Greg A. Gerhardt

Nafion-coated electrodes with high selectivity for CNS electrochemistry

A major improvement in the selectivity of small graphite electrodes used for in vivo electrochemistry is described. The electrodes are coated with Nafion, a perfluorosulfonated polymer. This coating is practically impermeable to ascorbic acid and anionic biogenic amine metabolites and only slightly responsive to neutral metabolites. Thus it becomes selective for the cationic primary neurotransmitters, dopamine, norepinephrine and 5-hydroxytryptamine. Responses of Nafion-coated and untreated electrodes in vivo are compared.

GLIAL CELL LINE- DERIVED NEUROTROPHIC FACTOR REVERSES TOXIN-INDUCED INJURY TO MIDBRAIN DOPAMINERGIC NEURONS IN VIVO

Fischer 344 rats were unilaterally injected into the medial forebrain bundle with 6-hydroxydopamine (6-OHDA). Apomorphine-induced rotational behavior was used to select animals whose rotation exceeded 300 turns/h, corresponding to greater than 95% dopamine (DA) depletion in the ipsilateral striatum. Four weeks later, glial cell line-derived neurotrophic factor (GDNF) or vehicle was injected intranigrally ipsilateral to the lesion (0.1-100 micrograms). The highest dose of GDNF tested produced a marked decrease in rotational behavior. This dose also produced levels of DA in the ipsilateral substantia nigra (SN) which were not statistically different from the contralateral side. Vehicle-treated animals showed a marked DA depletion in the ipsilateral SN. These results demonstrate neurochemical and behavioral improvements in unilaterally DA-lesioned rats following intranigral administration of GDNF, suggesting that GDNF may develop into a useful therapy for Parkinsons disease.

Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinsons disease, controversy exists concerning the use of apomorphine- or D-amphetamine-induced rotations as reliable indicators of nigrostriatal dopamine depletion. Our objective was to evaluate which, if either, drug-induced behavior is more predictive of the extent of nigrostriatal dopamine depletion. Fischer 344 and Sprague-Dawley rats were unilaterally injected with 9 micrograms/4 microliters/4 min 6-hydroxydopamine into the medial forebrain bundle. The animals were behaviorally tested with apomorphine (0.05 mg/kg, s.c.) and D-amphetamine (5.0 mg/kg, s.c.). Following testing, the brains were removed and the right and left striata, substantia nigra and ventral tegmental area were dissected free and quickly frozen at -70 degrees C for analysis of catecholamine content by high performance liquid chromatography coupled with electrochemical detection. Our results indicate that an animal which has greater than a 90% depletion of dopamine in the striatum might not rotate substantially on apomorphine, without a concomitant depletion of > 50% of the DA content in the corresponding substantia nigra. No correlations were seen involving depletions of the ventral tegmental area and the extent of the lesions to the striatum. Submaximally lesioned (75-90% depleted) rats were found to rotate on D-amphetamine but not on apomorphine. In addition, control rats that did not receive lesions were often seen to rotate extensively on D-amphetamine. We therefore conclude that maximal lesions of the striatum and substantia nigra are required to generate rotations demonstrable with low dose apomorphine but not with D-amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Chronic Antidepressant Treatments on Serotonin Transporter Function, Density, and mRNA Level

To investigate functional changes in the brain serotonin transporter (SERT) after chronic antidepressant treatment, several techniques were used to assess SERT activity, density, or its mRNA content. Rats were treated by osmotic minipump for 21 d with the selective serotonin reuptake inhibitors (SSRIs) paroxetine or sertraline, the selective norepinephrine reuptake inhibitor desipramine (DMI), or the monoamine oxidase inhibitor phenelzine. High-speedin vivo electrochemical recordings were used to assess the ability of the SSRI fluvoxamine to modulate the clearance of locally applied serotonin in the CA3 region of hippocampus in drug- or vehicle-treated rats. Fluvoxamine decreased the clearance of serotonin in rats treated with vehicle, DMI, or phenelzine but had no effect on the clearance of serotonin in SSRI-treated rats. SERT density in the CA3 region of the hippocampus of the same rats, assessed by quantitative autoradiography with tritiated cyanoimipramine ([3H]CN-IMI), was decreased by 80–90% in SSRI-treated rats but not in those treated with phenelzine or DMI. The serotonin content of the hippocampus was unaffected by paroxetine or sertraline treatment, ruling out neurotoxicity as a possible explanation for the SSRI-induced decrease in SERT binding and alteration in 5-HT clearance. Levels of mRNA for the SERT in the raphe nucleus were also unaltered by chronic paroxetine treatment. Based on these results, it appears that the SERT is downregulated by chronic administration of SSRIs but not other types of antidepressants; furthermore, the downregulation is not caused by decreases in SERT gene expression.

Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behavioral deficits in a primate model of Parkinson's disease

We report that a low-calorie diet can lessen the severity of neurochemical deficits and motor dysfunction in a primate model of Parkinsons disease. Adult male rhesus monkeys were maintained for 6 months on a reduced-calorie diet [30% caloric restriction (CR)] or an ad libitum control diet after which they were subjected to treatment with a neurotoxin to produce a hemiparkinson condition. After neurotoxin treatment, CR monkeys exhibited significantly higher levels of locomotor activity compared with control monkeys as well as higher levels of dopamine (DA) and DA metabolites in the striatal region. Increased survival of DA neurons in the substantia nigra and improved manual dexterity were noted but did not reach statistical significance. Levels of glial cell line-derived neurotrophic factor, which is known to promote the survival of DA neurons, were increased significantly in the caudate nucleus of CR monkeys, suggesting a role for glial cell line-derived neurotrophic factor in the anti-Parkinsons disease effect of the low-calorie diet.

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D2 dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D2 DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D2+/+), one (D2+/‐), or no (D2‐/‐) functional copies of the gene coding for the D2 DA receptor. In vivo microdialysis studies demonstrated that basal and K+‐evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D2‐/‐ mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D2+/+ mice. In D2+/+ mice, but not D2‐/‐ mice, local application of the D2‐like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [3H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D2 DA receptor subtype modulates activity of the striatal DAT.

Critical decline in fine motor hand movements in human aging

BACKGROUND Slowing of motor movements in human aging is a well-known occurrence, but its biologic basis is poorly understood. Reliable quantitation may refine observations of this phenomenon to better aid research on this entity. METHODS A panel equipped with timing sensors under computer control was used to measure upper extremity movement times in two groups of healthy individuals: adults younger than 60 years of age (n = 56; range, 18-58 years) and adults older than 60 years of age (n = 38; range, 61-94 years). RESULTS Fine motor performance was better in the dominant hand (p = 0.0007) regardless of age. Adult and aged groups differed on two basic timing measures, which reflect coarse motor and fine motor performance (p < 0.0001). There were no gender differences on either measure. There was a strong effect of task difficulty with age on coarse motor (p < 0.01) and fine motor (p < 0.0001) measures. The fine motor measure of hand performance in healthy individuals correlated in a nonlinear fashion with age for more difficult tasks (r2 = 0.63) but showed a simple linear relation for less-demanding tasks (r2 = 0.5). CONCLUSION This technique sensitively detects age-related motor performance decline in humans. There may be a critical period in late midlife when fine motor performance decline either begins or abruptly worsens.

Point source concentration of GDNF may explain failure of phase II clinical trial.

Significant differences have been reported in results from three clinical trials evaluating intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinsons disease. To determine if problems in drug bioavailability could have contributed to the discrepancies between studies, we have analyzed the distribution of intraputamenally infused GDNF in the rhesus monkey brain using the delivery system and infusion protocol followed in a phase 2 clinical trial that failed to achieve its primary endpoint. I125-GDNF was unilaterally infused into the putamen of three adult rhesus monkeys for 7 days. Three age- and sex-matched animals received vehicle infusions following identical procedures. GDNF levels in the brain, peripheral organs, blood and CSF were quantified and mapped by GDNF immunocytochemistry, GDNF ELISAs and I125 measurements. Infused GDNF was found to be unevenly concentrated around the catheter, with tissue levels dropping exponentially with increasing distance from the point source of the single opening in the catheter tip. The volume of distribution of GDNF around the catheter, as determined by immunocytochemistry, varied over four-fold between animals ranging from 87 to 369 mm3. The concentration of GDNF around the catheter tip and limited diffusion into surrounding brain parenchyma support the hypothesis that drug bioavailability was limited to a small portion (2-9%) of the human putamen in the clinical trial using this catheter and infusion protocol.

Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS.

This paper describes improvements and further characterization of a ceramic-based multisite microelectrode for in vivo measurements of L-glutamate. Improvements include increased recording area, insulation deposition using photolithography for more uniform recording sites and forming the microelectrodes using a diamond saw providing smoother microelectrode edges. The new microelectrodes are triangular in shape, 1 cm in length and taper from 1 mm to a 2-5 microm tip. Details on performing in vivo measurements are given, including microelectrode preparation, pitfalls of the recording method and approaches to enhance reproducibility of the technique. The detection limit for L-glutamate was lowered to approximately 0.5 microM and a self-referencing recording technique was utilized to remove interferents as well as decrease noise. Applications of the microelectrodes to study L-glutamate uptake and release in rat prefrontal cortex, cortex, cerebellum and striatum are included.