Glial cell line-derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non-human primates, in animal models of Parkinson's disease, and may improve motoric function in patients with advanced Parkinson's disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 lg of GDNF was injected into the striatum of normal aged (24-month-old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot-immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF-treated rats, TH phosphorylation at Ser31 increased 40% in striatum and 250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine-and potassiumevoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF-induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.

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Summary These experiments utilized an enzyme-based microelectrode selective for the second-by-second detection of extracellular glutamate to reveal the α7-based nicotinic modulation of glutamate release in the prefrontal cortex (PFC) of freely moving rats. Rats received intra-cortical infusions of the non-selective nicotinic agonist nicotine (1.0 μg/0.4 μL) or the selective α7 agonist choline (2.0 mM/0.4 μL). The selectivity of drug-induced glutamate release was assessed in subgroups of animals pre-treated with the α7 antagonist, α-bungarotoxin (α-BGT, 10 μM) or kynurenine (10 μM) the precursor of the astrocyte-derived, negative allosteric α7 modulator kynurenic acid. Local administration of nicotine increased glutamate signals (maximum amplitude = 4.3 ± 0.6 μM) that were cleared to baseline levels in 493 ± 80 sec. Pre-treatment with α-BGT or kynurenine attenuated nicotine-induced glutamate by 61% and 60%, respectively. Local administration of choline also increased glutamate signals (maximum amplitude = 6.3 ± 0.9 μM). In contrast to nicotine-evoked glutamate release, choline-evoked signals were cleared more quickly (28 ± 6 sec) and pre-treatment with α-BGT or kynurenine completely blocked the stimulated glutamate release. Using a method that reveals the temporal dynamics of in vivo glutamate release and clearance, these data indicate a nicotinic modulation of cortical glutamate release that is both α7 – and non-α7-mediated. Furthermore, these data may also provide a mechanism underlying the recent focus on α7 full and partial agonists as therapeutic agents in the treatment of cortically-mediated cognitive deficits in schizophrenia.

Using two in vivo methods, microdialysis and rapid in situ electrochemistry, this study examined the modulation of extracellular glutamate levels by endogenously produced kynurenic acid (KYNA) in the prefrontal cortex (PFC) of awake rats. Measured by microdialysis, intraperitoneal (i.p.) administration of KYNA's bioprecursor L-kynurenine dose-dependently elevated extracellular KYNA and reduced extracellular glutamate (nadir after 50 mg/kg kynurenine: 60% decrease from baseline values). This dose-dependent decrease in glutamate levels was also seen using a glutamate-sensitive microelectrode array (MEA) (31% decrease following 50 mg/kg kynurenine). The kynurenine-induced reduction in glutamate was blocked (microdialysis) or attenuated (MEA) by co-administration of galantamine (3 mg/kg, i.p.), a drug that competes with KYNA at an allosteric potentiating site of the α7 nicotinic acetylcholine receptor. In separate experiments, extracellular glutamate levels were measured by MEA following the local perfusion (45 min) of the PFC with kynurenine (2.5 μM) or the selective KYNA biosynthesis inhibitor S-ethylsulfonylbenzoylalanine (S-ESBA; 5 mM). In agreement with previous microdialysis studies, systemic kynurenine application produced a reversible reduction in glutamate (nadir: −29%), whereas perfusion with S-ESBA increased glutamate levels reversibly (maximum: +38%). Collectively, these results demonstrate that fluctuations in the biosynthesis of KYNA in the PFC bi-directionally modulate extracellular glutamate levels, and that qualitatively very similar data are obtained by microdialysis and MEA. Since KYNA levels are elevated in the PFC of individuals with schizophrenia, and since prefrontal glutamatergic and nicotinic transmission mediate cognitive flexibility, normalization of KYNA levels in the PFC may constitute an effective treatment strategy for alleviating cognitive deficits in schizophrenia.

Microdialysis has been widely used to measure acetylcholine (ACh) release in vivo and has provided important insights into the regulation of cholinergic transmission. However, microdialysis can be constrained by limited spatial and temporal resolution. The present experiments utilize a microelectrode array (MEA) to rapidly measure ACh release and clearance in anaesthetized rats. The array electrochemically detects, on a second-by-second basis, changes in current selectively produced by the hydrolysis of ACh to choline (Ch) and the subsequent oxidation of choline and hydrogen peroxidase (H(2)O(2)) at the electrode surface. In vitro calibration of the microelectrode revealed linear responses to ACh (R(2) = 0.9998), limit of detection of 0.08 microm, and signal-to-noise ratio of 3.0. The electrode was unresponsive to ascorbic acid (AA), dopamine (DA), or norepinephrine (NE) interferents. In vivo experiments were conducted in prefrontal cortex (PFC) of anaesthetized rats. Pressure ejections of ACh (10 mm; 40 nL) through an adjoining micropipette produced a rapid rise in current, reaching maximum amplitude in approximately 1.0 s and cleared by 80% within 4-11 s. Endogenously released ACh, following local depolarization with KCl (70 mm; 40, 160 nL), was detected at values as low as 0.05 microm. These signals were volume-dependent and cleared within 4-12 s. Finally, nicotine (1.0 mm, 80 nL) stimulated ACh signals. Nicotine-induced signals reflected the hydrolysis of ACh by endogenous acetylcholinesterase (AChE) as inhibition of the enzyme following perfusion with neostigmine (10 microm) attenuated the signal (40-94%). Collectively, these data validate a novel method for rapidly measuring cholinergic transmission in vivo with a spatial and temporal resolution that far exceeds conventional microdialysis.