Brain microdialysis and its applications in experimental neurochemistry

Microdialysis is one of the most powerful neurochemistry techniques, which allows the monitoring of changes in the extracellular content of endogenous and exogenous substances in the brain of living animals. The strength as well as wide applicability of this experimental approach are based on the bulk theory of brain neurotransmission. This methodological review introduces basic principles of chemical neurotransmission and emphasizes the difference in neurotransmission types. Clear understanding of their significance and degree of engagement in regulation of physiological processes is an ultimate prerequisite not only for choosing an appropriate method of monitoring for interneuronal communication via chemical messengers but also for accurate data interpretation. The focus on the processes of synthesis/metabolism, receptor interaction/ neuronal signaling or the behavioral relevance of neurochemical events sculpts the experiment design. Brain microdialysis is an important method for examining changes in the content of any substances, irrespective of their origin, in living animals. This article compares contemporary approaches and techniques that are used for monitoring neurotransmission (including in vivo brain microdialysis, voltammetric methods, etc). We highlight practical aspects of microdialysis experiments in particular to those researchers who are seeking to increase the repertoire of their experimental techniques with brain microdialysis.     

In vivo microdialysis for nonapeptides in rat brain--a practical guide

Microdialysis provides a direct approach to monitor changes in interneuronal communication by monitoring the fluctuation of local, extracellular concentrations of potential neurotransmitters/neuromodulators. The present article is based on more than 10 years experience in performing microdialysis experiments in freely moving animals with inexpensive self-made microdialysis probes and accessories for monitoring of intracerebral neuropeptide release. On the basis of this experience, we provide a guide for the construction of different types of microdialysis probes and their application. Furthermore, we give information about organizing and performing a microdialysis experiment that can easily be adapted to fit individual applications needs. Finally, on the basis of theoretical background information advantages as well as limitations of the microdialysis technique are discussed with the intent to provide help to potential users for designing an appropriate microdialysis experiment.     

Application of in vivo microdialysis to the study of cholinergic systems

The application of in vivo microdialysis to the study of acetylcholine (ACh) release has contributed greatly to our understanding of cholinergic brain systems. This article reviews standard experimental procedures for dialysis probe selection and implantation, perfusion parameters, neurochemical detection, and data analysis as they relate to microdialysis assessments of cholinergic function. Particular attention is focused on the unique methodological considerations that arise when in vivo microdialysis is dedicated expressly to the recovery and measurement of ACh as opposed to other neurotransmitters. Limitations of the microdialysis technique are discussed, as well as methodological adaptations that may prove useful in overcoming these limitations. This is followed by an overview of recent studies in which the application of in vivo microdialysis has been used to characterize the basic pharmacology and physiology of cholinergic neurons. Finally, the usefulness of the microdialysis approach for testing hypotheses regarding the cholinergic systems' involvement in cognitive processes is examined. It can be concluded that, in addition to being a versatile and practical method for studying the neurochemistry of cholinergic brain systems, in vivo microdialysis represents a valuable tool in our efforts to better comprehend ACh's underlying role in a variety of behavioral processes.     

N-methyl-D-aspartate receptor-mediated modulation of monoaminergic metabolites and amino acids in the chick forebrain: an in vivo microdialysis and electrophysiology study.

The associative avian forebrain region medio-rostral neostriatum/hyperstriatum ventrale (MNH) is involved in auditory filial imprinting and may be considered the avian analogue of the mammalian prefrontal cortex. In search of the neurochemical and physiological mechanisms which play a role in this learning process, we introduced microdialysis and a combined microdialysis/electrophysiological approach in domestic chicks a few days old. With this technique, we were able to follow changes of the extracellular levels of glutamate, taurine, 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin, and homovanillic acid (HVA), a metabolite of dopamine, and neuronal activity simultaneously in freely moving animals. We obtained first evidence of a modulatory interaction between glutamatergic and monoaminergic neurotransmission mediated by N-methyl-D-aspartate (NMDA) receptors. During local intracerebral infusion of 300 microM NMDA via reverse microdialysis, an increase of taurine and a decrease of 5-HIAA and HVA were detected, accompanied by enhanced extracellular spike rates. Glutamate was increased only during consecutive infusion of increasing NMDA concentrations, when higher (1 mM) NMDA concentrations were infused. The effects of NMDA were antagonized by D, L-2-amino-5-phosphonovaleric acid (1 mM). Infusion of high potassium induced similar changes in taurine, 5-HIAA, and HVA, as found during infusion of NMDA, but decreased extracellular spike rates, which indicates that different cellular mechanisms may underlie the observed neurochemical changes. Neither urethane anesthesia nor different delays between probe implantation and experiment influenced the neurochemical and electrophysiological results; however, changes of taurine were observed only in chronically implanted, awake animals. In summary, microdialysis in combination with electrophysiology provides a powerful tool to detect changes of neuronal activity and transmitter release in the avian brain, with which the role of transmitter interactions can be followed during and after different learning events.     

In vivo evidence against a role for adenosine in the exercise pressor reflex in humans.

The pressor response to exercise is of great importance in both physiology and pathophysiology. Whether endogenous adenosine is a trigger for this reflex remains controversial. Muscle interstitial adenosine concentration can be determined by microdialysis. However, there are indications that local muscle cell damage by the microdialysis probe confounds these measurements in exercising muscle. Therefore, we used the nucleoside uptake inhibitor dipyridamole as pharmacological tool to bypass this confounding. We used microdialysis probes to measure endogenous adenosine in forearm skeletal muscle of healthy volunteers during two cycles of 15 min of intermittent isometric handgripping. During the second contraction, dipyridamole (12 microg.min(-1).dl forearm(-1)) was administered into the brachial artery. Dipyridamole potentiated the exercise-induced increase in dialysate adenosine from 0.30 +/- 0.08 to 0.48 +/- 0.10 micromol/l (n = 9, P < 0.05), but it did not potentiate the exercise-induced increase in blood pressure. A time-control study without dipyridamole revealed no difference in exercise-induced increase in adenosine between both contractions (n = 8). To exclude the possibility that the dipyridamole-induced increase in dialysate adenosine originates from extravasation of increased circulating adenosine, we simultaneously measured adenosine with microdialysis probes in forearm muscle and antecubital vein. In a separate group of nine volunteers, simultaneous intrabrachial infusion of 100 microg.min(-1).dl(-1) dipyridamole and 5 microg.min(-1).dl(-1) adenosine increased dialysate adenosine from the intravenous but not the interstitial probe, indicating preserved endothelial barrier function for adenosine. We conclude that dipyridamole significantly inhibits uptake of interstitial adenosine without affecting the pressor response to exercise, suggesting that interstitial adenosine is not involved in the pressor response to rhythmic isometric exercise.     

In vivo Microdialysis of 5-Hydroxyindoleacetic Acid and Glutamic Acid in the Hamster Suprachiasmatic Nuclei

SYNOPSIS. The technique of in vivo brain microdialysis rapidlyis becoming a popular tool for research on the neurochemicalbasis of physiological and behavioral functions. The presentstudy describes the application of microdialysis to investigatethe endogenous release of 5-hydroxyindoleacetic acid (5-HIAA)and glutamic acid in the suprachiasmatic nuclei (SCN) of hamsters.There were apparent circadian patterns of release of both ofthese neurosecretions, with peak levels occurring during thedark phase. Pharmacological manipulations of serotonin releaseand reuptake, using tetrodotoxin and citalopram, respectively,provided evidence that the nocturnal increase in 5-HIAA reflectsan increase in serotonergic synaptic activity, rather than intraneuronalmetabolism of unreleased serotonin. These results illustratethe usefulness of the microdialysis technique for studies onthe neurochemistry of central pacemaker function.     

Microdialysis: intraoperative and posttraumatic applications in neurosurgery

Microdialysis offers a unique opportunity to study the extracellular human brain environment. Our aim was to improve the ability to detect neuronal injury in patients undergoing complicated neurovascular procedures and to detect secondary brain lesions after severe head injury with the use of in vivo microdialytic monitoring. We employed intraoperative microdialysis monitoring in patients with a variety of neurovascular procedures including high-flow and low-flow bypass surgery and aneurysm clipping. In the second group there were patients after severe brain injury where in vivo microdialysis was performed as a bedside monitoring in the intensive care unit. In this review we present our results for these various groups of patients, in which a variety of neurochemical parameters which are known to change in ischemia were studied. The advantages and disadvantages of the technique are discussed. Our results show that microdialysis is a useful tool for detecting ischemic changes intraoperatively and in severe head trauma. However, further studies are needed to get more data and to better refine the online microdialysis technique before we can recommend it for routine clinical use.     

GABA Uptake Inhibition Reduces In Vivo Extraction Fraction in the Ventral Tegmental Area of Long Evans Rats Measured by Quantitative Microdialysis Under Transient Conditions

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4?±?1.9 nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19?±?0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50 μM) significantly increased extracellular GABA concentration to 170?±?4 nM and reduced in vivo extraction fraction to 0.112?±?0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

N‐methyl‐D‐aspartate receptor–mediated modulation of monoaminergic metabolites and amino acids in the chick forebrain: An in vivo microdialysis and electrophysiology study

The associative avian forebrain region medio‐rostral neostriatum/hyperstriatum ventrale (MNH) is involved in auditory filial imprinting and may be considered the avian analogue of the mammalian prefrontal cortex. In search of the neurochemical and physiological mechanisms which play a role in this learning process, we introduced microdialysis and a combined microdialysis/electrophysiological approach in domestic chicks a few days old. With this technique, we were able to follow changes of the extracellular levels of glutamate, taurine, 5‐hydroxyindoleacetic acid (5‐HIAA), a metabolite of serotonin, and homovanillic acid (HVA), a metabolite of dopamine, and neuronal activity simultaneously in freely moving animals. We obtained first evidence of a modulatory interaction between glutamatergic and monoaminergic neurotransmission mediated by N‐methyl‐D ‐aspartate (NMDA) receptors. During local intracerebral infusion of 300 μM NMDA via reverse microdialysis, an increase of taurine and a decrease of 5‐HIAA and HVA were detected, accompanied by enhanced extracellular spike rates. Glutamate was increased only during consecutive infusion of increasing NMDA concentrations, when higher (1 mM) NMDA concentrations were infused. The effects of NMDA were antagonized by D , L ‐2‐amino‐5‐phosphonovaleric acid (1 mM). Infusion of high potassium induced similar changes in taurine, 5‐HIAA, and HVA, as found during infusion of NMDA, but decreased extracellular spike rates, which indicates that different cellular mechanisms may underlie the observed neurochemical changes. Neither urethane anesthesia nor different delays between probe implantation and experiment influenced the neurochemical and electrophysiological results; however, changes of taurine were observed only in chronically implanted, awake animals. In summary, microdialysis in combination with electrophysiology provides a powerful tool to detect changes of neuronal activity and transmitter release in the avian brain, with which the role of transmitter interactions can be followed during and after different learning events. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 116–135, 1999     

Microdialysis sampling of cytokines

Microdialysis sampling is a well-known method for collection of low molecular weight hydrophilic analytes. Due to the success of this sampling technique for these analytes, many researchers have wanted to extend the use of this method to a wider range of analytes-particularly proteins and peptides. These analytes pose unique challenges during microdialysis sampling. The primary challenges are the reduced recovery across the dialysis semi-permeable membrane and the volume limitations/requirements for the typical immunoassay methods used for detection of proteins. This review covers the practical and theoretical aspects needed for in vivo microdialysis sampling of cytokines, which are a vitally important class of signaling proteins. In addition to the basics of the microdialysis method for sampling cytokines, the use of the microdialysis device as a localized cytokine delivery method is also described. Since relative recovery of cytokines is often low during microdialysis sampling, methods to improve the membrane recovery are discussed for in vitro and in vivo applications.     

An Analytical Flow Injection System to Measure Glutamate in Microdialysis Samples Based on an Enzymatic Reaction and Electrochemical Detection

Glutamate (Glu) is the main excitatory neurotransmitter in the brain for which several methods have been developed to measure this compound in extracellular brain fluids. Most of these techniques are based on coupling microdialysis to HPLC and they have a resolution time of about 10 min. Here, we present a different approach to measure Glu with a resolution of about 1 min per microdialysis sample, enabling a better relationship to be established between EEG activity and biochemical changes. This new setup was used to determine the time delay between the tip of the microdialysis probe and the site of sample collection, and was accurate to within seconds. Indeed, the measurement of Glu concentrations was linear. Administration of 4-aminopyridine was used to provoke seizure convulsions and under these conditions, biochemical changes and EEG activity were evaluated. These experimental data support the key role of Glu in the initiation of a seizure convulsion. Special issue article in honor of Dr. Ricardo Tapia.     

