Chapter 2.5 Microdialysis as a platform for multidisciplinary strategies

CHAPTER 2.5

Microdialysis as a platform for multidisciplinary strategies J. Urenjak and T.P. Obrenovitch Pharmacy, School of Life Sciences, University of Bradford, Bradford, UK

Abstract: Microdialysis is a versatile in vivo sampling technique, with the unique capability to provide information on the biochemistry of the cellular microenvironment within a specific region of the brain or other organs. Microdialysis may also be used to deliver biochemicals, drugs and toxins directly to the site under study. As such, microdialysis by itself allows the combination of biochemistry, pharmacology, and experimental pathology in a single in vivo preparation. This basic multidisciplinary potential can be supplemented by a number of monitoring techniques, whether electrode based or optic fiber based, to collect information on other relevant biochemical or physiological variables. Finally, microdialysis can be associated with imaging methods such as magnetic resonance imaging (MRI). In this chapter, we make clear and illustrate with selected applications the unique capability of microdialysis to provide a versatile platform for the assembly of a wide range of multidisciplinary strategies that can be tailored to specific study objectives. In many cases, the interpretation of the data may be complicated by the fact that the different methods may not involve precisely the same tissue area or compartment. However, this potential difficulty can be dealt with, and once resolved it can lead to a better understanding of the biological processes under study. disciplines can be brought in, either applied locally (but independently from microdialysis) or more globally, to achieve a truly multidisciplinary and powerful experimental strategy. The primary purpose of this chapter is to make clear, and illustrate with selected applications, that microdialysis can be used as the core technique for a variety of in vivo multidisciplinary strategies. The monitoring of several variables from the same tissue site favors a more accurate and reliable interpretation of each separate data set, and reduces the number of animals required to test specific hypotheses. At the end of the chapter, we also discuss some limitations and pitfalls inherent to microdialysis within the context of multidisciplinary strategies. Notes:

Microdialysis is widely recognized as a versatile in vivo sampling technique, with the unique capability to provide information on the biochemistry of the cellular microenvironment (i.e., extracellular fluid composition) within a specific region of the brain and other organs. Microdialysis may also be\ used to deliver drugs directly to the site under study, which avoids any possible interference of the drugs with other organs, and in brain studies circumvents the potential impermeability of the blood-brain barrier to drug prototypes. As an extension to reverse microdialysis for drug delivery, by changing the composition of the perfusion medium or by adding specific agents to the medium, it may be possible to induce a relevant pathological condition, or to reproduce some aspect(s) of such a condition. Finally, a number of methods from various

 Corresponding author: E-mail: [email protected]

B.H.C. Westerink and T.I.F.H. Cremers (Eds.) Handbook of Microdialysis, Vol. 16 ISBN 0-444-52276-X

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Although some of the microdialysis-based multidisciplinary strategies may be applicable DOI: 10.1016/S1569-7339(06)16011-7 Copyright 2007 Elsevier B.V. All rights reserved

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to other organs, this chapter is essentially focused on in vivo studies of the central nervous system (CNS). Only selected methodological combinations based on microdialysis are considered and discussed herein. Many other combinations are possible, and it is clear that the optimal one for any given purpose will have to be selected carefully on the basis of the study objectives and practical considerations.

I. Pharmacology, biochemistry, and behavioral studies The combination of any two of these disciplines, or of the three together, is discussed only briefly here because other chapters in this handbook are dedicated to these topics (Sections on pharmacology and animal behavior). This combination has been very useful for investigations into the neurochemical control of feeding (Fig. 1), addiction (Fu et al., 2001), stress/anxiety (Swanson et al., 2004), and sexual/maternal behaviors (Da Costa et al., 1996).

I.A. Pharmacology – the drug delivery issue Although emphasis is placed on drug delivery via the perfusion medium in Fig. 1, this is only one way to bring a pharmacological component into the experimental strategy. Reverse microdialysis may be optimal for some studies, for example, when drugs are anticipated to act presynaptically or on the inactivation of released neurotransmitter

PHARMACOLOGY Microdialysis used for the delivery of drug(s) via the perfusion medium.

(i.e., enzymatic degradation and uptake). It also enables the sequential application of different drugs and concentrations to the site under study, and this can be carried out over an extended period, even in freely moving animals. However, reverse microdialysis may be inappropriate for a number of reasons as follows, whether separate or combined: (i) Behavioral effects may only occur when the drug acts on a brain region that is larger than the site that can be ‘influenced’ by microdialysis (i.e., up to 1 mm away from the probe axis; Benveniste and Huttemeier, 1990), and in some studies drug intake may be part of the behavioral test (e.g., self-administration of addictive drugs). Neurochemical changes are often to be expected in other brain regions than that challenged pharmacologically. In all these cases, systemic or intracerebroventricular drug administration may be considered. (Fu et al., 2001; Price and Lucki, 2001). In some studies, it may be pertinent to use two separate microdialysis probes, one for drug delivery and the other as a sampling device (Alex et al., 2005; Zackheim and Abercrombie, 2005). (ii) Some drugs may not be suitable for administration via the microdialysis probe because of their size relative to the dialysis membrane cutoff (e.g., neuropeptides), their adsorption to the dialysis fiber and other components of the microdialysis system (e.g., high-precision syringes and inlet tubing), their poor solubility in aqueous media or simply their cost because microdialysis

BIOCHEMISTRY Microdialysis sampling; Study of changes in the extracellular fluid composition.

BEHAVIORAL TESTS

Illustrative application – Serotonergic (5-HT) control of feeding: Delivery of a subtype selective 5-HT agonist to specific brain regions; monitoring of the extracellular changes in 5-HT and its metabolism in these regions; and measure of changes in food intake (See legend for reference).

Fig. 1. Diagram of one of the most common microdialysis-based multidisciplinary strategy. Reference for illustrative application Hikiji et al., 2004.

