Peter Huettl

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.

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.

Microelectrode array studies of basal and potassium-evoked release of l-glutamate in the anesthetized rat brain

l‐glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system. It plays major roles in normal neurophysiology and many brain disorders by binding to membrane‐bound Glu receptors. To overcome the spatial and temporal limitations encountered in previous in vivo extracellular Glu studies, we employed enzyme‐coated microelectrode arrays to measure both basal and potassium‐evoked release of Glu in the anesthetized rat brain. We also addressed the question of signal identity, which is the predominant criticism of these recording technologies. In vivo self‐referencing recordings demonstrated that our Glu signals were both enzyme‐ and voltage‐dependent, supporting the identity of l‐glutamate. In addition, basal Glu was actively regulated, tetrodotoxin (TTX)‐dependent, and measured in the low micromolar range (approximately 2 µm) using multiple self‐referencing subtraction approaches for identification of Glu. Moreover, potassium‐evoked Glu release exhibited fast kinetics that were concentration‐dependent and reproducible. These data support the hypothesis that Glu release is highly regulated, requiring detection technologies that must be very close to the synapse and measure on a second‐by‐second basis to best characterize the dynamics of the Glu system.

Prefrontal Cortex and Drug Abuse Vulnerability: Translation to Prevention and Treatment Interventions

Vulnerability to drug abuse is related to both reward seeking and impulsivity, two constructs thought to have a biological basis in the prefrontal cortex (PFC). This review addresses similarities and differences in neuroanatomy, neurochemistry and behavior associated with PFC function in rodents and humans. Emphasis is placed on monoamine and amino acid neurotransmitter systems located in anatomically distinct subregions: medial prefrontal cortex (mPFC); lateral prefrontal cortex (lPFC); anterior cingulate cortex (ACC); and orbitofrontal cortex (OFC). While there are complex interconnections and overlapping functions among these regions, each is thought to be involved in various functions related to health-related risk behaviors and drug abuse vulnerability. Among the various functions implicated, evidence suggests that mPFC is involved in reward processing, attention and drug reinstatement; lPFC is involved in decision-making, behavioral inhibition and attentional gating; ACC is involved in attention, emotional processing and self-monitoring; and OFC is involved in behavioral inhibition, signaling of expected outcomes and reward/punishment sensitivity. Individual differences (e.g., age and sex) influence functioning of these regions, which, in turn, impacts drug abuse vulnerability. Implications for the development of drug abuse prevention and treatment strategies aimed at engaging PFC inhibitory processes that may reduce risk-related behaviors are discussed, including the design of effective public service announcements, cognitive exercises, physical activity, direct current stimulation, feedback control training and pharmacotherapies. A major challenge in drug abuse prevention and treatment rests with improving intervention strategies aimed at strengthening PFC inhibitory systems among at-risk individuals.

Chronic second‐by‐second measures of l‐glutamate in the central nervous system of freely moving rats

l‐glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system (CNS) and is associated with motor behavior and sensory perception. While microdialysis methods have been used to record tonic levels of Glu, little is known about the more rapid changes in Glu signals that may be observed in awake rats. We have reported acute recording methods using enzyme‐based microelectrode arrays (MEA) with fast response time and low detection levels of Glu in anesthetized animals with minimal interference. The current paper concerns modification of the MEA design to allow for reliable measures in the brain of conscious rats. In this study, we characterized the effects of chronic implantation of the MEA into the brains of rats. We were capable of measuring Glu levels for 7 days without loss of sensitivity. We performed studies of tail‐pinch induced stress, which caused a robust biphasic increase in Glu. Histological data show chronic implantation of the MEAs caused minimal injury to the CNS. Taken together, our data show that chronic recordings of tonic and phasic Glu can be carried out in awake rats for up to 17 days in vivo allowing longer term studies of Glu regulation in behaving rats.

Rapid assessment of in vivo cholinergic transmission by amperometric detection of changes in extracellular choline levels.

Conventional microdialysis methods for measuring acetylcholine (ACh) efflux do not provide sufficient temporal resolution to relate cholinergic transmission to individual stimuli or behavioral responses, or sufficient spatial resolution to investigate heterogeneities in such regulation within a brain region. In an effort to overcome these constraints, we investigated a ceramic‐based microelectrode array designed to measure amperometrically rapid changes in extracellular choline as a marker for cholinergic transmission in the frontoparietal cortex of anesthetized rats. These microelectrodes exhibited detection limits of 300 nm for choline and selectivity (> 100 : 1) of choline over interferents such as ascorbic acid. Intracortical pressure ejections of choline (20 mm, 66–400 nL) and ACh (10 and 100 mm, 200 nL) dose‐dependently increased choline‐related signals that were cleared to background levels within 10 s. ACh, but not choline‐induced signals, were significantly attenuated by co‐ejection of the acetylcholinesterase inhibitor neostigmine (Neo; 100 mm). Pressure ejections of drugs known to increase cortical ACh efflux, potassium (KCl; 70 mm, 66, 200 nL) and scopolamine (Scop; 10 mm, 200 nL), also markedly increased extracellular choline signals, which again were inhibited by Neo. Scop‐induced choline signals were also found to be tetrodotoxin‐sensitive. Collectively, these findings suggest that drug‐induced increases in current measured with these microelectrode arrays reflect the oxidation of choline that is neuronally derived from the release and subsequent hydrolysis of ACh. Choline signals assessed using enzyme‐selective microelectrode arrays may represent a rapid, sensitive and spatially discrete measure of cholinergic transmission.

