Lawrence D. Middaugh

Homer Proteins Regulate Sensitivity to Cocaine

Drug addiction involves complex interactions between pharmacology and learning in genetically susceptible individuals. Members of the Homer gene family are regulated by acute and chronic cocaine administration. Here, we report that deletion of Homer1 or Homer2 in mice caused the same increase in sensitivity to cocaine-induced locomotion, conditioned reward, and augmented extracellular glutamate in nucleus accumbens as that elicited by withdrawal from repeated cocaine administration. Moreover, adeno-associated virus-mediated restoration of Homer2 in the accumbens of Homer2 KO mice reversed the cocaine-sensitized phenotype. Further analysis of Homer2 KO mice revealed extensive additional behavioral and neurochemical similarities to cocaine-sensitized animals, including accelerated acquisition of cocaine self-administration and altered regulation of glutamate by metabotropic glutamate receptors and cystine/glutamate exchange. These data show that Homer deletion mimics the behavioral and neurochemical phenotype produced by repeated cocaine administration and implicate Homer in regulating addiction to cocaine.

Homer2 Is Necessary for EtOH-Induced Neuroplasticity

Homer proteins are integral to the assembly of proteins regulating glutamate signaling and synaptic plasticity. Constitutive Homer2 gene deletion [knock-out (KO)] and rescue with adeno-associated viral (AAV) transfection of Homer2b was used to demonstrate the importance of Homer proteins in neuroplasticity produced by repeated ethanol (EtOH) administration. Homer2 KO mice avoided drinking high concentrations of EtOH and did not develop place preference or locomotor sensitization after repeated EtOH administration. The deficient behavioral plasticity to EtOH after Homer2 deletion was paralleled by a lack of augmentation in the rise in extracellular dopamine and glutamate elicited by repeated EtOH injections. The genotypic differences in EtOH-induced change in behavior and neurochemistry were essentially reversed by AAV-mediated transfection of Homer2b into accumbens cells including, differences in EtOH preference, locomotor sensitization, and EtOH-induced elevations in extracellular glutamate and dopamine. These data demonstrate a necessary and active role for accumbens Homer2 expression in regulating EtOH-induced behavioral and cellular neuroplasticity.

Ethanol Consumption by C57BL/6 Mice: Influence of Gender and Procedural Variables

Both sexes of C57BL/6 (C57) mice consumed substantial quantities of ethanol without food or water deprivation whether access was continuous or limited. Food deprivation increased the amount of ethanol consumed, and the amount consumed depended upon when the animals were tested with reference to their daily food allotment. Ethanol consumption was greater if the mice were tested postprandially, high thirst motivation, rather than preprandially (approximately 10 vs. approximately 4.5 g/kg/30 min). Preference for ethanol over water, however, was greater when mice were under low thirst motivation (i.e., tested preprandially or with water available during the test). Compared to males, female mice consumed more of a high-ethanol concentration solution (10%) when access was continuous or limited to the first hour of the dark (active) phase of the circadian cycle. Also, in contrast to males, female mice exhibited increased ethanol consumption across days of drinking experience. Finally, although ethanol consumption under the food deprivation conditions of this experiment did not differ according to sex, females had higher blood ethanol concentrations than male C57 mice, a finding not previously reported for rodents but common to humans.

Developmental neurotoxicity of anticonvulsants: human and animal evidence on phenytoin.

Most epileptic women delivering children each year take anticonvulsants throughout pregnancy. The teratogenic potential of anticonvulsants is most notable for phenytoin, trimethadione, valproic acid, and carbamazepine. This review focuses on the human and animal evidence for the teratogenicity of phenytoin, with emphasis on neurobehavioral end points. The Fetal Hydantoin Syndrome (FHS) consists of craniofacial defects and any two of the following: pre/postnatal growth deficiency, limb defects, major malformations, and mental deficiency. Available data suggest a prevalence of FHS of 10-30% in infants of women ingesting 100-800 mg/kg of phenytoin during the first trimester or beyond. Unfortunately, data on neurobehavioral development in FHS children is limited. Animal models of FHS have been developed and those focusing on neurobehavioral effects are reviewed. Phenytoin produces multiple behavioral dysfunctions in rat offspring at subteratogenic and nongrowth retarding doses. These behaviorally teratogenic doses produce maternal serum phenytoin concentrations in rats comparable to those found in humans. The dysfunctions in rats are dose-dependent and exposure-period-dependent, but independent of nutritional, maternal rearing, or seizure disorder confounds. Effects include vestibular dysfunction, hyperactivity and deficits in learning and memory. General comparability between the human and animal findings for phenytoin are apparent, however, difficulties with existing studies prevent precise comparisons. Animal studies have not dealt satisfactorily with the potential contribution of epileptic disease state to the FHS, with fetal brain drug concentration determinations, a complete dose-effect range, effects in multiple species (although limited nonhuman primate data exist), site of CNS injury, and the comparability of end points assessed. Human studies have not dealt satisfactorily with issues of the need for prospective study designs, separation of the effects of different anticonvulsants, or adequate long-term follow-up of cases, especially with attention to neuropsychological assessment.

Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines.

Abstract Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase in vitro . Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the in vivo effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.

A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for peripheral organs, spinal cord, and midbrain dopamine (DA) neurons. Levels of GDNF deteriorate in the substantia nigra in Parkinsons disease (PD). A heterozygous mouse model was created to assess whether chronic reductions in this neurotrophic factor impact motor function and the nigrostriatal dopamine system during the aging process. Due to the important role GDNF plays in kidney development, kidney function and histology were assessed and were found to be normal in both wild-type (WT) and GDNF+/- mice up to 22 months of age. Further, the animals of both genotypes had similar weights throughout the experiment. Locomotor activity was assessed for male WT and GDNF+/- mice at 4-month intervals from 4 to 20 months of age. Both GDNF+/- and WT mice exhibited an age-related decline in horizontal activity, although this was found 4 months earlier in GDNF+/- mice, at 12 months of age. Comparison of young (8 month old) and aged (20 month old) GDNF+/- and WT mice on an accelerating rotarod apparatus established a deficiency for aged but not young GDNF+/- mice, while aged WT mice performed as well as young WT mice on this task. Finally, both WT and GDNF+/- mice exhibited an age-related decrease in substantia nigra TH immunostaining, which was accelerated in the GDNF+/- mice. These behavioral and histological alterations suggest that GDNF may be an important factor for maintenance of motor coordination and spontaneous activity as well as DA neuronal function during aging, and further suggest that GDNF+/- mice may serve as a model for neuroprotective or rescue studies.

Chronic ethanol consumption by C57BL/6 mice promotes tolerance to its interoceptive cues and increases extracellular dopamine, an effect blocked by naltrexone.

BACKGROUND C57BL/6 (B6) mice voluntarily consume ethanol. Although preingestive factors might be accountable, the fact that B6 mice voluntarily consume sufficient ethanol to set the conditions for an ethanol-deprivation effect suggest that post-ingestive pharmacological induced changes also occur. In this study, we determined the amounts of ethanol voluntarily consumed by B6 mice and associated blood ethanol levels (BEL), the effects of this consumption on extracellular dopamine (DA) and how this was altered by naltrexone, as well as on its interoceptive discriminative cues. METHODS In experiment 1, the amounts of 12% ethanol consumed at 2, 4, and 6 hr into the active phase of the circadian cycle and associated BEL were determined. In experiment 2, dialysate samples were collected for 1 hr to establish basal DA levels. Mice were then injected with saline or naltrexone (6 mg/kg) and given access to water and 12% ethanol or to water only, and samples were collected at 20-min intervals for the next 2 hr. In experiment 3, mice were trained to discriminate ethanols interoceptive cues via operant techniques, and half were given 3 weeks access to ethanol and water, the other half water only. Ethanol-consuming and water control mice were again tested for their ability to discriminate the drugs interoceptive cues. RESULTS Mice ingested nearly 6 g/kg of ethanol and attained BEL near 100 mg/100 mL by 6 hr into the active phase. Ethanol intake at 2-hr into the dark phase was approximately 2.5 g/kg, and increased DA to approximately 100% above basal levels. Naltrexone reduced ethanol consumption and blocked the DA increase. Ethanol consumption for 3 weeks attenuated its discriminative cues. CONCLUSIONS B6 mice voluntarily consume sufficient ethanol (1) to produce intoxicating BEL; (2) to increase DA levels in nucleus accumbens, an effect blocked by naltrexone; and (3) to attenuate its discriminative cues.