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delivery implies that only a fraction of the drug in the perfusion medium is transferred to the tissue surrounding the dialysis fiber (estimated to be o10% of the perfusion medium content and dependent on the drug; Benveniste and Huttemeier, 1990). In any of these cases, and if systemic and intracerebroventricual administration are not suitable, a microinjection cannula positioned in a region adjacent to the microdialysis fiber may be used to microinject the drug precisely at the sampling site (Masuo et al., 1993). With both medium-sized molecules (e.g., endothelin-1, MW 2492; Fig. 4) and small molecules (e.g., glutamate receptor agonist AMPA, and MW 186) we have found that similar effects (assessed by local monitoring at the microdialysis site) can be achieved by microdialysis delivery and such a microinjection cannula. (iii) Inherently, reverse microdialysis does not allow a very sudden and brief rise in the effective drug concentration at tissue level. This potential problem, seldom envisaged, may be a pitfall in studies involving agonists designed to act on receptors that desensitize very rapidly (e.g., nicotinic acetylcholine receptors). A suitable pulse application of drug at the microdialysis site may be achievable by using a microcannula connected to a pressure ejection system (Rogers, 1985). With some compounds (e.g., nitric oxide) an alternative may be offered by ‘caged’ molecules, that is, compounds that are transformed to their biologically active state by illumination with appropriate wavelength and intensity (Godwin et al., 1997). However, in studies that combine pharmacology and biochemistry it is likely that microdialysis sampling would then become the limiting factor, despite the much better time resolution that is now achievable with advanced analytical techniques. Voltammetric methods may be more appropriate in these conditions (Sotty et al., 1998; Parikh et al., 2004). (iv) When biochemicals and drugs are perfused through a microdialysis probe, one often

assumes that, providing they permeate the dialysis membrane, they will reach their molecular target within the area ‘influenced’ by microdialysis. That is likely to occur with drugs designed to act on receptors distributed on the external side of cellular membranes, but not necessarily true when the expected site of drug action is intracellular. In addition, drug access via the extracellular fluid may not be optimal for some components of the brain cytoarchitecture (e.g., brain arterioles endothelium and smooth muscle, as these blood vessels are tightly surrounded by astrocyte endfeet).

II. Pharmacology and/or induction of a pathological condition, biochemistry and electrophysiology Recording some relevant electrophysiological variable(s) is most useful to verify, independently from the biochemical data, that the pharmacological treatment (e.g., concentration of drug in the perfusion medium) or the induced pathophysiological condition is effective and appropriate at the site studied by microdialysis. Conversely, the electrophysiological variable(s) may be the primary one, and the biochemical data used to verify the effectiveness of the pharmacological treatment (Fig. 2). Reverse microdialysis may be used also as a convenient way to apply different drugs, or several concentrations of a single drug, consecutively at the site studied by electrophysiology. For all such studies, optimal validity of the electrophysiological component implies that the electrode system should be located as close as possible to the dialysis fiber, which may be achieved as follows: (i) incorporation of the electrode within the microdialysis fiber (Obrenovitch et al., 1993a); (ii) a section of the metallic body of the microdialysis probe, just above the dialysis fiber, is used as electrode (Pena and Tapia, 1999); and (iii) the electrode system is adjacent to the external surface of the dialysis fiber (Bourne and Fosbraey, 2000). Some investigators successfully combined microdialysis and electrophysiological recordings in behaving animals (Ludvig et al., 1994, 2000; Kittner

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INDUCTION OF CNS PATHOLOGY This may be achieved via the microdialysis probe, or independently. PHARMACOLOGY Microdialysis used for drug delivery

BIOCHEMISTRY Microdialysis sampling

ELECTROPHYSIOLOGY

Illustrative application – Pharmacology of cortical spreading depression (CSD): Elicitation of CSD at the microdialysis site by perfusion of high-potassium medium; delivery of a phosphodiesterase inhibitor to increase the concentration of cGMP; monitoring of the extracellular changes in cGMP to verify the efficacy of the drug treatment; and recording of CSD as a negative shift of the DC-potential, using an electrode incorporated within the microdialysis fibre (See legend for reference). Fig. 2. Diagram illustrating the possibility of using microdialysis to combine four different disciplines, simultaneously, to the same specific brain region. Reference for illustrative application Wang et al., 2004.

et al., 2004), but this is only possible with some electrophysiological signals and electrode/microdialysis configurations, because the fine input line connected to the microdialysis probe enhances any electrical noise by acting as an effective high-impedance antenna (Obrenovitch et al., 1993a).

II.A. Electrophysiological variables Electroencephalography (EEG) is the most common electrophysiological variable recorded concurrently with microdialysis, especially for studies into epilepsies (Obrenovitch et al., 1996; Bourne and Fosbraey, 2000; Tian et al., 2005) and cerebral ischemia (Obrenovitch et al., 1993b). This strategy was also used successfully for the study of sleep, but in this case EEG electrodes were generally implanted remotely from the microdialysis probe (Penalva et al., 2003; Thakkar et al., 2003; Hong et al., 2005; Crouzier et al., 2006). Note that in a number of studies of epilepsies (Richards et al., 2000; Slezia et al., 2004) and brain ischemia (Martinez-Tica and Zornow, 2000) the EEG was also recorded remotely from the microdialysis probe, which may be appropriate depending on the study objectives and the pathophysiology investigated. The extracellular direct current (DC) potential is another pertinent electrophysiological variable, because this signal can provide relevant information on depolarization shifts occurring in the cell population surrounding the microdialysis fiber,

and negative DC potential shifts are key features of several important neurological conditions (Fig. 3). In cerebral ischemia, a sustained negative shift of the DC potential indicates anoxic depolarization (i.e., the loss of cellular ionic homeostasis subsequent to energy failure), which makes it a very useful variable to assess the severity of ischemia precisely at the microdialysis site (Obrenovitch et al., 1993b; Bruhn et al., 2003). The concurrent monitoring of this signal with microdialysis allowed showing that the sudden ischemic efflux of neurotransmitters and other compounds occurs with anoxic depolarization (Obrenovitch and Richards, 1995; Takata et al., 2005). A transient, negative DC shift is the hallmark of spreading depression (SD), that is, the propagating wave of cellular depolarization that is the underlying cause of the aura in classical migraine (Lauritzen, 1994), and an important component of the pathophysiology of focal brain injuries, whether ischemic or traumatic (Strong et al., 2002). In our hands, microdialysis electrodes constitute an unrivalled tool for investigations into the pharmacology and biochemistry of SD (Fig. 2). Monitoring the DC potential may be also pertinent in studies of epilepsies, as paroxysmal depolarization appears on the DC potential as brief negative shifts (Fig. 3). Finally, monitoring the DC potential may be a very useful signal for in vivo pharmacological studies of depolarizing drugs (e.g., agonists of ionotropic glutamate receptors; Obrenovitch et al., 1994). In comparison to EEG, single-unit extracellular recording is a more refined variable, which also

