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In JoVE (1)
Other Publications (6)
Articles by Donald A. Bruun in JoVE
JoVE Neuroscience
Inducing Dendritic Growth in Cultured Sympathetic Neurons
Department of Molecular Biosciences, University of California, Davis
We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.
Other articles by Donald A. Bruun on PubMed
Antigen Sensitization Influences Organophosphorus Pesticide-induced Airway Hyperreactivity
Recent epidemiologic studies have identified organophosphorus pesticides (OPs) as environmental factors potentially contributing to the increase in asthma prevalence over the last 25 years. In support of this hypothesis, we have demonstrated that environmentally relevant concentrations of OPs induce airway hyperreactivity in guinea pigs.
Organophosphorus Pesticides Decrease M2 Muscarinic Receptor Function in Guinea Pig Airway Nerves Via Indirect Mechanisms
Epidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms.
Epoxyeicosatrienoic Acids Enhance Axonal Growth in Primary Sensory and Cortical Neuronal Cell Cultures
It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.
Chlorpyrifos-oxon Disrupts Zebrafish Axonal Growth and Motor Behavior
Axonal morphology is a critical determinant of neuronal connectivity, and perturbation of the rate or extent of axonal growth during development has been linked to neurobehavioral deficits in animal models and humans. We previously demonstrated that the organophosphorus pesticide (OP) chlorpyrifos (CPF) inhibits axonal growth in cultured neurons. In this study, we used a zebrafish model to determine whether CPF, its oxon metabolite (CPFO), or the excreted metabolite trichloro-2-pyridinol (TCPy) alter spatiotemporal patterns of axonal growth in vivo. Static waterborne exposure to CPFO, but not CPF or TCPy, at concentrations ≥ 0.03 μM from 24- to 72-h post fertilization significantly inhibited acetylcholinesterase, and high-performance liquid chromatography detected significantly more TCPy in zebrafish exposed to 0.1 μM CPFO versus 1.0 μM CPF. These data suggest that zebrafish lack the metabolic enzymes to activate CPF during these early developmental stages. Consistent with this, CPFO, but not CPF, significantly inhibited axonal growth of sensory neurons, primary motoneurons, and secondary motoneurons at concentrations ≥ 0.1 μM. Secondary motoneurons were the most sensitive to axonal growth inhibition by CPFO, which was observed at concentrations that did not cause mortality, gross developmental defects, or aberrant somatic muscle differentiation. CPFO effects on axonal growth correlated with adverse effects on touch-induced swimming behavior, suggesting the functional relevance of these structural changes. These data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of CPF and demonstrate the relevance of zebrafish as a model for studying OP developmental neurotoxicity.
Spatiotemporal Pattern of Neuronal Injury Induced by DFP in Rats: a Model for Delayed Neuronal Cell Death Following Acute OP Intoxication
Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.
Characterization of Seizures Induced by Acute and Repeated Exposure to Tetramethylenedisulfotetramine
Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures and death. The CD(50) values for clonic and tonic seizures following oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 μg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days following a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.
