. The identified indicators of blast-mediated synaptopathy point to the need for early biomarkers of explosives altering synaptic integrity with links to dementia risk, to advance strategies for both cognitive health and therapeutic monitoring.

Modified adeno-associated virus targets the bacterial enzyme chondroitinase ABC to select mouse neuronal populations in vivo using the Cre-LoxP system.

Current methods of experimentally degrading the specialized extracellular matrix (ECM), perineuronal nets (PNNs) have several limitations. Genetic knockout of ECM components typically has only partial effects on PNNs, and knockout of the major ECM component aggrecan is lethal in mice. Direct injection of the chondroitinase ABC (ChABC) enzyme into the mammalian brain is effective at degrading PNNs in vivo but this method typically lacks consistent, localized spatial targeting of PNN degradation. PNNs also regenerate within weeks after a ChABC injection, thus limiting the ability to perform long-term studies. Previous work has demonstrated that viral delivery of ChABC in mammalian neurons can successfully degrade PNNs for much longer periods, but the effects are similarly diffuse beyond the injection site. In an effort to gain cell-specific targeting of ChABC, we designed an adeno-associated virus encoding ChABC under the control of the Cre-LoxP system. We show that this virus is effective at targeting the synthesis of ChABC to Cre-expressing mouse neurons in vivo. Although ChABC expression is localized to the Cre-expressing neurons, we also note that ChABC is apparently trafficked and secreted at projection sites, as was previously reported for the non-Cre dependent construct. Overall, this method allows for cell-specific targeting of ChABC and long-term degradation of PNNs, which will ultimately serve as an effective tool to study the function of cell-autonomous regulation of PNNs in vivo. This novel approach may also aid in determining whether specific, long-term PNN loss is an appropriate strategy for treatment of neurodevelopmental disorders associated with PNN pathology.

Novel role for mineralocorticoid receptors in control of a neuronal phenotype.

Mineralocorticoid receptors (MRs) in the brain play a role in learning and memory, neuronal differentiation, and regulation of the stress response. Within the hippocampus, the highest expression of MRs is in area CA2. CA2 pyramidal neurons have a distinct molecular makeup resulting in a plasticity-resistant phenotype, distinguishing them from neurons in CA1 and CA3. Thus, we asked whether MRs regulate CA2 neuron properties and CA2-related behaviors. Using three conditional knockout methods at different stages of development, we found a striking decrease in multiple molecular markers for CA2, an effect mimicked by chronic antagonism of MRs. Furthermore, embryonic deletion of MRs disrupted afferent inputs to CA2 and enabled synaptic potentiation of the normally LTP-resistant synaptic currents in CA2. We also found that CA2-targeted MR knockout was sufficient to disrupt social behavior and alter behavioral responses to novelty. Altogether, these results demonstrate an unappreciated role for MRs in controlling CA2 pyramidal cell identity and in facilitating CA2-dependent behaviors.

Hippocampal Subregions Express Distinct Dendritic Transcriptomes that Reveal Differences in Mitochondrial Function in CA2.

RNA localization is one mechanism neurons use to spatially and temporally regulate gene expression at synapses. Here, we test the hypothesis that cells exhibiting distinct forms of synaptic plasticity will have differences in dendritically localized RNAs. Indeed, we discover that each major subregion of the adult mouse hippocampus expresses a unique complement of dendritic RNAs. Specifically, we describe more than 1,000 differentially expressed dendritic RNAs, suggesting that RNA localization and local translation are regulated in a cell type-specific manner. Furthermore, by focusing Gene Ontology analyses on the plasticity-resistant CA2, we identify an enrichment of mitochondria-associated pathways in CA2 cell bodies and dendrites, and we provide functional evidence that these pathways differentially influence plasticity and mitochondrial respiration in CA2. These data indicate that differences in dendritic transcriptomes may regulate cell type-specific properties important for learning and memory and may influence region-specific differences in disease pathology.

Regulation of synaptic plasticity in hippocampal area CA2.

Synaptic plasticity in the hippocampus is thought to play a vital role in both the refinement of neuronal circuits during development and in learning in the mature brain. Synapses in hippocampal area CA1 are known for a robust capacity for long-term potentiation (LTP), whereas synapses in the stratum radiatum of hippocampal area CA2 are particularly resistant to such changes. Although we have yet to fully understand the mechanisms behind this resistance to plasticity, a number of genes and extracellular matrix components highly expressed in CA2 appear to function as molecular brakes on plasticity and develop postnatally in the rodent brain. Curiously, the developmental profile of several CA2-enriched molecules is suggestive of a still undefined critical window of plasticity in the hippocampus.

Ube3a reinstatement mitigates epileptogenesis in Angelman syndrome model mice.

Angelman syndrome (AS) is a neurodevelopmental disorder in which epilepsy is common (~90%) and often refractory to antiepileptics. AS is caused by mutation of the maternal allele encoding the ubiquitin protein ligase E3A (UBE3A), but it is unclear how this genetic insult confers vulnerability to seizure development and progression (i.e., epileptogenesis). Here, we implemented the flurothyl kindling and retest paradigm in AS model mice to assess epileptogenesis and to gain mechanistic insights owed to loss of maternal Ube3a. AS model mice kindled similarly to wild-type mice, but they displayed a markedly increased sensitivity to flurothyl-, kainic acid-, and hyperthermia-induced seizures measured a month later during retest. Pathological characterization revealed enhanced deposition of perineuronal nets in the dentate gyrus of the hippocampus of AS mice in the absence of overt neuronal loss or mossy fiber sprouting. This pro-epileptogenic phenotype resulted from Ube3a deletion in GABAergic but not glutamatergic neurons, and it was rescued by pancellular reinstatement of Ube3a at postnatal day 21 (P21), but not during adulthood. Our results suggest that epileptogenic susceptibility in AS patients is a consequence of the dysfunctional development of GABAergic circuits, which may be amenable to therapies leveraging juvenile reinstatement of UBE3A.

Perineuronal Nets Suppress Plasticity of Excitatory Synapses on CA2 Pyramidal Neurons.

UNLABELLED: Long-term potentiation of excitatory synapses on pyramidal neurons in the stratum radiatum rarely occurs in hippocampal area CA2. Here, we present evidence that perineuronal nets (PNNs), a specialized extracellular matrix typically localized around inhibitory neurons, also surround mouse CA2 pyramidal neurons and envelop their excitatory synapses. CA2 pyramidal neurons express mRNA transcripts for the major PNN component aggrecan, identifying these neurons as a novel source for PNNs in the hippocampus. We also found that disruption of PNNs allows synaptic potentiation of normally plasticity-resistant excitatory CA2 synapses; thus, PNNs play a role in restricting synaptic plasticity in area CA2. Finally, we found that postnatal development of PNNs on CA2 pyramidal neurons is modified by early-life enrichment, suggesting that the development of circuits containing CA2 excitatory synapses are sensitive to manipulations of the rearing environment. SIGNIFICANCE STATEMENT: Perineuronal nets (PNNs) are thought to play a major role in restricting synaptic plasticity during postnatal development, and are altered in several models of neurodevelopmental disorders, such as schizophrenia and Rett syndrome. Although PNNs have been predominantly studied in association with inhibitory neurons throughout the brain, we describe a dense expression of PNNs around excitatory pyramidal neurons in hippocampal area CA2. We also provide insight into a previously unrecognized role for PNNs in restricting plasticity at excitatory synapses and raise the possibility of an early critical period of hippocampal plasticity that may ultimately reveal a key mechanism underlying learning and memory impairments of PNN-associated neurodevelopmental disorders.