Long-Term Consequences of Methamphetamine Exposure in Young Adults Are Exacerbated in Glial Cell Line-Derived Neurotrophic Factor Heterozygous Mice

Methamphetamine abuse in young adults has long-term deleterious effects on brain function that are associated with damage to monoaminergic neurons. Administration of glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons from the toxic effects of methamphetamine in animal models. Therefore, we hypothesized that a partial GDNF gene deletion would increase the susceptibility of mice to methamphetamine neurotoxicity during young adulthood and possibly increase age-related deterioration of behavior and dopamine function. Two weeks after a methamphetamine binge (4 × 10 mg/kg, i.p., at 2 h intervals), GDNF+/− mice had a significantly greater reduction of tyrosine hydroxylase immunoreactivity in the medial striatum, a proportionally greater depletion of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum, and a greater increase in activated microglia in the substantia nigra than wild-type mice. At 12 months of age, methamphetamine-treated GDNF+/− mice exhibited less motor activity and lower levels of tyrosine hydroxylase-immunoreactivity, dopamine, DOPAC, and serotonin than wild-type mice. Greater striatal dopamine transporter activity in GDNF+/− mice may underlie their differential response to methamphetamine. These data suggest the possibility that methamphetamine use in young adults, when combined with lower levels of GDNF throughout life, may precipitate the appearance of parkinsonian-like behaviors during aging.

Operant ethanol reward in C57BL/6 mice : Influence of gender and procedural variables

Food-deprived C57BL/6 (C57) mice of either sex responded for oral ethanol rewards delivered on ratio schedules of reinforcement, thus extending to female C57 mice effects previously reported only for male members of the strain. Lever responding for ethanol reward was influenced by thirst motivation (post- vs. preprandial tests), time of access to ethanol reward, ethanol concentration, and reinforcement schedule. A particularly high response output for 12% ethanol delivered on a PR2 schedule (e.g., approximately 1400/15 min test session) indicates its efficacy as a reinforcer for C57 mice. Estimated consumption of ethanol differed from lever responding when reward access time was relatively long (10 s) and response demand of the reinforcement schedule was low, but paralleled lever responding when reward access time was restricted (3 s) and response demands were greater. Gender influenced lever responding for ethanol reward and its consumption, the difference depending upon reward access time and reinforcement schedule. When the response demands were low and the reward access time long, females tended to respond more than males for ethanol reward; with greater response demands and shorter reward access time, males tended to respond more. In conjunction with our companion report, the present study helps define the behavioral conditions under which ethanol is rewarding for C57 mice and establish the conditions under which ethanol reward differs for male and female mice.

SCID mice with HIV encephalitis develop behavioral abnormalities

Severe combined immunodeficient (SCID) mice inoculated intracerebrally (i.c.) with HIV-infected human monocytes develop brain pathology similar to that in humans with HIV encephalitis. This includes HIV-positive macrophages and multinucleated giant cells, astrogliosis, microglial nodules, and neuronal dropout. These xenografts survive about 1 month. To develop a model of chronic HIV encephalitis and to assay the resulting behavioral abnormalities, we reinoculated SCID mice i.c. every 4 weeks for 3 months with either HIV-infected human monocytes (n = 5) or uninfected human macrophages (n = 4) or administered no inoculation (n = 6); these three groups were monitored for behavioral abnormalities. Tests of cognitive function in a Morris water maze 3.5 months after the first inoculation suggested that HIV-infected mice performed poorly compared with controls. Following testing in the water maze on days 4 and 5 of acquisition, motor activity of infected mice was reduced in comparison with that of controls. Retention of goal location when tested 1 week later was impaired in HIV-infected mice compared with controls. Histopathologic analysis of brains revealed significant astrogliosis and strongly suggested higher numbers of major histocompatibility complex (MHC) class II-positive multinucleated macrophages in HIV-infected compared with control mice. Thus, our preliminary studies indicate that SCID mice with HIV encephalitis develop behavioral abnormalities reminiscent of human disease. These behavioral abnormalities are associated with significantly increased astrogliosis, the presence of HIV, and probably multinucleated giant cells. These studies further support the use of this SCID animal model system for studies of the pathogenesis of HIV encephalitis and for drug interventions.