In vivo electrochemical measurements of serotonin clearance in rat striatum: effects of neonatal 6-hydroxydopamine-induced serotonin hyperinnervation and serotonin uptake inhibitors.

Diffusion and clearance of extracellular serotonin (5-HT) was examined using in vivo chronoamperometry with "delayed-pulse" recordings after pressure ejections of 1 to 60 picomoles 5-HT into rat striatum at a fixed distance from a Nafion-coated carbon fiber electrode. Signals obtained were identified based on the signal characteristics to consist of 5-HT. Clearance times of 5-HT decreased, while amplitudes and rise times increased with serotonergic hyperinnervation induced by neonatal 6-hydroxydopamine (6-OHDA) lesions of dopamine (DA) neurons. Local applications of the 5-HT uptake inhibitors zimelidine or fluoxetine, in conjunction with 5-HT ejections, produced increased clearance times in both normal and 6-OHDA-treated animals. Thus, direct in vivo evidence was obtained for the importance of high affinity nerve terminal uptake as a key mechanism for clearance of 5-HT from the extracellular space. Inhibitors of 5-HT uptake appear to prolong the extracellular presence of 5-HT by increasing its clearance time.

o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide.

Nitric oxide (NO.) sensors were prepared using o-phenylenediamine (o-PD) and Nafion to modify the surface of 30 microns diameter carbon fiber electrodes. These electrodes were compared with nickel porphyrin-type NO. sensors that have already been described. High-speed chronoamperometry, amperometry, and differential pulse voltammetry were used to compare the performance of sensors modified with various combinations of Nafion, o-PD, or nickel(II) meso-tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin (Ni-TMPP), in order to determine which electrodes had the most sensitivity and selectivity for NO. Our findings showed that electrodes treated with Nafion first, followed by o-PD, were very sensitive to NO., with a detection limit of 35 +/- 7 nM. In addition, o-PD electrodes were also very selective against ascorbate (> 600:1), dopamine (> 300:1), and nitrite (> 900:1). Moreover, in the range of 0-6 microM NO., o-PD electrodes displayed excellent linearity (R2 > or = 0.997). In contrast, Ni-TMPP electrodes (with Nafion) had significantly poorer detection limits (76 +/- 12 nM) and were less selective against dopamine (< 5:1) and nitrite (< 200:1). Ni-TMPP electrodes were also less linear than o-PD electrodes (R2 > or = 0.911). Finally, we tested the in vitro and in vivo performance of the o-PD electrode in terms of its ability to detect NO. release from isolated rat renal arterioles and to measure NO. diffusion in the extracellular space of the rat brain.

In vivo electrochemical studies of the dynamic effects of locally applied excitatory amino acids in the striatum of the anesthetized rat.

The actions of locally applied excitatory amino acids (EAAs) on dopamine nerve terminals in the striatum of the urethane-anesthetized rat were investigated. Rapid (5 Hz) in vivo electrochemical recording was used to measure the amplitude and duration of dopamine (DA) overflow elicited by the local application of glutamate, N-methyl-D- aspartate (NMDA), kainate, quisqualate, quinolinate and DL- alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid. EAAs were pressure ejected into the striatum via a pipette positioned 300 +/- 30 mum away from the electrochemical working electrode. Brief (5s) applications of the EAA agonists directly elicited DA-like electrochemical signals with amplitudes averaging about 2 microM. In some instances, putative increases in ascorbic acid-like signals were detected. Repeated applications of glutamate agonists in the same brain site resulted in diminished electrochemical responses, compared to the complete reproducibility seen after repeated applications of 120 mM potassium. Low doses of NMDA (10 mM barrel conc), which did not cause a detectable increase in the electrochemical baseline signal, significantly potentiated (50%) potassium-evoked DA overflow. These results indicate that low levels of endogenously released glutamate may modulate overflow when DA nerve terminals are depolarized, while higher concentrations of glutamate may directly elicit increases in extracellular levels of DA and/or ascorbic acid.

Effects of the putative antidepressant, ABT 200, on the clearance of exogenous norepinephrine in rat cerebellum.