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( -)

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15-min 4-AP (3 mM) Fig. 3. Changes in the DC potential produced by perfusion of the potassium channel blocker 4-aminopyridine (4-AP, 3 mM) through a microdialysis probe (1 mm fiber length) implanted into the cortex of halothane-anesthetized rats. In this figure, the polarity of the DC potential signal was inverted (–). Relevant variables for the evaluation of the tissue susceptibility to 4-APinduced convulsant activity may be (i) latency for occurrence of the first paroxysmal depolarization shift; (ii) number of depolarizations induced by the 4-AP application; (iii) cumulative area of the depolarization(s) (see trace in insert; shaded area) (Urenjak, J. and Obrenovitch, T.P., unpublished data).

provides a better spatial match with microdialysisbased pharmacology and/or biochemistry. We have demonstrated that the DC signal recorded with a microdialysis electrode is primarily generated by cells adjacent to the dialysis membrane, but that a much larger region contributes to the EEG recorded with the same electrode (Obrenovitch et al., 1993a). Similarly, the firing of hippocampal neurons was completely abolished by perfusion of 1% lidocaine through the microdialysis probe (Ludvig et al., 1994), whereas the EEG recorded via a microdialysis electrode was only reduced by lidocaine and other manipulations that are known to suppress neuronal activity (Ludvig et al., 1994; Obrenovitch, T.P. and Urenjak, J., unpublished observations). Studying the firing pattern of neurons in a specific brain region, together with microdialysis-based pharmacology and/or biochemistry in the same or a different region, is a powerful strategy to determine how different brain regions interfere with each other (Page and Abercrombie, 1999; Lee et al., 2004) and how a specific neuronal activity influences a

particular behavior (Lodge and Grace, 2005) or brain function (Brazhnik et al., 2004). Evoked potentials also provide a variable that is more refine than the EEG. They are especially suitable for the investigation of afferent innervations between brain regions. In many studies, the recording electrode was in a region different from the microdialysis implantation site (Zhang de et al, 2005), but evoked potentials can be recorded within the area that can be influenced by reverse microdialysis (Oldford and Castro-Alamancos, 2003; Crochet et al., 2005) or sampled by microdialysis (Bronzino et al., 1999; Jay et al., 1999). Finally, simultaneous microdialysis and intracellular electrophysiological recording was also performed successfully (West and Grace, 2004). II.B. Induction of a pathological condition, or of a relevant experimental change Microdialysis can be used directly to produce or mimic a pathological condition at the site of study, simply by adding a relevant toxin or agent to the perfusion medium. A variety of neurological disorders have been investigated in this way: (i) Seizure activity, with picrotoxin or 4-aminopyridine as convulsant agents (SierraParedes and Sierra-Marcuno, 1996; Tian et al., 2005) (see also Fig. 3); (ii) Ischemia, with the potent vasoconstrictor endothelin-1 (ET-1) (see illustrative application in Fig. 4); (iii) Parkinson’s disease, with the mitochondrial complex I inhibitor, 1-methyl-4-phenylpyridinium ion (MPP+) (Rollema et al., 1990; Smith and Bennett, 1997; Staal and Sonsalla, 2000; Wu et al., 2000); (iv) Huntington’s disease, with the excitotoxin quinolinic acid or the mitochondrial toxin 3-nitropropionic acid (Reynolds et al., 1999; Blum et al., 2003); (v) Alzheimer’s disease, by reverse microdialysis of b-amyloid protein (Harkany et al., 2000). Reverse microdialysis may also be used to reproduce only one specific aspect of the neuropathology

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CNS PATHOLOGY Induced via the microdialysis probe PHARMACOLOGY Microdialysis used for drug delivery

ADDITIONAL VARIABLE(S) measured at the microdialysis site, but via independent method(s)

BIOCHEMISTRY Microdialysis sampling

ELECTROPHYSIOLOGY

Illustrative application – Pathophysiology and pharmacology of cerebral ischemia Focal ischemia was produced by perfusion of the potent vasoconstrictor, endothelin-1 (ET-1) through the microdialysis probe implanted in the cerebral cortex of an anesthetized rat. The data below show: Reduction in local cerebral blood flow (local CBF) monitored at ET-1 application site by laser Doppler flowmetry; changes in electrical activity (EEG) and occurrence of anoxic depolarization (AD) detected with microdialysis electrode; histological damage assessed radially from the microdialysis fiber axis.

% 100

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Histological damage produced by microdialysis perfusion of 100 µM ET-1 for 60 min, assessed by eosinhematoxylin staining. The extent of lesion was 582 ± 33 µm (mean ± SEM, n = 3)

20 min

Fig. 4. Superimposition of an independent method to examine another physiological or biochemical variable, within the region studied with microdialysis. In this illustrative application, the independent method was laser Doppler flowmetry (LDF), used to examine the change in local blood flow produced by perfusion of the potent vasoconstrictor ET-1 through the microdialysis probe (see Fig. 5, diagram B for a diagram of the probe/LDF combination). All the traces presented above were obtained from a single, representative experiment, with a 1-mm fiber length microdialysis probe implanted in the cortex of halothane-anesthetized rats. The histology was carried out in separate experiments. Note that biochemical changes (e.g., increased extracellular lactate, neurotransmitter efflux) and drugs effects could also be studied in these experiments. This model of ‘micro’ focal ischemia was designed to be used in conjunction with genetic tools that can produce discrete changes in gene expression, such as virus-mediated gene transfer and antisense oligonucleotides (Martin, M. et al., unpublished data).

under study. For example, if we consider brain ischemia, mitochondrial toxins such as malonate (Nixdorf et al., 2001) and rotenone (Santiago et al., 1995) may be used to inhibit energy metabolism

(i.e., chemical ischemia) at the sampling site, whereas the Na+/K+-ATPase blocker ouabain may be used to simulate anoxic depolarization (Fairbrother et al., 1990; Dobolyi et al., 2000).

