In-vitro and in-vivo performance studies of a porous infusion catheter designed for intraparenchymal delivery of therapeutic agents of varying size.

BACKGROUND: Limitations have previously existed for the use of brain infusion catheters with extended delivery port designs to achieve larger distribution volumes using convection-enhanced delivery (CED), due to poor transmittance of materials and uncontrolled backflow. The goal of this study was to evaluate a novel brain catheter that has been designed to allow for extended delivery and larger distribution volumes with limited backflow of fluid. It was characterized using a broad range of therapeutic pore sizes both for transmittance across the membranes to address possible occlusion and for distribution in short term infusion studies, both in-vitro in gels and in-vivo in canines. METHODS: Brain catheters with pore sizes of 10, 12, 15, 20 and 30 µm were evaluated using three infusates prepared in 0.9% sterile saline with diameters approximating 2, 5, and 30 nm, respectively. Magnevist™ was chosen as the small molecule infusate to mimic low-molecular weight therapeutics. Galbumin™ served as a surrogate for an assortment of proteins used for brain cancer and Parkinson's disease. Gadoluminate™ was used to assess the distribution of large therapeutics, such as adeno-associated viral particles and synthetic nanoparticles. The transmittance of the medium and large tracer particles through catheters of different pore size (15, 20 and 30 µm) was measured by MRI and compared with the measured concentration of the control. Infusions into 0.2% agarose gels were performed in order to evaluate differences in transmittance and distribution of the small, medium, and large tracer particles through catheters with different pore sizes (10, 12, 15, 20 and 30 µm). In-vivo infusions were performed in the canine in order to evaluate the ability of the catheter to infuse the small, medium, and large tracer particles into brain parenchyma at high flow rates through catheters with different pore sizes (10, 15, and 20 µm). Two catheters were stereotactically inserted into the brain for infusion, one per hemisphere, in each animal (N = 6). RESULTS: The transmittance of Galbumin and Gadoluminate across the catheter membrane surface was 100% to within the accuracy of the measurements. There was no evidence of any blockage or retardation of any of the infusates. Catheter pore size did not appear to significantly affect transmittance or distribution in gels of any of the molecule sizes in the range of catheter pore sizes tested. There were differences in the distributions between the different tracer molecules: Magnevist produced relatively large distributions, followed by Gadoluminate and Galbumin. We observed no instances of uncontrolled backflow in a total of 12 in-vivo infusions. In addition, several of the infusions resulted in substantial amounts remaining in tissue. We expect the in-tissue distributions to be substantially improved in the larger human brain. COMPARISON WITH EXISTING METHODS: The new porous brain catheter performed well in terms of both backflow and intraparenchymal infusion of molecules of varying size in the canine brain under CED flow conditions. CONCLUSIONS: Overall, the data presented in this report support that the novel porous brain catheter can deliver therapeutics of varying sizes at high infusion rates in the brain parenchyma, and resist backflow that can compromise the efficacy of CED therapy. Additional work is needed to further characterize the brain catheter, including animal toxicity studies of chronically implanted brain catheters to lay the foundation for its use in the clinic.

A Novel Occupancy Mapping Framework for Risk-Aware Path Planning in Unstructured Environments.

In the context of autonomous robots, one of the most important tasks is to prevent potential damage to the robot during navigation. For this purpose, it is often assumed that one must deal with known probabilistic obstacles, then compute the probability of collision with each obstacle. However, in complex scenarios or unstructured environments, it might be difficult to detect such obstacles. In these cases, a metric map is used, where each position stores the information of occupancy. The most common type of metric map is the Bayesian occupancy map. However, this type of map is not well suited for computing risk assessments for continuous paths due to its discrete nature. Hence, we introduce a novel type of map called the Lambda Field, which is specially designed for risk assessment. We first propose a way to compute such a map and the expectation of a generic risk over a path. Then, we demonstrate the benefits of our generic formulation with a use case defining the risk as the expected collision force over a path. Using this risk definition and the Lambda Field, we show that our framework is capable of doing classical path planning while having a physical-based metric. Furthermore, the Lambda Field gives a natural way to deal with unstructured environments, such as tall grass. Where standard environment representations would always generate trajectories going around such obstacles, our framework allows the robot to go through the grass while being aware of the risk taken.

Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures.

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical"" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Geometry Preserving Sampling Method Based on Spectral Decomposition for Large-Scale Environments.

In the context of 3D mapping, larger and larger point clouds are acquired with lidar sensors. Although pleasing to the eye, dense maps are not necessarily tailored for practical applications. For instance, in a surface inspection scenario, keeping geometric information such as the edges of objects is essential to detect cracks, whereas very dense areas of very little information such as the ground could hinder the main goal of the application. Several strategies exist to address this problem by reducing the number of points. However, they tend to underperform with non-uniform density, large sensor noise, spurious measurements, and large-scale point clouds, which is the case in mobile robotics. This paper presents a novel sampling algorithm based on spectral decomposition analysis to derive local density measures for each geometric primitive. The proposed method, called Spectral Decomposition Filter (SpDF), identifies and preserves geometric information along the topology of point clouds and is able to scale to large environments with a non-uniform density. Finally, qualitative and quantitative experiments verify the feasibility of our method and present a large-scale evaluation of SpDF with other seven point cloud sampling algorithms, in the context of the 3D registration problem using the Iterative Closest Point (ICP) algorithm on real-world datasets. Results show that a compression ratio up to 97 % can be achieved when accepting a registration error within the range accuracy of the sensor, here for large scale environments of less than 2 cm.

Modulation of epileptogenesis: A paradigm for the integration of enzyme-based microelectrode arrays and optogenetics.

BACKGROUND: Genesis of acquired epilepsy includes transformations spanning genetic-to- network-level modifications, disrupting the regional excitatory/inhibitory balance. Methodology concurrently tracking changes at multiple levels is lacking. Here, viral vectors are used to differentially express two opsin proteins in neuronal populations within dentate gyrus (DG) of hippocampus. When activated, these opsins induced excitatory or inhibitory neural output that differentially affected neural networks and epileptogenesis. In vivo measures included behavioral observation coupled to real-time measures of regional glutamate flux using ceramic-based amperometric microelectrode arrays (MEAs). RESULTS: Using MEA technology, phasic increases of extracellular glutamate were recorded immediately upon application of blue light/488 nm to DG of rats previously transfected with an AAV 2/5 vector containing an (excitatory) channelrhodopsin-2 transcript. Rats receiving twice-daily 30-sec light stimulation to DG ipsilateral to viral transfection progressed through Racine seizure stages. AAV 2/5 (inhibitory) halorhodopsin-transfected rats receiving concomitant amygdalar kindling and DG light stimuli were kindled significantly more slowly than non-stimulated controls. In in vitro slice preparations, both excitatory and inhibitory responses were independently evoked in dentate granule cells during appropriate light stimulation. Latency to response and sensitivity of responses suggest a degree of neuron subtype-selective functional expression of the transcripts. CONCLUSIONS: This study demonstrates the potential for coupling MEA technology and optogenetics for real-time neurotransmitter release measures and modification of seizure susceptibility in animal models of epileptogenesis. This microelectrode/optogenetic technology could prove useful for characterization of network and system level dysfunction in diseases involving imbalanced excitatory/inhibitory control of neuron populations and guide development of future treatment strategies.

Challenges of simultaneous measurements of brain extracellular GABA and glutamate in vivo using enzyme-coated microelectrode arrays.

BACKGROUND: Although GABA is the major inhibitory neurotransmitter in the CNS, quantifying in vivo GABA levels has been challenging. The ability to co-monitor both GABA and the major excitatory neurotransmitter, glutamate, would be a powerful tool in both research and clinical settings. NEW METHOD: Ceramic-based microelectrode arrays (MEAs) were used to quantify gamma-aminobutyric acid (GABA) by employing a dual-enzyme reaction scheme including GABase and glutamate oxidase (GluOx). Glutamate was simultaneously quantified on adjacent recording sites coated with GluOx alone. Endogenous glutamate was subtracted from the combined GABA and glutamate signal to yield a pure GABA concentration. RESULTS: Electrode sensitivity to GABA in conventional, stirred in vitro calibrations at pH 7.4 did not match the in vivo sensitivity due to diffusional losses. Non-stirred calibrations in agarose or stirred calibrations at pH 8.6 were used to match the in vivo GABA sensitivity. In vivo data collected in the rat brain demonstrated feasibility of the GABA/glutamate MEA including uptake of locally applied GABA, KCl-evoked GABA release and modulation of endogenous GABA with vigabatrin. COMPARISON WITH EXISTING METHODS: Implantable enzyme-coated microelectrode arrays have better temporal and spatial resolution than existing off-line methods. However, interpretation of results can be complicated due to the multiple recording site and dual enzyme approach. CONCLUSIONS: The initial in vitro and in vivo studies supported that the new MEA configuration may be a viable platform for combined GABA and glutamate measures in the CNS extending the previous reports to in vivo GABA detection. The challenges of this approach are emphasized.

Chronic Methylphenidate Alters Tonic and Phasic Glutamate Signaling in the Frontal Cortex of a Freely-Moving Rat Model of ADHD.

Glutamate dysfunction has been implicated in a number of substance of abuse studies, including cocaine and methamphetamine. Moreover, in attention-deficit/hyperactivity disorder (ADHD), it has been discovered that when the initiation of stimulant treatment occurs during adolescence, there is an increased risk of developing a substance use disorder later in life. The spontaneously hypertensive rat (SHR) serves as a phenotype for ADHD and studies have found increased cocaine self-administration in adult SHRs when treated with the stimulant methylphenidate (MPH) during adolescence. For this reason, we wanted to examine glutamate signaling in the pre-limbic frontal cortex, a region implicated in ADHD and drug addiction, in the SHR and its progenitor control strain, the Wistar Kyoto (WKY). We chronically implanted glutamate-selective microelectrode arrays (MEAs) into 8-week-old animals and treated with MPH (2 mg/kg, s.c.) for 11 days while measuring tonic and phasic extracellular glutamate concentrations. We observed that intermediate treatment with a clinically relevant dose of MPH increased tonic glutamate levels in the SHR but not the WKY compared to vehicle controls. After chronic treatment, both the SHR and WKY exhibited increased tonic glutamate levels; however, only the SHR was found to have decreased amplitudes of phasic glutamate signaling following chronic MPH administration. The findings from this study suggest that the MPH effects on extracellular glutamate levels in the SHR may potentiate the response for drug abuse later in life. Additionally, these data illuminate a pathway for investigating novel therapies for the treatment of ADHD and suggest that possibly targeting the group II metabotropic glutamate receptors may be a useful therapeutic avenue for adolescents diagnosed with ADHD.

GDNF revisited: A novel mammalian cell-derived variant form of GDNF increases dopamine turnover and improves brain biodistribution.

Parkinson's disease (PD) is a disorder affecting dopamine neurons for which there is no cure. Glial cell line-derived neurotrophic factor (GDNF) and the closely related protein neurturin are two trophic factors with demonstrated neuroprotective and neurorestorative properties on dopamine neurons in multiple animal species. However, GDNF and neurturin Phase-2 clinical trials have failed to demonstrate a significant level of improvement over placebo controls. Insufficient drug distribution in the brain parenchyma has been proposed as a major contributing factor for the lack of clinical efficacy in the Phase-2 trial patients. To address this issue, a novel mammalian cell-derived variant form of GDNF (GDNFv) was designed to promote better tissue distribution by reducing its heparin binding to the extracellular matrix and key amino acids were substituted to enhance its chemical stability. Administration of this fully glycosylated GDNFv in the normal rat striatum increased dopamine turnover and produced significantly greater brain distribution than E. coli-produced wildtype GDNF (GDNFwt). Intrastriatal GDNFv also protected midbrain dopamine neuron function in 6-hydroxydopamine-lesioned rats. Studies conducted in normal adult rhesus macaques support that GDNFv was well tolerated in all animals and demonstrated a greater volume of distribution than GDNFwt in the brain following intrastriatal infusion. Importantly, favorable physiological activity of potential therapeutic value was maintained in this variant trophic factor with significant target activation in GDNFv recipients as indicated by dopamine turnover modulation. These data suggest that GDNFv may be a promising drug candidate for the treatment of PD. Additional studies are needed in non-human primates with dopamine depletion. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

The role of glutamate signaling in incentive salience: second-by-second glutamate recordings in awake Sprague-Dawley rats.

The attribution of incentive salience to reward-predictive stimuli has been shown to be associated with substance abuse-like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N-methyl-D-aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second-by-second glutamate neurotransmission in the attribution of incentive salience to reward-predictive stimuli by measuring sign-tracking behavior during a Pavlovian conditioned approach procedure using ceramic-based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague-Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign-tracking behavior to a food-predictive conditioned stimulus (CS+) compared to the presentation of a non-predictive conditioned stimulus (CS-). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign-tracking behavior to a food-predictive CS+.

Acute treatment with doxorubicin affects glutamate neurotransmission in the mouse frontal cortex and hippocampus.

Doxorubicin (DOX) is a potent chemotherapeutic agent known to cause acute and long-term cognitive impairments in cancer patients. Cognitive function is presumed to be primarily mediated by neuronal circuitry in the frontal cortex (FC) and hippocampus, where glutamate is the primary excitatory neurotransmitter. Mice treated with DOX (25mg/kg i.p.) were subjected to in vivo recordings under urethane anesthesia at 24h post-DOX injection or 5 consecutive days of cognitive testing (Morris Water Maze; MWM). Using novel glutamate-selective microelectrode arrays, amperometric recordings measured parameters of extracellular glutamate clearance and potassium-evoked release of glutamate within the medial FC and dentate gyrus (DG) of the hippocampus. By 24h post-DOX injection, glutamate uptake was 45% slower in the FC in comparison to saline-treated mice. In the DG, glutamate took 48% longer to clear than saline-treated mice. Glutamate overflow in the FC was similar between treatment groups, however, it was significantly increased in the DG of DOX treated mice. MWM data indicated that a single dose of DOX impaired swim speed without impacting total length traveled. These data indicate that systemic DOX treatment changes glutamate neurotransmission in key nuclei associated with cognitive function within 24h, without a lasting impact on spatial learning and memory. Understanding the functional effects of DOX on glutamate neurotransmission may help us understand and prevent some of the debilitating side effects of chemotherapeutic treatment in cancer survivors.

Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis.

In screening a library of plant extracts from ~1000 species native to the Southeastern United States, Lobelia cardinalis was identified as containing nicotinic acetylcholine receptor (nicAchR) binding activity which was relatively non-selective for the α4ß2- and α7-nicAchR subtypes. This nicAchR binding profile is atypical for plant-derived nicAchR ligands, the majority of which are highly selective for α4ß2-nicAchRs. Its potential therapeutic relevance is noteworthy since agonism of α4ß2- and α7-nicAchRs is associated with anti-inflammatory and neuroprotective properties. Bioassay-guided fractionation of L. cardinalis extracts led to the identification of lobinaline, a complex binitrogenous alkaloid, as the main source of the unique nicAchR binding profile. Purified lobinaline was a potent free radical scavenger, displayed similar binding affinity at α4ß2- and α7-nicAchRs, exhibited agonist activity at nicAchRs in SH-SY5Y cells, and inhibited [(3)H]-dopamine (DA) uptake in rat striatal synaptosomes. Lobinaline significantly increased fractional [(3)H] release from superfused rat striatal slices preloaded with [(3)H]-DA, an effect that was inhibited by the non-selective nicAchR antagonist mecamylamine. In vivo electrochemical studies in urethane-anesthetized rats demonstrated that lobinaline locally applied in the striatum significantly prolonged clearance of exogenous DA by the dopamine transporter (DAT). In contrast, lobeline, the most thoroughly investigated Lobelia alkaloid, is an α4ß2-nicAchR antagonist, a poor free radical scavenger, and is a less potent DAT inhibitor. These previously unreported multifunctional effects of lobinaline make it of interest as a lead to develop therapeutics for neuropathological disorders that involve free radical generation, cholinergic, and dopaminergic neurotransmission. These include neurodegenerative conditions, such as Parkinson's disease, and drug abuse.

Randomized Trial Comparing the Primary Patency following Cutting Versus High-Pressure Balloon Angioplasty for Treatment of de Novo Venous Stenoses in Hemodialysis Arteriovenous Fistulae.

PURPOSE: A single-center randomized clinical trial was performed to compare postinterventional primary patency rates achieved by cutting balloon angioplasty and high-pressure balloon angioplasty in the treatment of de novo stenoses within autogenous arteriovenous (AV) fistulae for hemodialysis. MATERIALS AND METHODS: Forty-eight patients undergoing their first angioplasty were prospectively randomized to undergo angioplasty with a cutting balloon or high-pressure balloon 4-8 mm in diameter because cutting balloons larger than 8 mm are not available. Nine patients were excluded after angiography, with seven requiring balloons larger than 8 mm. In the remaining 39 patients, there were 42 stenoses in the following regions: juxtaanastomotic (38%), perianstomotic (38%), midcephalic (9%), and cephalic arch (14%). Patients in the cutting balloon group were younger (mean age difference, 9 y; P = .04), but other demographic variables were comparable (range, P = .08-.89). The mean follow-up period was 8.5 mo (range, 24 d to 32 mo). Kaplan-Meier analysis was used to compare duration of patency. Mann-Whitney rank-sum t test and χ2/Fisher exact tests were used to compare continuous and categoric variables, respectively. RESULTS: Technical success was achieved in all 39 patients. At 3, 6, and 12 months, the postinterventional primary patency rates for the cutting balloon group were 61.1% (95% confidence interval [CI], 35.75%-82.70%), 27.7% (95% CI, 9.69%-53.48%), and 11.1% (95% CI, 1.38%-34.71%), respectively, compared with 70.0% (95% CI, 45.72%-88.11%), 42.1% (95% CI, 20.25%-66.50%), and 26.3% (95% CI, 9.15%-51.20%), respectively, for the high-pressure balloon group (P < .3 at each interval). CONCLUSIONS: Compared with high-pressure balloon angioplasty, cutting balloon angioplasty does not improve postinterventional primary patency of de novo stenotic lesions in autogenous arteriovenous fistulae.

Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease.

BACKGROUND: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. NEW METHOD: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. RESULTS: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300µg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrease in DA turnover in the lesioned striatum; and an increased sparing of tyrosine hydroxylase (TH) positive (+) neurons in a specific sub-region of the lesioned substantia nigra pars compacta (SNpc). Finally, tracer studies showed (125)I-DNSP-11 distributed diffusely throughout the brain, including the striatum and SN, as quickly as 30min after a single intranasal dose. COMPARISON WITH EXISTING METHODS: The results of bilateral intranasal administration of DNSP-11 are compared to our unilateral single infusion studies to the brain in rats. CONCLUSIONS: These studies support that DNSP-11 can be delivered intranasally and maintain its neuroactive properties in both normal rats and in a unilateral 6-OHDA rat model of PD.

Simultaneous glutamate recordings in the frontal cortex network with multisite biomorphic microelectrodes: New tools for ADHD research.

BACKGROUND: The aberrant regulation of glutamate has been implicated in numerous psychiatric disorders including drug addiction and attention-deficit/hyperactivity disorder. To understand glutamate signaling and its role in facilitating disease, tools to directly measure glutamate in a complex, neural network are needed. NEW METHOD: The development of a ceramic-based, dual-sided, biomorphic microelectrode array with four recording sites on each side to facilitate a more detailed measurement of glutamate in awake, behaving rodents. RESULTS: In vitro calibrations of these biosensors showed selective and specific responses to glutamate. In awake rats, these biomorphic electrode arrays enabled the concurrent evaluation of glutamate in a network, the frontal cortex: including the cingulate, prelimbic, infralimbic and dorsal peduncle regions. Regions within the frontal cortex exhibited varying phasic glutamate patterns in awake animals.Comparison with existing method: Existing methodologies to measure glutamate neurotransmission employ single-sided biosensors or biosensors capable of measuring neurochemicals at only one location in space. CONCLUSIONS: Multi-site, biomorphic neurochemical biosensors provide a method for simultaneously measuring glutamate in multiple areas of a neural network in the brain.

Tonic glutamate in CA1 of aging rats correlates with phasic glutamate dysregulation during seizure.

OBJECTIVE: Characterize glutamate neurotransmission in the hippocampus of awake-behaving rodents during focal seizures in a model of aging. METHODS: We used enzyme-based ceramic microelectrode array technology to measure in vivo extracellular tonic glutamate levels and real-time phasic glutamate release and clearance events in the hippocampus of awake Fischer 344 rats. Local application of 4-aminopyridine (4-AP) into the CA1 region was used to induce focal motor seizures in different animal age groups representing young, late-middle aged and elderly humans. RESULTS: Rats with the highest preseizure tonic glutamate levels (all in late-middle aged or elderly groups) experienced the most persistent 4-AP-induced focal seizure motor activity (wet dog shakes) and greatest degree of acute seizure-associated disruption of glutamate neurotransmission measured as rapid transient changes in extracellular glutamate levels. SIGNIFICANCE: Increased seizure susceptibility was demonstrated in the rats with the highest baseline hippocampal extracellular glutamate levels, all of which were late-middle aged or aged animals. The manifestation of seizures behaviorally was associated with dynamic changes in glutamate neurotransmission. To our knowledge, this is the first report of a relationship between seizure susceptibility and alterations in both baseline tonic and phasic glutamate neurotransmission.

Aberrant glutamate signaling in the prefrontal cortex and striatum of the spontaneously hypertensive rat model of attention-deficit/hyperactivity disorder.

RATIONALE: Attention-deficit/hyperactivity disorder (ADHD) is thought to involve hypofunctional catecholamine systems in the striatum, nucleus accumbens, and prefrontal cortex (PFC); however, recent clinical evidence has implicated glutamate dysfunction in the pathophysiology of ADHD. Recent studies show that increased stimulation of dopamine D2 and D4 receptors causes inhibition of N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, respectively. The spontaneously hypertensive rat (SHR) model of ADHD combined type (C) has been found to have a hypofunctional dopamine system in the ventral striatum, nucleus accumbens, and PFC compared to the control Wistar Kyoto (WKY) strain. OBJECTIVES: Based on the current understanding of typical dopamine-glutamate interactions, we hypothesized that the SHR model of ADHD would have a hyperfunctional glutamate system terminating in the striatum, nucleus accumbens, and PFC. RESULTS: High-speed amperometric recordings combined with four-channel microelectrode arrays to directly measure glutamate dynamics showed increased evoked glutamate release in the PFC (cingulate and infralimbic cortices, p < 0.05) and also in the striatum (p < 0.05) of the SHR (ADHD-C) as compared to the WKY. Finally, glutamate uptake was discovered to be aberrant in the PFC, but not the striatum, of the SHR when compared to the control WKY strain. CONCLUSIONS: These results suggest that the glutamatergic system in the PFC of the SHR model of ADHD is hyperfunctional and that targeting glutamate in the PFC could lead to the development of novel therapeutics for the treatment of ADHD.

Glutaraldehyde cross-linked glutamate oxidase coated microelectrode arrays: selectivity and resting levels of glutamate in the CNS.

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.

The spontaneously hypertensive and Wistar Kyoto rat models of ADHD exhibit sub-regional differences in dopamine release and uptake in the striatum and nucleus accumbens.

The most widely used animal model of attention-deficit/hyperactivity disorder (ADHD) is the spontaneously hypertensive rat (SHR/NCrl), which best represents the combined subtype (ADHD-C). Recent evidence has revealed that a progenitor strain, the Wistar Kyoto from Charles River Laboratories (WKY/NCrl), is useful as a model of the inattentive subtype (ADHD-PI) and the Wistar Kyoto from Harlan Laboratories (WKY/NHsd) and the Sprague Dawley (SD) have been suggested as controls. Dopamine (DA) dysfunction in the striatum (Str) and nucleus accumbens core (NAc) is thought to play a significant role in the pathophysiology of ADHD but data obtained with the SHR is equivocal. Using high-speed chronoamperometric recordings with carbon fiber microelectrodes, we found that the SHR/NCrl displayed decreased KCl-evoked DA release versus the WKY/NCrl model of ADHD-PI in the dorsal Str. The WKY/NCrl and the WKY/NHsd control did not differ from each other; however, the control SD released less DA than the WKY/NCrl model of ADHD-PI in the dorsal Str and less than the control WKY/NHsd in the intermediate Str. The SHR/NCrl had faster DA uptake in the ventral Str and NAc versus both control strains, while the WKY/NCrl model of ADHD-PI exhibited faster DA uptake in the NAc versus the SD control. These results suggest that increased surface expression of DA transporters may explain the more rapid uptake of DA in the Str and NAc of these rodent models of ADHD.

An allosteric modulator of metabotropic glutamate receptors (mGluR2), (+)-TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex.

The potential anxiolytic effects of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor subgroup 2 (mGluR2) were investigated using a self-referencing recording technique with enzyme-based microelectrode arrays (MEAs) that reliably measures tonic and phasic changes in extracellular glutamate levels in awake rats. Studies involved glutamate measures in the rat prefrontal cortex during subcutaneous injections of the following: vehicle, a mGluR2/3 agonist, LY354740 (10 mg/kg), or a mGluR2 PAM, 1-Methyl-2-((cis-(R,R)-3-methyl-4-(4-trifluoromethoxy-2-fluoro)phenyl)piperidin-1-yl)methyl)-1H-imidazo[4,5-b]pyridine ((+)-TFMPIP; 1.0 or 17.8 mg/kg). Studies assessed changes in tonic glutamate levels and the glutamatergic responses to a 5-min restraint stress. Subcutaneous injection of (+)-TFMPIP at a dose of 1.0 mg/kg (day 3: -7.1 ± 15.1 net AUC; day 5: -24.8 ± 24.9 net AUC) and 17.8 mg/kg (day 3: -46.5 ± 33.0 net AUC; day 5: 34.6 ± 36.8 net AUC) significantly attenuated the stress-evoked glutamate release compared to vehicle controls (day 3: 134.7 ± 50.6 net AUC; day 5: 286.6 ± 104.5 net AUC), whereas the mGluR2/3 agonist LY354740 had no effect. None of the compounds significantly affected resting glutamate levels, which we have recently shown to be extensively derived from neurons. Taken together, these data support that systemic administration of (+)-TFMPIP produces phasic rather than tonic release of glutamate that may play a major role in the effects of stress on glutamate neuronal systems in the prefrontal cortex.

Enhanced dopamine transporter activity in middle-aged Gdnf heterozygous mice.

Glial cell line-derived neurotrophic factor (GDNF) supports the viability of midbrain dopamine (DA) neurons that degenerate in Parkinson's disease. Middle-aged, 12 month old, Gdnf heterozygous (Gdnf(+/-)) mice have diminished spontaneous locomotor activity and enhanced synaptosomal DA uptake compared with wild type mice. In this study, dopamine transporter (DAT) function in middle-aged, 12 month old Gdnf(+/-) mice was more thoroughly investigated using in vivo electrochemistry. Gdnf(+/-) mice injected with the DAT inhibitor, nomifensine, exhibited significantly more locomotor activity than wild type mice. In vivo electrochemistry with carbon fiber microelectrodes demonstrated enhanced clearance of DA in the striatum of Gdnf(+/-) mice, suggesting greater surface expression of DAT than in wild type littermates. Additionally, 12 month old Gdnf(+/-) mice expressed greater D(2) receptor mRNA and protein in the striatum than wild type mice. Neurochemical analyses of striatal tissue samples indicated significant reductions in DA and a faster DA metabolic rate in Gdnf(+/-) mice than in wild type mice. Altogether, these data support an important role for GDNF in the regulation of uptake, synthesis, and metabolism of DA during aging.