Effects of Different Semipermeable Membranes on In Vitro and In Vivo Performance of Microdialysis Probes

The in vitro and in vivo performance of three different semipermeable microdialysis membranes was compared: a proprietary polycarbonate-ether membrane made by Carnegie Medecin; cuprophan, a regenerated cellulose membrane; and polyacrylonitrile. When microdialysis probes were tested in a stirred in vitro solution, large and statistically significant differences among the three membranes in extraction of acid metabolites (3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, and homovanillic acid) and acetaminophen were found. Polyacrylonitrile had the highest extractions in vitro. In contrast, when microdialysis probes were implanted in vivo (in rat striatum), extraction of acid metabolites and acetaminophen did not differ significantly among the different membranes. These results are consistent with predictions made by a mathematical model of microdialysis and can be explained by the fact that in vitro the main factor limiting extraction is membrane resistance to diffusion, whereas tissue resistance to diffusion plays a more dominant role in vivo. These findings suggest that (aside from differences in surface area), the choice of semipermeable membrane will generally have little effect on in vivo microdialysis results. Furthermore, in vitro measurements of microdialysis probe extractions are not a reliable way of calibrating in vivo performance.     

Effect of fluoxetine on serotonin and dopamine concentration in microdialysis fluid from rat striatum.

Fluoxetine injected i.p. into rats at a dose of 10 mg/kg rapidly increased serotonin concentration in microdialysis fluid from the striatum by at least 4-fold, an increase that was maintained throughout the 3 hr observation period. Dopamine concentration in the microdialysis fluid did not change. The concentration of the two dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was not changed in the microdialysis fluid, whereas the concentration of the serotonin metabolite, 5-hydroxyindoleacetic acid, was significantly decreased after fluoxetine injection. The increased extracellular concentration of serotonin no doubt resulted from inhibition of the serotonin uptake carrier by fluoxetine, and the lack of change in dopamine is evidence for the specificity of action of this uptake inhibitor.     

Intracerebral Microdialysis: Electrophysiological Evidence of a Critical Pitfall

Abstract: Using microdialysis probes incorporating an electrode for the recording of extracellular field potentials, we have found that microdialysis markedly inhibited the propagation of spreading depression. This effect was independent of the microdialysis flow rate and did not result from tissue injury following probe implantation. Increasing the K⁺ concentration in the perfused artificial CSF dose-dependently restored the propagation of spreading depression and revealed a large, synchronous transient increase in extracellular glutamate. These findings clearly illustrate that microdialysis can influence the experimental or pathological conditions under study, by buffering transient changes in the extracellular fluid composition. Epileptic seizures and ischaemia are two important conditions that may be prone to such a detrimental interaction.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Extracellular glutamate changes in rat striatum during ischemia determined by a novel dialysis electrode and conventional microdialysis

Our newly developed method using a dialysis electrode has made it possible to perform real time monitoring of extracellular glutamate concentration ([Glu]e) utilizing the oxygen-independent reaction with glutamate oxidase and ferrocene. In this study, we therefore, investigated [Glu]e changes during brain ischemia using both the conventional microdialysis method and the dialysis electrode method. A comparison between our newly developed dialysis electrode and conventional microdialysis methods provided the following results. When the conventional microdialysis method was employed: (1) the elevation of [Glu]e during complete global ischemia was delayed; and (2) the elevation of concentration and reuptake of glutamate were delayed during 10-min transient ischemia, and the elevation of [Glu]e reached a maximum later using conventional microdialysis than using our dialysis electrode. (3) The biphasic [Glu]e elevation of glutamate concentration detected using the dialysis electrode method was not observed using the conventional microdialysis method. It was additionally investigated why the conventional microdialysis method provides inferior time resolution. In this study, we also demonstrated with the chromatographic SMART procedure coupled to UV detection that biogenic substances, i.e. low molecular weight proteins and peptides, are released during ischemic injury, and they may cause a delay in the time resolution in the microdialysis method.     

Quantitative dual-probe microdialysis: mathematical model and analysis

Steady-state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual-probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual-probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual-probe microdialysis.     

Endogenous Release of γ-Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.