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Simple changes of the perfusion medium composition may also be relevant, such as a buffered, acidic perfusion medium to impose extracellular acidosis (Waterfall et al., 1996; Urenjak et al., 1997), or a hypo-osmolar medium to induce cell swelling (Taylor et al., 1995; Scheller, 2000). For some studies, it may be pertinent to use microdialysis for the local delivery of selective neurotoxins, such as the dopaminergic toxin 6-hydroxydopamine (6-OHDA; Lorrain et al., 1998; Ferger et al., 2001) and the serotonergic toxin 3,4-methylenedioxymethamphetamine (MDMA or ecstasy; Nair and Gudelsky, 2006). The latter approach, however, is essentially suitable for investigations into the early effects of the neurotoxin. Another pertinent application of reverse microdialysis, likely to become increasingly popular as our understanding of the genome improves rapidly, is the possibility to apply antisense oligonucleotides (ODNs), selectively to the site under study. Antisense ODNs are short chains of 20 nucleotides that target a specific mRNA sequence of complementary bases, to ultimately block the synthesis of a specific protein. This application of microdialysis is well described and discussed by Thakkar et al. (2003). According to these investigators, although only o1% of the antisense diffused through the dialysis membrane, reverse microdialysis of antisense ODNs has several advantages over microinjection techniques, including the ability to deliver very low and constant concentrations of antisense, which is likely to reduce the probability of any neurotoxic damage. All these elements clearly indicate that reverse microdialysis is a versatile approach to reproduce a neurological condition, or a specific abnormality that is associated with such a condition, in a relevant brain region. However, the validity of the ensuing preparation still very much depends on the hypothesis to be tested, and most of the time it will not reflect the complexity and the true neuropathogenesis of the corresponding disease. Beside the possibility offered by microdialysis to produce or mimic a neurological abnormality, microdialysis can obviously be applied to a wide range of models in which a neurological condition is produced independently (Handbook section on

models of CNS pathology). This includes a variety of models of neurological disorders, which are increasingly supplemented by the availability of mice with targeted mutations. Several studies using microdialysis have been carried out in mice with a mutation that is specific to a human disease, such as the following: mice transfected with a mutant Cu,Zn-superoxide dismutase (SOD1) gene from humans with familial amyotrophic lateral sclerosis (ALS) (Bogdanov et al., 1998; Tovar-Y-Romo and Tapia, 2006); transgenic models of Huntingon’s disease (Petersen et al., 2002; Gianfriddo et al., 2004); transgenic model of early-onset familial Parkinson’s disease (Goldberg et al., 2003); mice overexpressing amyloid-b, as a model of early-onset forms of familial Alzheimer’s disease (Cirrito et al., 2003). Many more studies used knockout mice to examine whether a specific protein is involved in a particular brain function or CNS pathology (only as examples, see Shimizu-Sasamata et al., 1998; Bortolozzi et al., 2004; Morishima et al., 2005). Although constitutive gene knockout mice are an invaluable research tool, it is important to recognize their potential limitations, which include (Beglopoulos and Shen, 2004): (i) the biological effects of the genetic modification may be compensated by adaptive changes as the organism develops; (ii) lack of specificity to brain areas of interest; and (iii) as the targeted gene(s) are altered throughout the organism and in all cell types, peripheral effects may occur and result in unwanted interferences. Inducible knockouts avoid some of these constraints, but their availability is limited. Accordingly, for microdialysis-based experiments, it may be pertinent to consider using as alternative genetic tools the antisense technology outlined above, or virus-mediated gene transfer, that is, viral vectors that can be used to introduce or upregulate specific gene(s) in a selected region of the brain of adult mice. As the antisense method, viral gene transfer reduces the risk of compensation for the genetic change, and it is organ specific. In addition, cell type specificity is easier to achieve with viral vectors than with antisense. With the viral gene transfer method, the distribution of the genetic change is restricted to a small brain region (Gerdes et al., 2000), but it is large enough for

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microdialysis applications as the studies of Sanchez-Pernaute et al. (2001) and Hirooka and Sakai (2004) showed.

III. Monitoring of physiological and/or biochemical variables, with methods independent of microdialysis III.A. Measurements focused on the microdialysis site Microdialysis can be supplemented by a wide range of methods that can provide independent information on the site under study. In vivo electrochemistry may be used to examine changes in electroactive compounds, essentially neurotransmitters and their metabolites (i.e., monoamines), ascorbic acid and uric acid. This strategy allowed investigators to examine how neurotransmitter release and uptake may be altered by microdialysis (Borland et al., 2005), and how relevant compounds administered by reverse microdialysis influenced extracellular monoamine levels monitored by in vivo voltammetry (Moghaddam et al., 1990). Within this context, the advantage of voltammetry over microdialysis is that it can provide a better time and spatial resolution. In the future, one would expect microbiosensors (or enzyme-selective microelectrodes) to extend this strategy to a wider range of endogenous compounds such as glucose, lactate, glutamate, and choline. Within this category, electrochemical nitric oxide (NO) sensors are noteworthy because of the rapid developments in the field of NO-cGMP signaling (Cherian et al., 2000; Heinzen and Pollack, 2002, 2004). Finally, local changes in tissue oxygen (PtO2) can be monitored with amperometry (Lowry et al., 1998), and Osborne et al. (2001) used a gold collar electroplated directly onto the metal shaft of the microdialysis probe as working electrode. Ion-selective electrodes (i.e., microelectrodes for the recording of changes in extracellular K+, Ca2+, H+ etc.) have also been combined successfully with microdialysis, for example, in studies of spreading depression (Moghaddam et al., 1990; Herreras and Somjen, 1993) and anoxic depolarization (Perez-Pinzon et al., 1993; Dohmen et al., 2005). They were also used to determine how

far from the dialysis fiber the perfusion of Ca2+-free medium impacts on extracellular Ca2+ (Benveniste et al., 1989). Electrochemical techniques and ion-selective electrodes may be used also to monitor exogenous compounds administered as relevant tracers. In the field of cerebral ischemia, several studies have combined microdialysis with repeated measures of local cerebral blood flow by the hydrogen clearance technique (i.e., amperometric detection of local brain tissue hydrogen with platinum electrodes) (Lowry et al., 1998; Bhardwaj et al. 2000). Tetramethylammonium (TMA+) is a useful tracer to investigate changes in extracellular space diffusion parameters as its concentration can be monitored with TMA+-selective electrodes (Nicholson, 1993; Nicholson et al., 2000). The current optical fiber technology and associated light reflectance imaging/monitoring already provide methods that can be combined with microdialysis. So far, the most relevant and readily available is laser Doppler flowmetry (LDF; see Fig. 4), designed to give information on (relative) local changes in cerebral blood flow (Bogaert et al., 2000). The imaging of intrinsic optical signals (i.e., changed in the reflectance of the tissue itself) can provide information on the neuronal activity in the vicinity of a microdialysis probe (Poe et al., 1996), but one would expect this approach to be improved by using novel, voltage(i.e., cell membrane potential) sensitive fluorescent tracers (Grinvald and Hildesheim, 2004).

III.B. Microdialysis and imaging of the living brain The imaging methods to which we refer in this section are essentially magnetic resonance imaging (MRI) and positron emission tomography (PET). The obvious advantage of combining microdialysis with one of these brain imaging techniques is that both strategies allow investigators to acquire sequential data over an extended period of time. In some studies, MRI was used to examine changes in relevant variables (e.g., ADC, apparent diffusion coefficient; fMRI, functional MRI through imaging of regional blood flow or deoxyhemoglobin) specifically in the region sampled

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with microdialysis (Forman et al., 1998; Harris et al., 2000), or in the brain area where drugs were applied by reverse microdialysis (Benveniste et al., 1992). In other studies, pharmacological MRI (phMRI) was used to collect information while the effectiveness of a systemic pharmacological challenge was tested at brain level with microdialysis (Schwarz et al., 2004). Although the combination of microdialysis with proton nuclear magnetic resonance spectroscopy (1H-MRS) appears less versatile than that with MRI, it is a pertinent strategy for the examination of sequential changes in the level of 1H-MRS-visible metabolites in both total tissue (MRS) and extracellular fluid (microdialysis). This strategy is especially relevant to N-acetylaspartate (NAA) as this compound is the most visible in 1H-MR brain spectra, and NAA may play an important role in the volume regulation of neurons (Alessandri et al., 2000). The same view and comments apply to 31P-MRS and 15N-MRS (Kintner et al., 1999; Kanamori and Ross, 2005). Note that nonmetallic probes need to be used whenever microdialysis is to be combined with magnetic resonance methods in vivo (Mason and Romano, 1995). The combination of microdialysis and PET has been used in a number of studies of ischemic or traumatic brain injury in nonhuman primates (Frykholm et al., 2005), presumably because of the relatively low spatial resolution of PET, and the relevance of these studies to multimodal brain monitoring in neurocritical care units (Vespa et al., 2005). Similarly, as PET may be used to assess the distribution of neurotransmitter receptors and their alteration by neurological disorders in the human brain, the microdialysis/PET combination was also applied to this field of study (Zimmer et al., 2002; Tsukada et al., 2004).

IV. Limitations and pitfalls of multidisciplinary strategies centered on microdialysis It is clear that any multidisciplinary strategy including microdialysis will be limited by the potential problems associated with this method, which include (i) the area of study encompasses the fiber/ tissue injury interface; (ii) difficulties in measuring ‘true’ extracellular concentrations; (iii) marked

concentration gradient of the pharmacological challenge when the drug is delivered by reverse microdialysis; (iv) low time and spatial resolution relative to synaptic events and brain cytoarchitecture, respectively. As all these limitations of microdialysis are dealt with in other chapters, we focus here on a single question that is specific to multidisciplinary strategies when they are applied together to the same brain region: Can one correlate all the information gathered with the different methods? Or, in other words: Do all the variables considered reflect the status of the same tissue area? This is a critical issue within the context of multidisciplinary strategies, because the primary aim of this approach is actually to monitor signals and gather data from the same tissue area. Unfortunately, as the following examples illustrate, that is seldom the case. We have already mentioned that, with microdialysis electrodes, the region contributing to the EEG is much larger than that contributing to the DC potential (Obrenovitch et al., 1993a). In our hands, the best combination with regard to similarity of the tissue involved in associated methods is drug delivery by reverse microdialysis and DC potential recording, because the DC potential reflects primarily the cellular ionic homeostasis of a ring of cells that are adjacent or very close to the fiber surface. Although the very selective genesis of the DC potential signal is favorable to the microdialysis electrode/drug delivery combination, this is not the case when microdialysis is used as a sampling technique in a brain region where heterogeneous changes may develop. For example, with a microdialysis electrode (4-mm fiber length) implanted into the striatum of rats subjected to middle cerebral artery occlusion (i.e., a model of focal ischemia) the microdialysis glutamate data indicated a severe ischemia with anoxic depolarization in this region – paradoxically, the DC potential showed recurrent, peri-lesion spreading depression indicating that anoxic depolarization did not occur in the ventral striatum (Wahl et al., 1994). Accordingly, probes with a short fiber-length (1 mm) should be favored when microdialysis sampling is to be combined with DC potential recording.

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LDF field pSD

Fig. 5. Illustration of the problem that may occur when several methods are applied to the same brain region; that is, data interpretation may be difficult because the different methods do not actually involve identical tissue areas or compartments. In these experiments, 100 mM of the potent vasoconstrictor endothelin-1 (ET-1) was perfused via a microdialysis probe implanted in the cortex of halothane-anesthetized rats to produce a ‘micro’ focal ischemia (see Fig. 4 for complementary data). Diagram B, shows that the microdialysis probe incorporated an electrode for the recording of the EEG and DC potential, and that local cerebral blood flow (lCBF) was monitored at the same site by LDF. Trace A, shows the changes in lCBF as ET-1 was applied to the region under study, with an unexpected, multiphasic change occurring with anoxic depolarization (AD). Peri-lesion spreading depression is known to occur in the vicinity of the ischemic core, and this was confirmed with this model. Trace C, shows the pattern of change in lCBF produced by spreading depression (SD) when it is elicited in normal cortex (transient reduction of lCBF immediately followed by hyperemia; Obrenovitch et al., 2004). On the basis of this information, we propose that the lCBF changes shown in trace A reflect the combination of two different components that occur in adjacent areas of the small brain region explored by LDF (see diagram D for the cross section of tissue studied by microdialysis and LDF). The progressive reduction of lCBF reflects the vasoconstriction produced by ET-1 in the tissue adjacent to the microdialysis fiber (MD), leading to the formation of an ischemic core (IC) around the MD. When anoxic depolarization occurs in this region (IC), a wave of perilesion spreading depression (pSD) is elicited in the area adjacent to IC, and detected by LDF as the biphasic change in lCBF that is circled in trace A.

An interesting illustration of how a difference in the areas contributing to the monitored signals may complicate their interpretation is presented in Fig. 5; that is, the complex pattern of changes in local blood flow monitored by LDF when anoxic depolarization occurred with focal ischemia produced by microdialysis delivery of the vasoconstrictor endothelin-1 (ET-1) (see also Fig. 4). These data clearly show that even with an LDF probe designed to provide information on changes in blood flow in a very small region (0.25 mm optic fiber separation) the LDF signal was markedly influenced by a larger region than the ischemic core at the time of anoxic depolarization. Although we place emphasis on this potential problem, it should not deter investigators to embark in multidisciplinary strategies assembled around microdialysis. Indeed, we have found consistently that such a multidisciplinary approach constitutes a powerful and robust tool, and interpretation difficulties can be resolved by running pertinent control experiments. V. Conclusions Microdialysis, by itself, allows investigators to combine neurochemistry, neuropharmacology, and experimental neurology in a single in vivo preparation, by sampling the extracellular fluid of the region under study while exposing it to relevant agents delivered via the probe. This basic multidisciplinary strategy can be supplemented by a number of monitoring techniques, whether electrode based or optic fiber based. Finally, microdialysis can be associated with imaging methods such as MRI and PET. As such, microdialysis constitutes a versatile platform for the assembly of a wide range of truly multidisciplinary strategies that can be tailored to specific study objectives. In many cases, the interpretation of the data may be complicated by the fact that the different methods may not involve precisely the same brain region or tissue compartment, but this potential difficulty can be dealt with, and once resolved it often leads to a better understanding of the biological processes under study.

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References Alessandri, B., al-Samsam, R., Corwin, F., Fatouros, P., Young, H.F. and Bullock, R.M. (2000) Acute and late changes in N-acetyl-aspartate following diffuse axonal injury in rats: an MRI spectroscopy and microdialysis study. Neurol. Res., 22: 705–712. Alex, K.D., Yavanian, G.J., McFarlane, H.G., Pluto, C.P. and Pehek, E.A. (2005) Modulation of dopamine release by striatal 5-HT2C receptors. Synapse, 55: 242–251. Beglopoulos, V. and Shen, J. (2004) Gene-targeting technologies for the study of neurological disorders. Neuromolecular Med., 6: 13–30. Benveniste, H., Hansen, A.J. and Ottosen, N.S. (1989) Determination of brain interstitial concentrations by microdialysis. J. Neurochem., 52: 1741–1750. Benveniste, H., Hedlund, L.W. and Johnson, G.A. (1992) Mechanism of detection of acute cerebral ischemia in rats by diffusion-weighted magnetic resonance microscopy. Stroke, 23: 746–754. Benveniste, H. and Huttemeier, P.C. (1990) Microdialysis: theory and application. Prog. Neurobiol., 35: 195–215. Bhardwaj, A., Northington, F.J., Carhuapoma, J.R., Falck, J.R., Harder, D.R., Traystman, R.J. and Koehler, R.C. (2000) P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate. Am. J. Physiol. Heart Circ. Physiol., 279: H1616–H1624. Blum, D., Galas, M.C., Pintor, A., Brouillet, E., Ledent, C., Muller, C.E., Bantubungi, K., Galluzzo, M., Gall, D., Cuvelier, L., Rolland, A.S., Popoli, P. and Schiffmann, S.N. (2003) A dual role of adenosine A2A receptors in 3-nitropropionic acid-induced striatal lesions: implications for the neuroprotective potential of A2A antagonists. J. Neurosci., 23: 5361–5369. Bogaert, L., Scheller, D., Moonen, J., Sarre, S., Smolders, I., Ebinger, G. and Michotte, Y. (2000) Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia. Brain Res., 887: 266–275. Bogdanov, M.B., Ramos, L.E., Xu, Z. and Beal, M.F. (1998) Elevated ‘‘hydroxyl radical’’ generation in vivo in an animal model of amyotrophic lateral sclerosis. J. Neurochem., 71: 1321–1324. Borland, L.M., Shi, G., Yang, H. and Michael, A.C. (2005) Voltammetric study of extracellular dopamine near microdialysis probes acutely implanted in the striatum of the anesthetized rat. J. Neurosci. Methods, 146: 149–158. Bortolozzi, A., Amargos-Bosch, M., Toth, M., Artigas, F. and Adell, A. (2004) In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice. J. Neurochem., 88: 1373–1379. Bourne, J.A. and Fosbraey, P. (2000) Novel method of monitoring electroencephalography at the site of microdialysis during chemically evoked seizures in a freely moving animal. J. Neurosci. Methods, 99: 85–90. Brazhnik, E., Borgnis, R., Muller, R.U. and Fox, S.E. (2004) The effects on place cells of local scopolamine dialysis are mimicked by a mixture of two specific muscarinic antagonists. J. Neurosci., 24: 9313–9323.

