The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-Daspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A) ؉ The striatum receives dense dopaminergic afferents from midbrain areas (1, 2) and glutamatergic projections from all areas of the cerebral cortex (3, 4) and thalamus (5). Ionotropic glutamate receptors have been traditionally classified into three families on the basis of pharmacological and electrophysiological data: N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) receptors.In the striatum excessive activation of NMDA receptors has been associated with neurological disorders such as Huntington's (6, 7) and Parkinson's diseases (8).Phosphorylation of neurotransmitter receptors plays an important role in the modulation of their function. NMDA receptors contain multiple consensus sites for phosphorylation by several protein kinases (9). Protein kinase C (PKC) and tyrosine kinases have been shown to phosphorylate NMDA receptor subunits directly (10-12). Moreover, forskolin, dopamine, and a D 1 agonist increased the phosphorylation of the NMDAR1 receptor apparently through activation of cAMPdependent protein kinase (PKA) (13). In rat neostriatal brain slices NMDA-induced excitatory synaptic transmission was potentiated by the PKA activators forskolin and S P -cAMPS (14). Furthermore, activation of D 1 receptors potentiated NMDA-induced responses (15), whereas stimulation of metabotropic glutamate receptors had inhibitory effects on NMDA receptor function (16). In both cases, it was suggested that a cAMP͞PKA-dependent mechanism was involved. However, it could not be concluded from those experiments whether the NMDA receptor complex and͞or an additional intracellular regulatory protein was the target of PKA phosphorylation.In the present study, we used the Xenopus oocyte expression system to further unravel the molecular mechanisms underlying the modulation of striatal NMDA receptor function by PKA. In initial experiments, we recorded NMDA-induced currents from Xenopus oocytes expressing NR1b, NR1b͞ NR2A, NR1b͞NR2B, or NR1b͞NR2A͞NR2B NMDA receptor subunits, the main subunits found in rat striatum (17, 18). These currents were not affected by PKA stimulation (T.B. and I.N., unpublished data) suggesting the involvement of an indirect mechanism underlying the PKA-dependent modulation of striatal NMDA receptor function. MATERIALS AND METHODS RNA Preparation and Expression in XenopusOocytes. Total RNA was isolated from striatum and hippocampus of adult Wistar rats by extraction of fresh tissue with guanidine thiocyanate and precipitation with LiCl (19). Poly(A) ϩ mRNA was purified by oligo(dT)-cellulose chromatography (Pharmacia mRNA Purification Kit) and dissolved in RNase-free water at a concentration of 0.5 g͞l.Plasmid pET3A-DARPP-32 containing the cDNA coding for the rat M r 32,000 dopamine and adenosine 3Ј,5Ј-monopho...

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (β-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed ‘modified risk’. On 4–6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air–Liquid Interface- In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/ In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.