Search

Abstract: Facial grimacing is used to quantify spontaneous pain in mice and other mammals, but scoring relies on humans with different levels of proficiency. Here, we developed a cloud-based software platform called PainFace ( http://painface.net ) that uses machine learning to detect 4 facial action units of the mouse grimace scale (orbitals, nose, ears, whiskers) and score facial grimaces of black-coated C57BL/6 male and female mice on a 0 to 8 scale. Platform accuracy was validated in 2 different laboratories, with 3 conditions that evoke grimacing-laparotomy surgery, bilateral hindpaw injection of carrageenan, and intraplantar injection of formalin. PainFace can generate up to 1 grimace score per second from a standard 30 frames/s video, making it possible to quantify facial grimacing over time, and operates at a speed that scales with computing power. By analyzing the frequency distribution of grimace scores, we found that mice spent 7x more time in a "high grimace" state following laparotomy surgery relative to sham surgery controls. Our study shows that PainFace reproducibly quantifies facial grimaces indicative of nonevoked spontaneous pain and enables laboratories to standardize and scale-up facial grimace analyses., (Copyright © 2024 International Association for the Study of Pain.)

Background: The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 ( Trpm8 ) knock-out ( Trpm8 -/- ) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin., Methods: Mice were placed in the open field at 4°C and 23°C for 1 hour. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded., Results: The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8 -/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8 -/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8 -/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8 -/- mice., Conclusions: Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Jimenez JA et al.)

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3A T485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3A T503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3A T503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3A T503A , irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3a T503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Azoxystrobin (AZ) is a broad-spectrum strobilurin fungicide that is used in agriculture and was recently added to mold- and mildew-resistant wallboards. AZ was found to have toxic effects in animals at embryonic stages and was listed as a frontline target for biomonitoring in children., Objectives: This study investigated exposure to AZ in pregnant women and young children, whether AZ could be transferred from an exposed mother to offspring, and whether AZ or one of its primary metabolites, AZ-acid, was neurotoxic in vitro ., Methods: We quantified AZ-acid, a sensitive indicator of AZ exposure, in urine samples collected from 8 pregnant women (12 urine samples) and 67 children (40-84 months old; 96 urine samples) with high-resolution mass spectrometry. Gestational and lactational transfer was assessed in C57Bl/6 mice. Neurotoxicity of AZ and AZ-acid was investigated in vitro with mouse cortical neuron cultures., Results: AZ-acid was present above the limit of quantification ( 0.01  ng / mL ) in 100% of the urine samples from pregnant women and in 70% of the urine samples from children, with median concentration of 0.10 and 0.07  ng / mL , and maximal concentration of 2.70 and 6.32  ng / mL , respectively. Studies in mice revealed that AZ transferred from the mother to offspring during gestation by crossing the placenta and entered the developing brain. AZ was also transferred to offspring via lactation. High levels of cytotoxicity were observed in embryonic mouse cortical neurons at concentrations that modeled environmentally relevant exposures., Discussion: Our study suggested that pregnant women and children were exposed to AZ, and at least 10% of the children (2 out of 20 that were evaluated at two ages) showed evidence of chronic exposure. Future studies are warranted to evaluate whether chronic AZ exposure affects human health and development. https://doi.org/10.1289/EHP9808.

The development of the mammalian cerebral cortex depends on careful orchestration of proliferation, maturation, and migration events, ultimately giving rise to a wide variety of neuronal and non-neuronal cell types. To better understand cellular and molecular processes that unfold during late corticogenesis, we perform single-cell RNA-seq on the mouse cerebral cortex at a progenitor driven phase (embryonic day 14.5) and at birth-after neurons from all six cortical layers are born. We identify numerous classes of neurons, progenitors, and glia, their proliferative, migratory, and activation states, and their relatedness within and across age. Using the cell-type-specific expression patterns of genes mutated in neurological and psychiatric diseases, we identify putative disease subtypes that associate with clinical phenotypes. Our study reveals the cellular template of a complex neurodevelopmental process, and provides a window into the cellular origins of brain diseases.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the maternal UBE3A allele. The maternal UBE3A allele is expressed in nearly all neurons of the brain and spinal cord, whereas the paternal UBE3A allele is repressed by an extremely long antisense transcript ( UBE3A-ATS ). Little is known about expression of UBE3A in the peripheral nervous system, where loss of maternal UBE3A might contribute to AS phenotypes. Here we sought to examine maternal and paternal Ube3a expression in DRGs neurons and to evaluate whether nociceptive responses were affected in AS model mice (global deletion of maternal Ube3a allele; Ube3a m -/ p + ). We found that most large-diameter proprioceptive and mechanosensitive DRG neurons expressed maternal Ube3a and paternal Ube3a-ATS In contrast, most small-diameter neurons expressed Ube3a biallelically and had low to undetectable levels of Ube3a-ATS Analysis of single-cell DRG transcriptomes further suggested that Ube3a is expressed monoallelically in myelinated large-diameter neurons and biallelically in unmyelinated small-diameter neurons. Behavioral responses to some noxious thermal and mechanical stimuli were enhanced in male and female AS model mice; however, nociceptive responses were not altered by the conditional deletion of maternal Ube3a in the DRG. These data suggest that the enhanced nociceptive responses in AS model mice are due to loss of maternal Ube3a in the central, but not peripheral, nervous system. Our study provides new insights into sensory processing deficits associated with AS. SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss or mutation of the maternal UBE3A allele. While sensory processing deficits are frequently associated with AS, it is currently unknown whether Ube3a is expressed in peripheral sensory neurons or whether maternal deletion of Ube3a affects somatosensory responses. Here, we found that Ube3a is primarily expressed from the maternally inherited allele in myelinated large-diameter sensory neurons and biallelically expressed in unmyelinated small-diameter neurons. Nociceptive responses to select noxious thermal and mechanical stimuli were enhanced following global, but not sensory neuron-specific, deletion of maternal Ube3a in mice. These data suggest that maternal loss of Ube3a affects nociception via a central, but not peripheral mechanism, with implications for AS., (Copyright © 2017 the authors 0270-6474/17/3710230-10$15.00/0.)

Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders.

Background: Calcitonin gene-related peptide-α (CGRPα) is a classic marker of peptidergic nociceptive neurons and is expressed in myelinated and unmyelinated dorsal root ganglia (DRG) neurons. Recently, we found that ablation of Cgrpα-expressing sensory neurons reduced noxious heat sensitivity and enhanced sensitivity to cold stimuli in mice. These studies suggested that the enhanced cold responses were due to disinhibition of spinal neurons that receive inputs from cold-sensing/TRPM8 primary afferents; although a direct role for TRPM8 was not examined at the time., Results: Here, we ablated Cgrpα-expressing sensory neurons in mice lacking functional TRPM8 and evaluated sensory responses to noxious heat, cold temperatures, and cold mimetics (acetone evaporative cooling and icilin). We also evaluated thermoregulation in these mice following an evaporative cold challenge. We found that ablation of Cgrpα-expressing sensory neurons in a Trpm8-/- background reduced sensitivity to noxious heat but did not enhance sensitivity to cold stimuli. Thermoregulation following the evaporative cold challenge was not affected by deletion of Trpm8 in control or Cgrpα-expressing sensory neuron-ablated mice., Conclusions: Our data indicate that the enhanced behavioral responses to cold stimuli in CGRPα sensory neuron-ablated mice are dependent on functional TRPM8, whereas the other sensory and thermoregulatory phenotypes caused by CGRPα sensory neuron ablation are independent of TRPM8.

Calcitonin gene-related peptide (CGRP) is a classic molecular marker of peptidergic primary somatosensory neurons. Despite years of research, it is unknown whether these neurons are required to sense pain or other sensory stimuli. Here, we found that genetic ablation of CGRPα-expressing sensory neurons reduced sensitivity to noxious heat, capsaicin, and itch (histamine and chloroquine) and impaired thermoregulation but did not impair mechanosensation or β-alanine itch-stimuli associated with nonpeptidergic sensory neurons. Unexpectedly, ablation enhanced behavioral responses to cold stimuli and cold mimetics without altering peripheral nerve responses to cooling. Mechanistically, ablation reduced tonic and evoked activity in postsynaptic spinal neurons associated with TRPV1/heat, while profoundly increasing tonic and evoked activity in spinal neurons associated with TRPM8/cold. Our data reveal that CGRPα sensory neurons encode heat and itch and tonically cross-inhibit cold-responsive spinal neurons. Disruption of this crosstalk unmasks cold hypersensitivity, with mechanistic implications for neuropathic pain and temperature perception., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5'-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well-based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC(1280)) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide the pathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)