GDNF improves dopamine function in the substantia nigra but not the putamen of unilateral MPTP-lesioned rhesus monkeys

Microdialysis measurements of dopamine (DA) and DA metabolites were carried out in the putamen and substantia nigra of unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rhesus monkeys that received intraventricular injections of vehicle or glial-derived neurotrophic factor (GDNF, 300 microg) 3 weeks prior to the microdialysis studies. Following behavioral measures in the MPTP-lesioned monkeys, they were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging (MRI)-guided sterile stereotaxic procedures were used for implantations of the microdialysis probes. Basal extracellular levels of DA and the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were found to be decreased by >95% in the right putamen of the MPTP-lesioned monkeys as compared to normal animals. In contrast, basal DA levels were not significantly decreased, and DOPAC and HVA levels were decreased by only 65% and 30%, respectively, in the MPTP-lesioned substantia nigra. Significant reductions in d-amphetamine-evoked DA release were also observed in the MPTP-lesioned substantia nigra and putamen of the monkeys as compared to normal animals. A single intraventricular administration of GDNF into one group of MPTP-lesioned monkeys elicited improvements in the parkinsonian symptoms in these animals at 2-3 weeks post-administration. In addition, d-amphetamine-evoked overflow of DA was significantly increased in the substantia nigra but not the putamen of MPTP-lesioned monkeys that had received GDNF. Moreover, post-mortem brain tissue studies showed increases in whole tissue levels of DA and DA metabolite levels primarily within the substantia nigra in MPTP-lesioned monkeys that had received GDNF. Taken together, these data support that single ventricular infusions of GDNF produce improvements in motoric behavior in MPTP-lesioned monkeys that correlate with increases in DA neuronal function that are localized to the substantia nigra and not the putamen.

Ceramic-based multisite microelectrode arrays for simultaneous measures of choline and acetylcholine in CNS

A ceramic-based microelectrode array (MEA) with enzyme coatings for the accurate measurement of acetylcholine (ACh) in brain tissues is presented. Novel design features allow for self-referencing recordings for improved limits of detection and highly selective measurements of ACh and choline (Ch), simultaneously. Design and fabrication features also result in minimal tissue damage during implantation and improved enzyme coatings due to isolated recording sites. In these studies we have used a recombinant human acetylcholinesterase enzyme coating, which has better reproducibility than other commercially available enzymes. The precisely patterned recording site dimensions, low limit of detection (0.2 micro M) and fast response time ( approximately 1s) allow for second-by-second measurements of ACh and Ch in brain tissues. An electropolymerized meta-phenylenediamine (mPD) layer was used to exclude interfering substances from being recorded at the platinum recording sites. Our studies support that the mPD layer was stable for over 24h under in vitro and in vivo recording conditions. In addition, our work supports that the current configuration of the MEAs produces a robust design, which is suited for measures of ACh and Ch in rat brain.

Auditory P50 in Schizophrenics on Clozapine: Improved Gating Parallels Clinical Improvement and Changes in Plasma 3-Methoxy-4-Hydroxyphenylglycol

Schizophrenic patients have decreased inhibition of the P50 auditory evoked potential response to the second of two paired click stimuli delivered 500 ms apart. This deficit in inhibitory gating does not change during treatment with typical neuroleptics. We recently reported that neuroleptic-resistant schizophrenics had enhanced P50 gating after 1 month of clozapine treatment, if they responded with decreased clinical symptoms. This study reports the outcome of more prolonged treatment. Ten treatment-refractory schizophrenic patients were studied at baseline, after 1 month on clozapine, and again after 15 ± 6.1 (SD) months of clozapine treatment. Eight subjects reached a clinically stable improved state, at which time they had significantly improved P50 auditory gating. One patient had a return of impaired gating after stopping clozapine, as did another during a clinical relapse. Decreasing plasma 3-methoxy-4-hydroxyphenylglycol levels with clozapine treatment were correlated with improved P50 gating and improved Brief Bsychiatric Rating Scale-positive scores. This study provides further evidence that improved P50 gating in schizophrenic patients treated with clozapine coincides with clinical improvement and that this improvement can be sustained for at least 1 year.

Diffuse Brain Injury Elevates Tonic Glutamate Levels and Potassium-Evoked Glutamate Release in Discrete Brain Regions at Two Days Post-Injury: An Enzyme-Based Microelectrode Array Study

Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI.