Dialysis Probe Research Articles

Microdialysis-integrated HPLC system with dual-electrode detection using track-etched membrane electrodes for in vivo monitoring of dopamine dynamics

A capillary high-performance liquid chromatography (HPLC) system equipped with a dual-electrode detector utilizing track-etched membrane electrodes (TEMEs) was combined with a microdialysis sampling setup. The electrochemical detector benefits from the high electrolysis efficiency of TEMEs, allowing for calibration-free coulometric detection and simplifying data analysis to determine the dopamine recovery through a dialysis probe. Additionally, this system was used for in vivo monitoring of dopamine in the right striatum of a mouse brain. Temporal changes in dopamine levels, including an exponential decay immediately after the dialysis probe insertion and an excess release of dopamine induced by a high concentration of potassium ions, confirmed the system’s proper operation. Furthermore, subsequent measurements following the intraperitoneal injection of mirtazapine showed no increase in dopamine levels in the right dorsal striatum. The dual-electrode system displayed characteristic dopamine detection behavior, with anodic and cathodic peak pairs indicative of reversible electrochemical reactions. This capability facilitated the identification of the dopamine peak within the complex chromatogram of the mouse brain dialysate. The consistency between dopamine collection efficiency from standard solutions and dialysate indicated the absence of interfering electroactive substances overlapping with the dopamine peak in the chromatogram. This integrated analysis system successfully tracked temporal fluctuations in dopamine concentration within the mouse brain.

Effects of dapagliflozin on myoglobin efflux from cardiomyocyte during myocardial ischemia/reperfusion in anesthetized rats

It has been suggested that sodium-glucose cotransporter 2 (SGLT2) inhibitors have cardioprotective effects during myocardial ischemia/reperfusion (I/R) independent of glucose-lowering action. However, the effects of SGLT2 inhibitors on structural damage to cardiomyocytes in the ischemic region during I/R remain unknown. We applied a microdialysis technique to the heart of anesthetized rats and investigated the effects of an SGLT2 inhibitor, dapagliflozin, on myocardial interstitial myoglobin levels in the ischemic region during coronary occlusion followed by reperfusion. Dapagliflozin was administered systemically (40 μg/body iv) or locally via a dialysis probe (100 μM and 1 mM) 30 min before coronary occlusion. In the vehicle group, coronary occlusion increased the dialysate myoglobin concentration in the ischemic region. Reperfusion further increased the dialysate myoglobin concentration. Intravenous administration of dapagliflozin reduced dialysate myoglobin concentration during ischemia and at 0–15 min after reperfusion, but local administration (100 μM and 1 mM) did not. Therefore, acute systemic administration of dapagliflozin prior to ischemia has cardioprotective effects on structural damage during I/R.

Rapid and Online Microvolume Flow-Through Dialysis Probe for Sample Preparation in Veterinary Drug Residue Analysis.

A rapid and online microvolume flow-through dialysis probe designed for sample preparation in the analysis of veterinary drug residues is introduced. This study addresses the need for efficient and green sample preparation methods that reduce chemical waste and reagent use. The dialysis probe integrates with liquid chromatography and mass spectrometry (LC-MS) systems, facilitating automated, high-throughput analysis. The dialysis method utilizes minimal reagent volumes per sample, significantly reducing the generation of solvent waste compared to traditional sample preparation techniques. Several veterinary drugs were spiked into tissue homogenates and analyzed to validate the probe's efficacy. A diagnostic sensitivity of >97% and specificity of >95% were obtained for this performance evaluation. The results demonstrated the effective removal of cellular debris and particulates, ensuring sample integrity and preventing instrument clogging. The automated dialysis probe yielded recovery rates between 27 and 77% for multiple analytes, confirming its potential to streamline veterinary drug residue analysis, while adhering to green chemistry principles. The approach highlights substantial improvements in both environmental impact and operational efficiency, presenting a viable alternative to conventional sample preparation methods in regulatory and research applications.

Plasmonic Optical Fiber Based Continuous in-Vivo Glucose Monitoring for ICU/CCU Setup.

This paper reports a sensor architecture for continuous monitoring of biomarkers directly in the blood, especially for ICU/CCU patients requiring critical care and rapid biomarker measurement. The sensor is based on a simple optical fiber that can be inserted through a catheter into the bloodstream, wherein gold nanoparticles are attached at its far distal end as a plasmonic material for highly sensitive opto-chemical sensing of target biomolecules (glucose in our application) via the excitation of surface plasmon polaritons. For specificity, the nanoparticles are functionalized with a specific receptor enzyme that enables the localized surface plasmon resonance (LSPR)-based targeted bio-sensing. Further, a micro dialysis probe is introduced in the proposed architecture, which facilitates continuous monitoring for an extended period without fouling the sensor surface with cells and blood debris present in whole blood, leading to prolonged enhanced sensitivity and limit of detection, relative to existing state-of-the-art continuous monitoring devices that can conduct direct measurements in blood. To establish this proof-of-concept, we tested the sensor device to monitor glucose in-vivo involving an animal model, where continuous monitoring was done directly in the circulation of living rats. The sensor's sensitivity to glucose was found to be 0.0354 a.u./mg.dl-1 with a detection limit of 50.89 mg/dl.

Influence of NO Synthase Inhibitors on Serotonin Release in the Medial Prefrontal Cortex during the Formation and Generalization of the Conditioned Fear Response in Rats

Serotonergic and nitrergic systems of the medial prefrontal cortex (mPFC) are involved in the control of fear generalization, but their local interaction during this function has been little studied. The aim of the work was to study the effect of blockade of endogenous nitrergic signals on serotonin release in the mPFC during the acquisition of a conditioned fear response (CFR – a fear model) and on the dynamics of its generalization. In male Sprague-Dawley rats using intracranial microdialysis in vivo and high-performance liquid chromatography with electrochemical detection, we found that the intra-mPFC infusion through the dialysis probe of the NO synthase inhibitor N-ω-nitro-L-arginine (NA, 0.5 mM) and selective neuronal NO synthase inhibitor N-ω-propyl-L-arginine (NPLA, 2mM) decreased the basal level of extracellular serotonin in the mPFC and reduced its rise, caused by the CFR acquisition (a paired presentation of a conditioned cue (CS+) and inescapable footshock). The intra-mPFC infusion of NA and NPLA increased animals’ freezing to a differential cue (CS–) not associated with footshock, during the first test, carried out 70 minutes after the CFR acquisition, but reduced it during repeated testing a day after the infusion, without changing freezing of the same animals to the potentially dangerous CS+. The data obtained indicate the involvement of endogenous NO in the activation of serotonin release in the mPFC, caused by the CFR acquisition. In addition, they show that the blockade of endogenous nitrergic signals of the mPFC, which enhances the initial generalization of the fear reaction, contributes to the extinction of the generalized fear, possibly due to inhibition of the serotonin release in the mPFC.

In vivo microdialysis reveals that blockade of accumbal orexin OX2 but not OX1 receptors enhances dopamine efflux in the nucleus accumbens of freely moving rats.

The nucleus accumbens contain orexinergic neural inputs and orexin OX1 - and OX2 -receptors. Behavioural studies suggest that accumbal orexin receptors modulate accumbal dopaminergic activity-dependent locomotion in rats. We studied the effects of intra-accumbal injection of orexin receptor ligands on accumbal extracellular dopamine levels in freely moving rats, using in vivo microdialysis and analysed the roles of OX1 - and OX2 -receptors in the regulation of basal accumbal dopamine efflux. The orexin receptor ligands were applied intra-accumbally though a microinjection needle attached with a dialysis probe. Neither the nonselective OX1 - and OX2 -receptor agonist orexin-A nor the preferential OX2 -receptor agonist orexin-B (500.0pg and 5.0ng) altered accumbal dopamine levels. The nonselective OX1 - and OX2 -receptor antagonist MK-4305 (suvorexant, 500.0pg, 2.5 and 5.0ng) enhanced dopamine efflux. A 2-h tetrodotoxin infusion into nucleus accumbens through the probe or co-administration of orexin-A (500.0pg) strongly inhibited MK-4305 (5.0ng)-induced accumbal dopamine efflux. The selective OX2 -receptor antagonist EMPA (90.0 and 900.0pg, 9.0ng) increased dopamine efflux. Intra-accumbal infusion of tetrodotoxin abolished EMPA (9.0ng)-induced dopamine efflux. The selective OX1 -receptor antagonist SB-334867 (10.0 and 20.0ng) failed to alter dopamine efflux. Co-administration of orexin-B (500.0pg) inhibited both EMPA (9.0ng)- and MK-4305 (5.0ng)-induced dopamine efflux. Intraperitoneal injection of MK-4305 (10.0mg/kg) did not affect accumbal dopamine efflux. The present study provides in vivo neuropharmacological evidence that accumbal OX2 - but not OX1 -receptors exert inhibitory regulation of basal accumbal dopamine efflux and that blockade of accumbal OX2 -receptors enhances dopamine efflux in nucleus accumbens of freely moving rats.

In Vivo Monitoring of Acetylcholine Release from Nerve Endings in Salivary Gland.

Simple SummaryStimulation of the parasympathetic nervous system results in the secretion of saliva. Previous studies have demonstrated acetylcholine content in homogenate obtained from salivary glands. Acetylcholine in homogenate, however, includes that stored in the cells, as well as that released in the interstitial fluid. The activity of the parasympathetic nervous system is mainly determined by the amount of acetylcholine released. We established an in vivo microdialysis method for monitoring the acetylcholine released from nerve endings in the salivary glands in rats. The results of the present study demonstrated that acetylcholine levels in the dialysate reflect acetylcholine levels in the interstitial fluid of the submandibular gland, and that an increase in the acetylcholine level in the dialysate depends predominantly on the release of acetylcholine from the parasympathetic nerve endings.A microdialysis technique was used to monitor acetylcholine levels in the local interstitial fluid in rat submandibular glands, with the aim of determining parasympathetic nerve activity in vivo. The dialysis probe housed a 10 × 0.22 mm semipermeable membrane (molecular weight cutoffs: 50,000 Da). When the probe was perfused at 2 μL/min in vitro, the mean relative recovery of acetylcholine was 41.7% ± 2.5%. The dialysis probes were implanted in the submandibular glands of anesthetized rats and perfusion with Ringer’s solution, at 2 μL/min, was performed. Acetylcholine concentrations in the dialysate were measured by high-performance liquid chromatography and electrochemical detection. The results revealed the following: (1) that mixing Eserine with Ringer’s solution allowed acetylcholine in the salivary glands to be quantified; (2) that acetylcholine concentrations in the dialysate were highly variable and unstable over the first 120 min after probe implantation, but reached a nearly stable level (4.8 ± 2.7 nM) thereafter in the presence of 100 µM of Eserine; and (3) that electrical stimulation of the chorda tympani nerve, or perfusion with high potassium Ringer’s solution, significantly increased acetylcholine concentrations in the dialysate. These results indicate that the present microdialysis technique offers a powerful tool for detecting changes in parasympathetic activity within the salivary glands.

Insulin restores the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats.

This study examined whether insulin modulates the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats. The rats received vehicle or streptozotocin (STZ) to induce hypoinsulinemia. A subset of STZ-treated rats was implanted with insulin pellets that rapidly normalized glucose levels. Two-weeks later, dialysis probes were implanted into the nucleus accumbens (NAc) and ipsilateral ventral tegmental area (VTA). The next day, dialysate samples were collected during baseline and then following systemic administration of nicotine. Samples were also collected following intra-VTA administration of the gamma-aminobutyric acid (GABA)A receptor antagonist, bicuculline. Dopamine, GABA, glutamate, and acetylcholine (ACh) levels were assessed using liquid chromatography/mass spectrometry (LC/MS). The results revealed that vehicle-treated rats displayed a nicotine-induced increase in NAc dopamine levels. In contrast, STZ-treated rats did not display any changes in NAc dopamine following nicotine administration, an effect that was likely related to a concomitant increase in GABA and decrease in glutamate levels in both the NAc and VTA. Intra-VTA administration of bicuculline increased NAc dopamine in vehicle-treated rats, and this effect was absent in STZ-treated rats. Vehicle-treated rats displayed a nicotine-induced increase in ACh levels in the NAc (but not VTA), an effect that was lower in the NAc of STZ-treated rats. Insulin supplementation normalized the neurochemical effects of nicotine in the NAc and VTA of STZ-treated rats, suggesting that insulin modulates the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats.

3D-Printed Microfluidic Devices for Enhanced Online Sampling and Direct Optical Measurements.

3D printing has emerged as a robust technique to fabricate reliable and reproducible microfluidic devices. However, a limitation of 3D-printed devices has been the low transparency even when printed in a "clear" material. There are currently no reports regarding direct optical measurements through a 3D-printed device. Here, we present for the first time that the printing orientation can affect the transparency of a 3D-printed object. With the optimal orientation, we printed a microfluidic detector that was sufficiently transparent (transmittance ≈ 80%) for optical quantitation. This finding is significant because it shows the feasibility to directly 3D-print optical components for analytical applications. In addition, we created a novel microfluidic dialysis device via 3D printing, which enabled higher flow rates (for sampling with high temporal resolution) and increased extraction efficiency than commercially available ones. By coupling the microfluidic detector and dialysis probe, we successfully measured the release kinetics of indole from biofilms in a continuous, automated, and near real-time fashion. Indole is an intercellular signaling molecule in biofilms, which may regulate antibiotic resistance. The release kinetics of this molecule had not been quantitated likely because of the lack of a suitable analytical tool. Our results fill this knowledge gap.

Local administration of the orexin receptor antagonist MK-4305, but not orexin-A, into the nucleus accumbens increases accumbal dopamine efflux in freely moving rats

The nucleus accumbens (NAc), a major terminal area of mesolimbic dopaminergic projections, receives inputs from orexin neurons. Behavioural studies suggest that accumbal orexin receptors (-Rs) play modulatory roles in the regulation of accumbal dopaminergic activity-dependent locomotion in rats (Kotani et al., 2008). We studied the effects of intra-accumbal application of orexin-R ligands on accumbal dopamine (DA) efflux in freely moving rats, using in vivo microdialysis, to analyse the roles of orexin-Rs in regulating accumbal dopaminergic neural activity. Orexin-R ligands were applied into NAc though a microinjection needle attached with a dialysis probe. The OX1- and OX2-R agonist orexin-A failed to alter DA levels in NAc. However, the OX1- and OX2-R antagonist MK-4305 induced a dose-related increase in DA levels. This MK-4305-induced increase in accumbal DA levels was suppressed by infusion of TTX through the probe and was inhibited by local co-administration of orexin-A. These results suggest that intra-accumbal application of MK-4305 could enhance accumbal DA efflux by antagonism of OX1- and/or OX2-Rs. The present study also indicates that accumbal OX1- and/or OX2-Rs could play inhibitory roles in the regulation of accumbal dopaminergic neural activity.

Rapid determination of majority cations in yoghurts using on-line connection of capillary electrophoresis with mini-dialysis

An analyser was constructed on the basis of on-line connection of capillary electrophoresis over a short separation path with continuous mini-dialysis sample collection. The developed instrument was employed for simultaneous determination of the majority minerals K+, Ca2+, Na+ and Mg2+ (and possibly NH4+ ions) in commercially available unflavoured yoghurts. The cations are released from the organic structures by digestion with boiling 6 mol/L HCl. They were separated from residues of the organic matrix by a dialysis probe and were transferred to a stream of water. From the continuous stream, the dialysate was injected into the separation capillary through a flow-gating interface. Within the reliability interval, the determined total mineral content was equal to their contents stated on the yoghurt labels and the content determined by flame atomic absorption spectrometry and complexometric titration. The relative standard deviation of the electrophoretic determination is mostly about 5%.

Deuterium oxide protects against myocardial injury induced by ischemia and reperfusion in rats

Objectives. Although deuterium oxide (D2O) has preservative property on the extracted organ, whether D2O also protects the in situ myocardial injury remains unknown. Using cardiac microdialysis, local administration of D2O through dialysis probe was applied in situ rat heart. We examined the effect of the D2O on the myocardial injury induced ischemia, reperfusion, and chemical hypoxia. Methodology. We measured dialysate myoglobin levels during 30 min of coronary occlusion and reperfusion in the absence and presence of D2O. Furthermore, to confirm the effect of D2O on NaCN induced myocardial injury, we measured the dialysate myoglobin levels with local perfusion of NaCN in the absence and presence of D2O. Results. The dialysate myoglobin levels increased from 177 ± 45 ng/mL at baseline to 3030 ± 1523 ng/mL during 15–30 min of coronary occlusion and further increased to 8588 ± 1684ng/mL at 0–15 min of reperfusion. The dialysate myoglobin levels with 60 min local perfusion of NaCN increased to 1214 ± 279 ng/mL. D2O attenuated myocardial myoglobin release during 15–30 min of coronary occlusion and 0-30 min of reperfusion and 15–60 min of local perfusion of NaCN. Conclusions. D2O might have a beneficial effect of myocardium against ischemia, reperfusion and chemical hypoxia.

A Microdialysis Assay for Assessing sGC Stimulator Target Engagement in the Rat Liver

Dysfunction of the nitric oxide (NO)‐soluble guanylate cyclase (sGC)‐cyclic guanosine monophosphate (cGMP) pathway has been implicated in numerous cardiovascular, metabolic, and fibrotic diseases. sGC stimulators are small molecules that bind sGC and synergize with NO to increase the enzymatic formation of cGMP and amplify downstream signaling. In preclinical models of liver disease, sGC stimulators have been shown to attenuate liver steatosis, inflammation, and fibrosis. To demonstrate their engagement in the sGC‐cGMP pathway in vivo, a unique microdialysis assay was developed to directly measure changes in cGMP levels in the rat liver.A midline laparotomy was performed on anesthetized male, Sprague Dawley rats (225–250 g) to expose the liver. A 25‐gauge needle was used to place a dialysis probe within the right medial lobe. The probe was perfused with saline at a rate of 2.5 μL/min and dialysate collected in one‐hour periods. Following baseline collection, test compound was administered intravenously, and dialysate collected for the following 2 hours. The NO donor sodium nitroprusside (SNP, 300 nM) was dosed via retrodialysis at study's end as a positive control. cGMP in the dialysate was measured using an enzyme immunoassay.In a study investigating our clinical‐stage sGC stimulator, praliciguat, an oral 3‐mg/kg dose produced a 3.9 ± 1.7‐fold increase (p<.0001) in cGMP from baseline levels of 0.8 ± 0.1 nM. A 1‐mg/kg dose caused a 1.7 ± 0.1‐fold increase in cGMP (p<.001), while the effect of a 0.3‐mg/kg dose was not significant. On average, SNP induced a 4.7 ± 1‐fold increase in cGMP (p<.0001).In summary, our microdialysis assay enabled in vivo monitoring of sGC stimulation in the rat liver. Beyond demonstrating the effects of praliciguat, this method can be used to evaluate and characterize other sGC stimulators for their capacity to amplify liver cGMP production. Further, this assay helps to link previously observed hepatic effects of sGC stimulators in disease models with an enhancement of sGC signaling.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vivo neurochemical evidence that stimulation of accumbal GABAA and GABAB receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats.

Cholinergic neurons in the nucleus accumbens contain GABAA and GABAB receptors that are thought to inhibit neural activity. We analyzed the roles of GABAA and GABAB receptors in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. The effects of GABA receptor ligands on the accumbal dopamine efflux were also analyzed because accumbal cholinergic and dopaminergic neurons could mutually interact. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 30-60min infusions. To monitor basal acetylcholine, a low concentration of physostigmine (50nM) was added to the perfusate. GABAA receptor agonist muscimol (3 and 30pmol) induced a dose-related decrease in accumbal acetylcholine. GABAB receptor agonist baclofen (30 and 300pmol) also produced a dose-related decrease in acetylcholine. GABAA receptor antagonist bicuculline (60pmol) which failed to alter baseline acetylcholine counteracted the muscimol (30pmol)-induced decrease in acetylcholine. GABAB receptor antagonist 2-hydroxysaclofen (12nmol) which failed to change baseline acetylcholine, counteracted the baclofen (300pmol)-induced decrease in acetylcholine. Neither muscimol (30pmol) nor baclofen (300pmol) which reduced accumbal acetylcholine altered baseline accumbal dopamine. Neither bicuculline (60pmol) nor 2-hydroxysaclofen (12nmol) also affected the baseline dopamine. These results show that GABAA and GABAB receptors each exert inhibitory roles in the regulation of accumbal cholinergic neural activity. The present results also provides in vivo neurochemical evidence that stimulation of GABAA and GABAB receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats.

Stimulation of accumbal GABAB receptors inhibits delta1- and delta2-opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats

The nucleus accumbens contains delta-opioid receptors that may decrease inhibitory neurotransmission. As GABAB receptors inhibit dopamine release, decrease in activation of GABAB receptors may be a mediator of delta-opioid receptor-induced accumbal dopamine efflux. If so, accumbal dopamine efflux induced by delta-opioid receptor activation should be suppressed by stimulating GABAB receptors. As delta-opioid receptors are further subdivided into delta1- and delta2-opioid receptors, we analysed the effects of the GABAB receptor agonist baclofen on delta1- and delta2-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds show total amount administered (mol) during 25–50 min infusions. Baclofen (2.5 and 5.0 nmol), which did not alter basal dopamine levels, inhibited the delta1-opioid receptor agonist DPDPE (5.0 nmol)-induced dopamine efflux. Baclofen (2.5 and 5.0 nmol) also inhibited the delta2-opioid receptor agonist deltorphin II (25.0 nmol)-induced dopamine efflux. A low dose of the GABAB receptor antagonist 2-hydroxysaclofen (100.0 pmol), which failed to alter basal accumbal dopamine levels, counteracted the inhibitory effects of baclofen (5.0 nmol) on DPDPE (5.0 nmol)- and deltorphin II (25.0 nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABAB receptor-mediated inhibition of accumbal dopaminergic activity facilitates activation of delta1- and delta2-opioid receptor-induced increases in accumbal dopamine efflux. This study suggests that activation of delta1- and delta2-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABAB receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux.

Abstract 3872: Quantitative measurement of immune-modulatory mediators within tumors of freely moving mice utilizing in vivo microdialysis

Abstract Monitoring changes in biochemical elements within tumors is crucial to understanding cancer biology and to helping with the development of novel therapies. Yet to date, experimental techniques enabling sensitive and quantitative measurement of the levels of small molecules contributing to tumor development have been limited in preclinical oncology models. In the current set of experiments, we implemented in vivo microdialysis to measure signaling molecules and oncometabolites in the tumor microenvironment of freely moving rodents. To this aim, a small dialysis probe is implanted in syngeneic tumors in mice. Probes are perfused with artificial dialysate fluid and dialysate containing molecules from the tumor microenvironment is collected at regular intervals. Levels of biochemicals in the dialysate are then quantified by liquid chromatography-mass spectrometry (LC-MS), a robust and sensitive quantitative technique. We report here proof-of-concept results supporting the use of in vivo microdialysis coupled to LC-MS in cancer research. First, we tested whether implantation of dialysis probe affects in vivo tumor growth. To do so, tumor volume from probe-implanted and non-implanted MC38 allografts in mice was monitored daily for up to 10 days following probe implantation. We found that the growth curve from probe-bearing MC38 tumors was similar to probe-free tumors, revealing that probe implantation did not influence tumor size progression. Next, the levels of metabolites that are known to be involved in tumor development were measured in tumor allografts of freely-moving mice using in vivo microdialysis. Relevant levels of arginine, ornithine and putrescine were quantified by LC-MS. Levels of key molecules of the adenosine signaling and tryptophan/kynurenine pathway metabolites in MC38 tumors were also quantified. To better understand the homogeneity of this particular allograft model, we measured the concentration of the above-mentioned molecules in different regions of a tumor. To this end, two separate probes were implanted in the center and the peripheral area of the same 300 mm3 MC38 allograft. We found that levels of measured molecules were similar in dialysates collected from both probe locations, suggesting homogenous levels of biochemicals throughout the microenvironment of these non-necrotic tumors. Our proposed experimental approach allows for a quantitative and sensitive measurement of key biochemical mediators of tumor progression. In vivo microdialysis in murine tumor models may be used to elucidate the mechanisms by which therapies, such as chemotherapy and immune checkpoint inhibitors, modulate the tumor microenvironment. Our efforts in preclinical cancer research has the potential to bring new insights on the mechanisms underlying cancer development and help the discovery of next-generation therapies for cancer Citation Format: Nadege Morisot, Julien Roeser, Hajo Schiewe, Holden Brown Janssens, Marieke van der Hart, Arash Rassoulpour. Quantitative measurement of immune-modulatory mediators within tumors of freely moving mice utilizing in vivo microdialysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3872.

P.2.023 - Impairment of learning and memory in mice overexpressing α- and γ-synuclein in dopaminergic neurons- implication in Parkinson’s disease

Introduction Idiopathic Parkinson’s disease (PD) is characterized by progressive brain pathology affecting several neurotransmitter systems, leading to a diverse profile of autonomic, motor, cognitive and psychiatric dysfunctions. Multiple convergent lines of evidence implicate the aggregation and accumulation of α-synuclein in the PD pathogenesis [1]. Moreover, post-translational changes of γ-synuclein increase the α-synuclein aggregation [2]. α- and γ-synuclein are members of the synuclein family of small cytoplasmic neuron-specific proteins, and have been suggested to modulate the function of monoamine transporters (DAT, SERT, NET) [3]. Previously, we reported that α-synuclein knockdown mice showed increased extracellular dopamine and serotonin levels –compared to control mice– in caudate-putamen (CPu) and medial prefrontal cortex (mPFC) after DAT or SERT blockade with nomifensine or citalopram, respectively (Alarcon-Aris et al., in review). Aims Given the role of monoamine neurotransmission in cognitive processes, in the present study we assessed the effects of over-expressing α- and γ-synuclein in mouse dopaminergic neurons on spatial and working memory. Methods We generated a mouse model overexpressing human wild-type α-synucleinor endogenous γ-synuclein in dopaminergic neurons from the substantia nigra compacta (SNc) and ventral tegmental area (VTA) using adeno-associated virus type-5 (AAV5-human-α-synuclein) or type-10 (AAV10-mouse-γ-synuclein) unilaterally injected in SNc/VTA. Behavioral tests were performed in mice at 4, 8 and 16 weeks post-infection using cognitive (novel object recognition, Morris water maze, passive avoidance tests) and motor paradigms (cylinder test, rotarod). Extracellular striatal dopamine levels were measured by in vivo microdialysis after implanting concentric dialysis probes, and analyzed by high precision liquid chromatography with electrochemical detection (+0.75 V). In situ hybridization was performed on fixed mice tissues, with 33P-dATP labeled probes in order to detect α- and γ-synuclein transcripts through autoradiography. Results AAV5-α-synuclein mice showed increased human α-synuclein mRNA levels in the ipsilateral SNc over time reaching a 278% of sham mice at 8 weeks post-infection. Similarly, AAV10-γ-synuclein mice reached to a maximal of 250% of sham mice. Overexpression of α- or γ-synuclein led to lower extracellular dopamine levels in CPu and mPFC compared to sham mice after local veratridine (sodium chanel activator agent, 50 µM) or nomifensine (DAT inhibitor, 1-10-50 µM) administration at 4 weeks post-infection. Moreover, overexpression of α- or γ-synuclein led to the impairment of working memory in mice as revealed by passive avoidance test (retention memory 35.5% and 45.9% shorter in AAV5-α-synuclein andAAV10-γ-synuclein mice, respectively) and novel object recognition test (reduction of 10% in time spent exploring novel object in both infected models) at 4-8 weeks post-infection. Likewise, these mice showed deficits in the spatial learning using the Morris water maze (F(1,28)=5.711, P=0.02 in AAV5-α-synuclein; F(1,28)=11.43, P=0.0021 in AAV10-γ-synuclein). These non-motor symptoms appeared earlier than motor deficits assessed by the cylinder test and rotarod. Conclusions Overall, these findings indicate that 1) α- and γ-synuclein are directly involved in the regulation of cognitive functions, likely through changes in monoamine functions, and 2) overexpression of α- or γ-synuclein in dopaminergic neurons mimics the temporal progression of clinical symptoms in PD, with early cognitive deficits appearing before motor symptoms.

Evaluation of a flow-through dialysis probe for sampling and sample preparation: Hyphenation to ion chromatography

A commercially available dialysis probe has, for the first time, been evaluated for sampling and sample preparation in combination with ion chromatography (IC). In operation the probe is immersed into the sample solution and a receiver liquid is propelled through the probe behind a suitable dialysis membrane. Small inorganic anions (fluoride, chloride, nitrite, bromide, nitrate, sulfate, phosphate) are transferred across the membrane into the receiver liquid and carried towards the injection valve of the IC system. Two different modes of operation, i.e. constantly flowing receiver liquid and a temporarily halted receiver liquid have been investigated. The influence of different parameters affecting the recovery of ions with respect to the initial concentration in the sample solutions was examined. These parameters were the flow rate of the receiver liquid, the hydrodynamic conditions in the sample solution, the kind of dialysis membrane and the chemical composition of the sample solution. Under dynamic dialysis conditions with constantly flowing receiver liquid the recoveries for different ions differ and – even under optimized experimental conditions – remain far away from concentration equilibrium between sample solution and receiver liquid. As a consequence of the different transfer rates Donnan potential effects occur in the presence of varying ion concentrations which make calibration and quantification in real sample analysis difficult. By temporarily halting the receiver liquid almost concentration equilibrium can be achieved so that Donnan potential effects are absent. A new concept, termed “total dialysate injection technique”, was combined with the stopped-flow approach to improve the sensitivity of the dialysis procedure.

Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats

The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABAA receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABAA receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABAA receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25–50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABAA receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABAA receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABAA receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux.

(Invited) Conjoint Measurement of Brain Electrophysiology and Neurochemistry

Purpose: We routinely perform conjoint measurement of intracranial electrophysiology and brain neurochemistry in patients with medically intractable epilepsy undergoing intracranial EEG (icEEG) monitoring for possible epilepsy surgery. The conjoint measurement of these two modalities can be revealing of the presumed neurochemical basis of electrophysiological markers of human brain excitation and inhibition. This study focused on interictal electroencephalography (EEG) spikes. Interictal EEG spikes have traditionally been considered to be a marker of brain excitability in epilepsy, though some recent research has questioned this assumption. Here we correlated co-localized measurements of intracranially recorded interictal EEG spikes and basal extracellular glutamate concentrations, a well-established marker of excitation, in patients with intractable epilepsy. Methods: Glutamate was quantified from brain microdialysate obtained from 30 combined depth icEEG electrode and microdialysis catheters placed in 15 patients. EEG spikes were counted, at the time when glutamate was documented, in two separate manners: (1) for the two icEEG electrode contacts on either side of the microdialysis membrane, and (2) for all contacts within the brain region where the dialysis probe was located. Results:We observed the interictal icEEG spike rate was not directly proportional to basal glutamate; rather the icEEG spike rate was low when basal glutamate was high, and vice versa. A joint analysis of brain electrophysiology and neurochemistry thus indicates an inverse, rather than a direct, relationship between interictal icEEG spikes and brain extracellular glutamate. Discussion: Interictal EEG spikes have traditionally been considered to be a marker of brain excitability. The lack of a direct relation between basal glutamate and icEEG spike rate, however, supports the suggestion that EEG spikes may not be reflective of brain excitability, but rather may be reflective of inhibition. To support the findings of this study and underscore the importance of the conjoint measurement of brain electrophysiology and neurochemistry we discuss observations of other studies of interictal EEG spikes, and observations from other studies of conjoint brain electrophysiology and neurochemistry performed by our group. Conclusions: This study suggests that there is considerable value for in-vivo co-localized measurement of human brain electrophysiology and neurochemistry to improve our understanding of these two modalities, and for understanding the mechanism underlying medically intractable epilepsy.