a molecular complex with primarily nuclear localization (Kupfer et al., 1997; Garcia-Higuera et al., 1999; Waisfisz et al., 1999a). FANCC also localizes to the cytoplasm,

Background: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca2+) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca2+ signaling. Ca2+ signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known.Objective: We determined whether RyR activity is required for PCB effects on dendritic growth.Methods and Results: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2´,3,5´6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4´,4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs.Conclusions: Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.

Background: Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) promote dendritic growth in hippocampal neurons via ryanodine receptor (RyR)-dependent mechanisms; however, downstream signaling events that link enhanced RyR activity to dendritic growth are unknown. Activity-dependent dendritic growth, which is a critical determinant of neuronal connectivity in the developing brain, is mediated by calcium ion (Ca2+)-dependent activation of Ca2+/calmodulin kinase-I (CaMKI), which triggers cAMP response element binding protein (CREB)-dependent Wnt2 transcription. RyRs regulate the spatiotemporal dynamics of intracellular Ca2+ signals, but whether RyRs promote dendritic growth via modulation of this signaling pathway is not known.Objective: We tested the hypothesis that the CaMKI–CREB–Wnt2 signaling pathway couples NDL PCB-enhanced RyR activity to dendritic arborization.Methods and Results: Ca2+ imaging of dissociated cultures of primary rat hippocampal neurons indicated that PCB-95 (2,2´,3,5´6-pentachlorobiphenyl; a potent RyR potentiator), enhanced synchronized Ca2+ oscillations in somata and dendrites that were blocked by ryanodine. As determined by Western blotting and quantitative polymerase chain reaction, PCB-95 also activated CREB and up-regulated Wnt2. Blocking CaMKK, CaMKIα/γ, MEK/ERK, CREB, or Wnt2 prevented PCB-95–induced dendritic growth. Antagonism of γ-aminobutyric acid (GABA) receptors with bicuculline (BIC) phenocopied the dendrite-promoting effects of PCB-95, and pharmacological antagonism or siRNA knockdown of RyR blocked BIC-induced dendritic growth in dissociated and slice cultures of hippocampal neurons.Conclusions: RyR activity contributes to dynamic remodeling of dendritic architecture in response to NDL PCBs via CaMKI–CREB–Wnt2 signaling in rats. Our findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling associated with autism.

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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 (20 mg/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 1 h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by FluoroJade-B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4 h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24 h 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.

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 after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 g) 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 after 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.

The Rit GTPase is widely expressed in developing and adult nervous systems, and our previous data with pheochromocytoma cells implicate Rit signaling in NGF-induced neurite outgrowth. In this study, we investigated a role for Rit in neuronal morphogenesis. Expression of a dominant-negative (dn) Rit mutant in hippocampal neurons inhibited axonal growth but potentiated dendritic growth. Conversely, a constitutively active (ca) Rit mutant promoted axonal growth but inhibited dendritic growth. Dendritogenesis is regulated differently in sympathetic neurons versus hippocampal neurons in that sympathetic neurons require NGF and bone morphogenetic proteins (BMPs) to trigger dendritic growth. Despite these differences, dnRit potentiated and caRit blocked BMP7-induced dendritic growth in sympathetic neurons. Biochemical studies indicated that BMP7 treatments that caused dendritic growth also decreased Rit GTP loading. Additional studies demonstrate that caRit increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and pharmacological inhibition of MEK1 (mitogen-activated protein kinase/ERK 1) blocked the axon-promoting and dendrite-inhibiting effects of caRit. These observations suggest that Rit is a convergence point for multiple signaling pathways and it functions to promote axonal growth but inhibit dendritic growth via activation of ERK1/2. Modulation of the activational status of Rit may therefore represent a generalized mechanism across divergent neuronal cell types for regulating axonal versus dendritic growth modes.

BackgroundAcute intoxication with organophosphorus (OP) cholinesterase inhibitors can trigger convulsions that progress to life-threatening status epilepticus. Survivors face long-term morbidity including mild-to-severe decline in memory. It is posited that neuroinflammation plays a key role in the pathogenesis of OP-induced neuropsychiatric deficits. Rigorous testing of this hypothesis requires preclinical models that recapitulate relevant phenotypic outcomes. Here, we describe a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) that exhibits persistent neuroinflammation and cognitive impairment.MethodsNeuroinflammation, neurodegeneration, and cognitive function were compared in adult male Sprague Dawley rats injected with an acutely toxic dose of DFP vs. vehicle controls at multiple time points up to 36 days post-exposure. Neuroinflammation was quantified using immunohistochemical biomarkers of microglia (ionized calcium-binding adapter molecule 1, IBA1) and activated astrocytes (glial fibrillary acidic protein, GFAP) and positron emission tomography (PET) imaging of [11C]-(R)-PK11195, a ligand for the 18-kDa mitochondrial membrane translocator protein (TSPO). FluoroJade-B staining was used to assess neurodegeneration; Pavlovian conditioning, to assess cognitive function.ResultsAnimals exhibited moderate-to-severe seizures within minutes of DFP injection that continued for up to 6 h post-injection. As indicated by IBA1 and GFAP immunoreactivity and by PET imaging of TSPO, acute DFP intoxication triggered neuroinflammation in the hippocampus and cortex during the first 3 days that peaked at 7 days and persisted to 21 days post-exposure in most animals. Neurodegeneration was detected in multiple brain regions from 1 to 14 days post-exposure. All DFP-intoxicated animals exhibited significant deficits in contextual fear conditioning at 9 and 20 days post-exposure compared to vehicle controls. Whole-brain TSPO labeling positively correlated with seizure severity score, but did not correlate with performance in the contextual fear-conditioning task.ConclusionsWe describe a preclinical model in which acute DFP intoxication causes seizures, persistent neuroinflammation, neurodegeneration, and memory impairment. The extent of the neuroinflammatory response is influenced by seizure severity. However, the observation that a subset of animals with moderate seizures and minimal TSPO labeling exhibited cognitive deficits comparable to those of animals with severe seizures and significant TSPO labeling suggests that DFP may impair learning and memory circuitry via mechanisms independent of seizures or neuroinflammation.