DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood–brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1–6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid–induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)–packaged clones were administered directly into brain or i.v. 1 day after kainic acid–induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells.

Methimazole is a compound administered to humans for the treatment of hyperthyroidism and is used experimentally as a model substrate for the flavin-containing monooxygenase (FMO) system. Previous results from this laboratory demonstrated that methimazole is an olfactory system toxicant, causing nearly complete destruction of the olfactory epithelium in the male Long-Evans rat following a single ip dose of 300 mg/kg.The present studies were undertaken to determine the dose-response relationship for methimazole-induced olfactory mucosal damage and to determine whether or not similar damage occurs as a result of oral administration, mimicking the relevant route of human exposure. We also investigated the mechanism of olfactory toxicity of methimazole by means of a structure-activity study and began the characterization of the form(s) of FMO present in the olfactory mucosa of the male Long-Evans rat. Dose-response analysis demonstrated that methimazole causes olfactory mucosal damage at doses of 25 mg/kg ip and greater. The results of gavage studies showed that a single oral dose of 50 mg/kg also caused olfactory mucosal damage. Two structurally related compounds, methylimidazole and methylpyrrole, were not olfactory toxicants, suggesting that a reactive intermediate generated in the course of metabolizing methimazole to an S-oxide is the olfactory toxic species. Microsomal incubation studies revealed the presence ofmethimazole S-oxidation activity in olfactory mucosal microsomes at levels comparable to those in liver. An anti-mouse liver FMO antibody reacted on Western blots with olfactory mucosal microsomes. These findings demonstrate a doseresponse for the olfactory toxicity of methimazole and suggest that characterization of human olfactory mucosal FMO activity may be necessary to assess the potential for human risk associated with therapeutic exposure to methimazole.

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D 2 -like dopamine receptors mediate functional changes via activation of inhibitory G proteins, including those that affect adenylate cyclase activity, and potassium and calcium channels. Although it is assumed that the binding of a drug to a single isoform of a D 2 -like receptor will cause similar changes in all receptor-mediated functions, it has been demonstrated in brain that the dopamine agonists dihydrexidine (DHX) and N-npropyl-DHX are "functionally selective". The current study explores the underlying mechanism using transfected MN9D cells and D 2 -producing anterior pituitary lactotrophs. Both dopamine and DHX inhibited adenylate cyclase activity in a concentrationdependent manner in both systems, effects blocked by D 2 , but not D 1 , antagonists. In the MN9D cells, quinpirole and R-(Ϫ)-N-propylnorapomorphine (NPA) also inhibited the K ϩ -stimulated release of [ 3 H]dopamine in a concentration-responsive, antagonist-reversible manner. Conversely, neither DHX, nor its analogs, inhibited K ϩ -stimulated [ 3 H]dopamine release, although they antagonized the effects of quinpirole. S-(ϩ)-NPA actually had the reverse functional selectivity profile from DHX (i.e., it was a full agonist at D 2L receptors coupled to inhibition of dopamine release, but a weak partial agonist at D 2L receptormediated inhibition of adenylate cyclase). In lactotrophs, DHX had little intrinsic activity at D 2 receptors coupled to G proteincoupled inwardly rectifying potassium channels, and actually antagonized the effects of dopamine at these D 2 receptors. Together, these findings provide compelling evidence for agonist-induced functional selectivity with the D 2L receptor. Although the underlying molecular mechanism is controversial (e.g., "conformational induction" versus "drug-active state selection"), such data are irreconcilable with the widely held view that drugs have "intrinsic efficacy".The dopamine receptors, members of the G protein-coupled receptor superfamily, are generally divided into two classes, "D 1 -like" and "D 2 -like" (Jaber et al., 1996;Huff, 1997) In the central nervous system, dopamine receptors exist on both dopamine neurons (on soma, dendrites, and terminals) and postsynaptically on target cells. Presynaptic receptors (autoreceptors) fall under the D 2 -like class of dopamine re-

Resected epileptic tissues exhibit elements of chronic neuroinflammation that include elevated TNFα and increased TNFα receptor activation, but the seizure related consequences of chronic TNFα expression remain unknown. Twenty four hours after acute limbic seizures the rat hippocampus exhibited a rapid upregulation of TNFR1, but a simultaneous downregulation of TNFR2. These limbic seizures also evoked significant increases in measures of neuroinflammation and caused significant neuronal cell death in both the hilus and CA3 of the hippocampus. In order to mimic a state of chronic TNFα exposure, adeno-associated viral vectors were packaged with a TNF receptor 1 (TNFR1) specific agonist, human TNFα, or a TNF receptor 1/2 agonist, rat TNFα. Subsequently, chronic hippocampal overexpression of either TNFR ligand caused microglial activation and blood-brain barrier compromise, a pattern similar to limbic seizure-induced neuroinflammation. However, no evidence was found for neuronal cell death or spontaneous seizure activity. Thus, chronic, in vivo TNFα expression and the subsequent neuroinflammation alone did not cause cell death or elicit seizure activity. In contrast, chronic hippocampal activation of TNFR1 alone significantly increased limbic seizure sensitivity in both amygdala kainic acid and electrical amygdala kindling models, while chronic activation of both TNFR1 and TNFR2 significantly attenuated the amygdala kindling rate. With regard to endogenous TNFα, chronic hippocampal expression of a TNFα decoy receptor significantly reduced seizure-induced cell death in the hippocampus, but did not alter seizure susceptibility. These findings suggest that blockade of endogenous TNFα could attenuate seizure related neuropathology, while selective activation of TNFR2 could exert beneficial therapeutic effects on in vivo seizure sensitivity.