ABT 200 [(RR,SS)-3-phenyl-1-[1',2',3',4'-tetrahydro-5',6'-methylenedioxy- 1'-naphthalenyl-methyl]-pyrrolidine methanesulfonate] is a potent alpha 2-adrenoceptor antagonist (Ki = 1.2 nM) with modest norepinephrine uptake-blocking activity (IC50 = 841 nM) that is currently under clinical evaluation as an antidepressant. The effects of ABT 200, nomifensine (an inhibitor of catecholamine uptake), and rauwolscine (a selective alpha 2-adrenoceptor antagonist) on the clearance of exogenous norepinephrine in the cerebellum of urethane-anesthetized rats was investigated using a vivo electrochemistry. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated, single carbon fiber electrodes. When a calibrated amount of norepinephrine was pressure-ejected at 5-min intervals from a micropipette adjacent (290-330 microM) to the electrode, transient and reproducible norepinephrine signals were detected. In response to systemic ABT 200 (30 mg/kg i.p.) or nomifensine (30 mg/kg i.p.), the signals increased in both amplitude and time course, indicating significant inhibition of the norepinephrine transporter. A lower dose (15 mg/kg i.p.) of either ABT 200 or nomifensine had no effect in this paradigm. Local application of ABT 200 (400 microM) or nomifensine (400 microM) prior to pressure-ejection of norepinephrine also significantly increased the amplitude and time course of the norepinephrine signals. In contrast, systemic administration of rauwolscine (30 mg/kg i.p.) or vehicle solution, and local application of vehicle solution, had no effect on the norepinephrine signals. These data indicate that at the higher dose evaluated, both ABT 200 and nomifensine inhibit cerebellar norepinephrine uptake in vivo.

In vivo electrochemical measurements of exogenous dopamine clearance in normal and neonatal 6-hydroxydopamine-treated rat striatum.

The regulation of extracellular dopamine (DA) levels was studied in rat striatum after neonatal dopamine lesions and enhanced serotonin (5-HT) fiber ingrowth, induced with 6-hydroxydopamine (6-OHDA). We used rapid in vivo chronoamperometry combined with local DA applications. DA was pressure ejected in doses of 5 to 100 pmol at a distance of approximately 300 microns from the recording electrode, using single- or multibarrel glass micropipettes. Almost twice as much DA had to be applied in control rat dorsal and ventral striatum to obtain signals comparable to those recorded in the neonatal 6-OHDA-treated animals. In addition, in the dorsal striatum, the later portions of the DA clearance signals were significantly prolonged in the 6-OHDA group. Some clearance decay times in ventral striatum were also significantly prolonged in the neonatal 6-OHDA-treated rats. Concomitant application of the DA uptake inhibitor, nomifensine, in conjunction with the DA ejections, produced signal characteristics in the control striatum that were similar to those seen in the neonatal 6-OHDA-treated striatum. Taken together, these data support the hypothesis that a major component of the clearance of extracellular DA is dependent on intact terminals with high-affinity DA uptake, and that loss of DA afferents from neonatal 6-OHDA treatment results in a slowed clearance time of extracellular DA which is not apparently compensated by the enhanced 5-HT fiber ingrowth.

Regional effects of aging on dopaminergic function in the Fischer-344 rat.

To examine the consequences of aging on nigrostriatal (A9) and mesolimbic (A10) dopamine (DA) function, neurochemical and behavioral measurements were performed in male Fischer-344 rats 6, 12, 18, 24, and 30 months old. Regional analyses of dopaminergic overflow and uptake processes were examined using high-speed (5 Hz) in vivo electrochemical recordings and local drug application techniques. When potassium was used to elicit the presynaptic overflow of dopamine (DA) in striatal areas predominantly innervated by the substantia nigra, the amplitude of DA overflow was significantly lower in 24- and 30-month-old rats (p less than 0.05 and p less than 0.01 vs. 6 months, respectively). Furthermore, in ventral striatum (including the nucleus accumbens) which is innervated primarily by A10 DA cell bodies, potassium-evoked DA overflow release amplitudes were significantly lower in the 18, 24, and 30 month groups (p less than 0.01 vs. 6 months). In addition, age-related differences between the dorsal and ventral striatum were found in a preliminary investigation of DA diffusion and clearance. Local application of nomifensine, a DA uptake inhibitor, significantly increased (p less than 0.05 vs. control) the amplitude of the signal recorded after local application of 25-30 pmol DA in the ventral striatum of 6 month-old but not 24-month-old rats. High-performance liquid chromatography coupled with electrochemical detection (HPLC-EC) was used to analyze whole striatal DA levels, DA metabolite levels and turnover indices. However, no age-related differences in any of these variables were observed. Finally, a rod walking test was used to measure motor coordination and balance in animals prior to in vivo electrochemical recording.(ABSTRACT TRUNCATED AT 250 WORDS)