Opposing actions of hippocampus TNFα receptors on limbic seizure susceptibility.

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.

Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).

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.

Changes in apical dendritic structure correlate with sustained ERK1/2 phosphorylation in medial prefrontal cortex of a rat model of dopamine D1 receptor agonist sensitization.

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia, and other developmental disorders of frontostriatal circuit dysfunction. In these animals a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the postpubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo.

Nifedipine suppresses self-injurious behaviors in animals.

Self-injurious behavior is a common problem in many developmental disorders. The neurobiology of this behavior is not well understood, but the differing behavioral manifestations and associations with different disorders suggest that the underlying biological mechanisms are heterogeneous. The behavioral and biological heterogeneity is also evident in several animal models, where different manifestations can be provoked under different experimental conditions. Identifying commonalities among the different mechanisms is likely to be helpful in the design of treatments useful for the broadest populations of patients. The current studies reveal that nifedipine suppresses self-injurious behavior in 4 unrelated animal models: acute administration of high doses of +/-BayK 8644 or methamphetamine in mice, dopamine agonist treatment in rats with lesions of dopamine pathways during early development and repeated administration of pemoline in rats. The effect of nifedipine does not appear to be due to nonspecific mechanisms, such as sedation, since other classes of behaviors are unaffected or exaggerated. These results suggest that nifedipine may target a common biological mechanism in the expression of self-injurious behavior, and they suggest it should be considered in the treatment of self-injury in humans.

The role of dopamine receptors in the neurobehavioral syndrome provoked by activation of L-type calcium channels in rodents.

In rodents, activation of L-type calcium channels with +/-BayK 8644 causes an unusual behavioral syndrome that includes dystonia and self-biting. Prior studies have linked both of these behaviors to dysfunction of dopaminergic transmission in the striatum. The current studies were designed to further elucidate the relationship between +/-BayK 8644 and dopaminergic transmission in the expression of the behavioral syndrome. The drug does not appear to release presynaptic dopamine stores, since microdialysis of the striatum revealed dopamine release was unaltered by +/-BayK 8644. In addition, the behaviors were preserved or even exaggerated in mice or rats with virtually complete dopamine depletion. On the other hand, pretreatment of mice with D(3) or D(1/5) dopamine receptor antagonists attenuated the behavioral effects of +/-BayK 8644, while pretreatment with D(2) or D(4) antagonists had no effect. In D(3) receptor knockout mice, +/-BayK 8644 elicited both dystonia and self-biting, but these behaviors were less severe than in matched controls. In D(1) receptor knockout mice, behavioral responses to +/-BayK 8644 appeared exaggerated. These results argue that the behavioral effects of +/-BayK 8644 are not mediated by a presynaptic influence. Instead, the behaviors appear to result from a postsynaptic activation of the drug, which does not require but can be modified by D(3) or D(1/5) receptors.

The neonate-6-hydroxydopamine-lesioned rat: a model for clinical neuroscience and neurobiological principles.

In 1973, a technique of administering 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine) intracisternally to neonate rats was introduced to selectively reduce brain dopamine (neonate-lesioned rat). This neonate treatment proved unique when compared to rats lesioned as adults with 6-hydroxydopamine--prompting the discovery of differing functional characteristics resulting from the age at which brain dopamine is reduced. A realization was that neonate-lesioned rats modeled the loss of central dopamine and the increased susceptibility for self-injury in Lesch-Nyhan disease, which allowed identification of drugs useful in treating self-injury in mentally retarded patients. The neonate-lesioned rat has also been proposed to model the hyperactivity observed in attention-deficit hyperactivity disorder. Because the neonate-lesioned rat exhibits enhanced sensitization to repeated NMDA receptor antagonist administration and has functional changes characteristic of schizophrenia, the neonate lesioning is believed to emulate the hypothesized NMDA hypofunction in this psychiatric disorder. Besides modeling features of neurological and psychiatric disorders, important neurobiological concepts emerged from pharmacological studies in the neonate-lesioned rats. One was the discovery of coupling of D1/D2-dopamine receptor function. Another was the progressive increase in responsiveness to repeated D1-dopamine agonist administration referred to as "priming" of D1-dopamine receptor function. Additionally, a unique profile of signaling protein expression related to neonate reduction of dopamine has been identified. Thus, from modeling characteristics of disease to defining adaptive mechanisms related to neonatal loss of dopamine, the neonate-lesioned rat has had a persisting influence on neuroscience. Despite an extraordinary legacy from studies of the neurobiology of this treatment, a host of unknowns remain that will inspire future investigations.

Sustained extracellular signal-regulated kinase 1/2 phosphorylation in neonate 6-hydroxydopamine-lesioned rats after repeated D1-dopamine receptor agonist administration: implications for NMDA receptor involvement.

Extracellular signal-regulated kinase (ERK) 1/2, a well known regulator of gene expression, is likely to contribute to signaling events underlying enduring neural adaptations. Phosphorylated (phospho)-ERK was examined immunohistochemically after both single and repeated (i.e., sensitizing) doses of the partial D1-dopamine (DA) receptor agonist SKF-38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benazepine HCl) to adult rats lesioned as neonates (neonate lesioned) with 6-hydroxydopamine. Remarkably, prolonged phospho-ERK accumulated primarily in layers II-III of medial prefrontal cortex (MPC), where it declined gradually yet remained significantly elevated for at least 36 d after repeated doses of SKF-38393. Sustained (> or =7 d) phospho-ERK was observed for shorter periods in various other cortical regions but was not detectable in striatum or nucleus accumbens. At 36 d, an additional injection of SKF-38393 to sensitized rats restored phospho-ERK to maximal levels only in MPC when examined 7 d later. Phosphorylated cAMP response element-binding protein (CREB), examined 7 d after the sensitizing regimen, was observed exclusively in MPC, where it was abundant throughout all layers. Systemic injections of SL327 (alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile), an inhibitor of the upstream ERK activator mitogen ERK kinase, attenuated both ERK and CREB phosphorylation in layers II-III of MPC. Pretreatment with the D1 antagonist SCH-23390 ((R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-OL maleate) inhibited the prolonged increase in MPC phospho-ERK, whereas the 5-HT2 receptor antagonist ketanserin (3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione tartrate) was ineffective. Competitive and noncompetitive NMDA receptor antagonists also blocked sustained ERK phosphorylation. Collectively, the present results demonstrate coupling of D1 and NMDA receptor function reflected in sustained activation of the ERK signaling pathway in MPC of SKF-38393-sensitized neonate-lesioned rats. Ultimately, long-lasting phosphorylation of ERK and CREB in MPC may play a pivotal role in any permanent adaptive change(s) in these animals.

Functional selectivity of dopamine receptor agonists. II. Actions of dihydrexidine in D2L receptor-transfected MN9D cells and pituitary lactotrophs.

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-n-propyl-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 concentration-dependent manner in both systems, effects blocked by D(2), but not D(1), antagonists. In the MN9D cells, quinpirole and R-(-)-N-propylnorapomorphine (NPA) also inhibited the K(+)-stimulated release of [(3)H]dopamine in a concentration-responsive, antagonist-reversible manner. Conversely, neither DHX, nor its analogs, inhibited K(+)-stimulated [(3)H]dopamine release, although they antagonized the effects of quinpirole. S-(+)-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) receptor-mediated inhibition of adenylate cyclase). In lactotrophs, DHX had little intrinsic activity at D(2) receptors coupled to G protein-coupled 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".