Bronzino, J.D., Kehoe, P., Hendriks, R., Vita, L., Golas, B., Vivona, C. and Morgane, P.J. (1999) Hippocampal neurochemical and electrophysiological measures from freely moving rats. Exp. Neurol., 155: 150–155. Bruhn, T., Christensen, T. and Diemer, N.H. (2003) Uptake of glutamate is impaired in the cortical penumbra of the rat following middle cerebral artery occlusion: an in vivo microdialysis extraction study. J. Neurosci. Res., 71: 551–558. Cherian, L., Goodman, J.C. and Robertson, C.S. (2000) Brain nitric oxide changes after controlled cortical impact injury in rats. J. Neurophysiol., 83: 2171–2178. Cirrito, J.R., May, P.C., O’Dell, M.A., Taylor, J.W., Parsadanian, M., Cramer, J.W., Audia, J.E., Nissen, J.S., Bales, K.R., Paul, S.M., DeMattos, R.B. and Holtzman, D.M. (2003) In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J. Neurosci., 23: 8844–8853. Crochet, S., Chauvette, S., Boucetta, S. and Timofeev, I. (2005) Modulation of synaptic transmission in neocortex by network activities. Eur. J. Neurosci., 21: 1030–1044. Crouzier, D., Baubichon, D., Bourbon, F. and Testylier, G. (2006) Acetylcholine release, EEG spectral analysis, sleep staging and body temperature studies: a multiparametric approach on freely moving rats. J. Neurosci. Methods. Da Costa, A.P., Guevara-Guzman, R.G., Ohkura, S., Goode, J.A. and Kendrick, K.M. (1996) The role of oxytocin release in the paraventricular nucleus in the control of maternal behaviour in the sheep. J. Neuroendocrinol., 8: 163–177. Dobolyi, A., Reichart, A., Szikra, T., Nyitrai, G., Kekesi, K.A. and Juhasz, G. (2000) Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus. Neurochem. Int., 37: 71–79. Dohmen, C., Kumura, E., Rosner, G., Heiss, W.D. and Graf, R. (2005) Extracellular correlates of glutamate toxicity in short-term cerebral ischemia and reperfusion: a direct in vivo comparison between white and gray matter. Brain Res., 1037: 43–51. Fairbrother, I.S., Arbuthnott, G.W., Kelly, J.S. and Butcher, S.P. (1990) In vivo mechanisms underlying dopamine release from rat nigrostriatal terminals: I. Studies using veratrine and ouabain. J. Neurochem., 54: 1834–1843. Ferger, B., Themann, C., Rose, S., Halliwell, B. and Jenner, P. (2001) 6-Hydroxydopamine increases the hydroxylation and nitration of phenylalanine in vivo: implication of peroxynitrite formation. J. Neurochem., 78: 509–514. Forman, S.D., Silva, A.C., Dedousis, N., Barbier, E.L., Fernstrom, J.D. and Koretsky, A.P. (1998) Simultaneous glutamate and perfusion fMRI responses to regional brain stimulation. J. Cereb. Blood Flow Metab., 18: 1064–1070. Frykholm, P., Hillered, L., Langstrom, B., Persson, L., Valtysson, J. and Enblad, P. (2005) Relationship between cerebral blood flow and oxygen metabolism, and extracellular glucose and lactate concentrations during middle cerebral artery occlusion and reperfusion: a microdialysis and positron emission tomography study in nonhuman primates. J. Neurosurg., 102: 1076–1084.

212 Fu, Y., Matta, S.G., Kane, V.B. and Sharp, B.M. (2001) Norepinephrine secretion in the hypothalamic paraventricular nucleus of rats during unlimited access to self-administered nicotine: an in vivo microdialysis study. J. Neurosci., 21: 8979–8989. Gerdes, C.A., Castro, M.G. and Lowenstein, P.R. (2000) Strong promoters are the key to highly efficient, noninflammatory and noncytotoxic adenoviral-mediated transgene delivery into the brain in vivo. Mol. Ther., 2: 330–338. Gianfriddo, M., Melani, A., Turchi, D., Giovannini, M.G. and Pedata, F. (2004) Adenosine and glutamate extracellular concentrations and mitogen-activated protein kinases in the striatum of Huntington transgenic mice. Selective antagonism of adenosine A2A receptors reduces transmitter outflow. Neurobiol. Dis., 17: 77–88. Godwin, D.W., Che, D., O’Malley, D.M. and Zhou, Q. (1997) Photostimulation with caged neurotransmitters using fiber optic light guides. J. Neurosci. Methods, 73: 91–106. Goldberg, M.S., Fleming, S.M., Palacino, J.J., Cepeda, C., Lam, H.A., Bhatnagar, A., Meloni, E.G., Wu, N., Ackerson, L.C., Klapstein, G.J., Gajendiran, M., Roth, B.L., Chesselet, M.F., Maidment, N.T., Levine, M.S. and Shen, J. (2003) Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons. J. Biol. Chem., 278: 43628–43635. Grinvald, A. and Hildesheim, R. (2004) VSDI: a new era in functional imaging of cortical dynamics. Nat. Rev. Neurosci., 5: 874–885. Harkany, T., Abraham, I., Timmerman, W., Laskay, G., Toth, B., Sasvari, M., Konya, C., Sebens, J.B., Korf, J., Nyakas, C., Zarandi, M., Soos, K., Penke, B. and Luiten, P.G. (2000) Beta-amyloid neurotoxicity is mediated by a glutamatetriggered excitotoxic cascade in rat nucleus basalis. Eur. J. Neurosci., 12: 2735–2745. Harris, N.G., Zilkha, E., Houseman, J., Symms, M.R., Obrenovitch, T.P. and Williams, S.R. (2000) The relationship between the apparent diffusion coefficient measured by magnetic resonance imaging, anoxic depolarization, and glutamate efflux during experimental cerebral ischemia. J. Cereb. Blood Flow Metab., 20: 28–36. Heinzen, E.L. and Pollack, G.M. (2002) Use of an electrochemical nitric oxide sensor to detect neuronal nitric oxide production in conscious, unrestrained rats. J. Pharmacol. Toxicol. Methods, 48: 139–146. Heinzen, E.L. and Pollack, G.M. (2004) Pharmacodynamics of morphine-induced neuronal nitric oxide production and antinociceptive tolerance development. Brain Res., 1023: 175–184. Herreras, O. and Somjen, G.G. (1993) Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K+ in hippocampus of anesthetized rats. Brain Res., 610: 283–294. Hikiji, K., Inoue, K., Iwasaki, S., Ichihara, K. and Kiriike, N. (2004) Local perfusion of mCPP into ventromedial hypothalamic nucleus, but not into lateral hypothalamic area and frontal cortex, inhibits food intake in rats. Psychopharmacology (Berl.), 174: 190–196.

Hirooka, Y. and Sakai, K. (2004) Adenovirus-mediated nitric oxide synthase gene transfer into the nucleus tractus solitarius in conscious rats. Methods Mol. Biol., 279: 187–200. Hong, Z.Y., Huang, Z.L., Qu, W.M., Eguchi, N., Urade, Y. and Hayaishi, O. (2005) An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats. J. Neurochem., 92: 1542–1549. Jay, T.M., Zilkha, E. and Obrenovitch, T.P. (1999) Long-term potentiation in the dentate gyrus is not linked to increased extracellular glutamate concentration. J. Neurophysiol., 81: 1741–1748. Kanamori, K. and Ross, B.D. (2005) Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain. J. Neurochem., 94: 74–85. Kintner, D.B., Anderson, M.E., Sailor, K.A., Dienel, G., Fitzpatrick, J.H. Jr. and Gilboe, D.D. (1999) In vivo microdialysis of 2-deoxyglucose 6-phosphate into brain: a novel method for the measurement of interstitial pH using 31PNMR. J. Neurochem., 72: 405–412. Kittner, H., Krugel, U., Hoffmann, E. and Illes, P. (2004) Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study. Eur. J. Neurosci., 19: 396–404. Lauritzen, M. (1994) Pathophysiology of the migraine aura. The spreading depression theory. Brain, 117: 199–210. Lee, C.R., Abercrombie, E.D. and Tepper, J.M. (2004) Pallidal control of substantia nigra dopaminergic neuron firing pattern and its relation to extracellular neostriatal dopamine levels. Neuroscience, 129: 481–489. Lodge, D.J. and Grace, A.A. (2005) Acute and chronic corticotropin-releasing factor 1 receptor blockade inhibits cocaine-induced dopamine release: correlation with dopamine neuron activity. J. Pharmacol. Exp. Ther., 314: 201–206. Lorrain, D.S., Matuszewich, L. and Hull, E.M. (1998) 8-OHDPAT influences extracellular levels of serotonin and dopamine in the medial preoptic area of male rats. Brain Res., 790: 217–223. Lowry, J.P., Demestre, M. and Fillenz, M. (1998) Relation between cerebral blood flow and extracellular glucose in rat striatum during mild hypoxia and hyperoxia. Dev. Neurosci., 20: 52–58. Ludvig, N., Nguyen, M.C., Botero, J.M., Tang, H.M., Scalia, F., Scharf, B.A. and Kral, J.G. (2000) Delivering drugs, via microdialysis, into the environment of extracellularly recorded hippocampal neurons in behaving primates. Brain Res. Brain Res. Protoc., 5: 75–84. Ludvig, N., Potter, P.E. and Fox, S.E. (1994) Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method. J. Neurosci. Methods, 55: 31–40. Martinez-Tica, J.F. and Zornow, M.H. (2000) Effects of adenosine agonists and an antagonist on excitatory transmitter release from the ischemic rabbit hippocampus. Brain Res., 872: 110–115.

213 Mason, P.A. and Romano, W.F. (1995) Recovery characteristics of a rigid, nonmetallic microdialysis probe for use in an electromagnetic field. Bioelectromagnetics, 16: 113–118. Masuo, Y., Matsumoto, Y., Tokito, F., Tsuda, M. and Fujino, M. (1993) Effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) on the spontaneous release of acetylcholine from the rat hippocampus by brain microdialysis. Brain Res., 611: 207–215. Moghaddam, B., Gruen, R.J., Roth, R.H., Bunney, B.S. and Adams, R.N. (1990) Effect of L-glutamate on the release of striatal dopamine: in vivo dialysis and electrochemical studies. Brain Res., 518: 55–60. Morishima, Y., Miyakawa, T., Furuyashiki, T., Tanaka, Y., Mizuma, H. and Nakanishi, S. (2005) Enhanced cocaine responsiveness and impaired motor coordination in metabotropic glutamate receptor subtype 2 knockout mice. Proc. Natl. Acad. Sci. U.S.A., 102: 4170–4175. Nair, S.G. and Gudelsky, G.A. (2006) 3,4-Methylenedioxymethamphetamine enhances the release of acetylcholine in the prefrontal cortex and dorsal hippocampus of the rat. Psychopharmacology (Berl.), 184: 182–189. Nicholson, C. (1993) Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment. J. Neurosci. Methods, 48: 199–213. Nicholson, C., Chen, K.C., Hrabetova, S. and Tao, L. (2000) Diffusion of molecules in brain extracellular space: theory and experiment. Prog. Brain Res., 125: 129–154. Nixdorf, W.L., Burrows, K.B., Gudelsky, G.A. and Yamamoto, B.K. (2001) Enhancement of 3,4-methylenedioxymethamphetamine neurotoxicity by the energy inhibitor malonate. J. Neurochem., 77: 647–654. Obrenovitch, T.P. and Richards, D.A. (1995) Extracellular neurotransmitter changes in cerebral ischaemia. Cerebrovasc. Brain Metab. Rev., 7: 1–54. Obrenovitch, T.P., Richards, D.A., Sarna, G.S. and Symon, L. (1993a) Combined intracerebral microdialysis and electrophysiological recording: methodology and applications. J. Neurosci. Methods, 47: 139–145. Obrenovitch, T.P., Urenjak, J., Richards, D.A., Ueda, Y., Curzon, G. and Symon, L. (1993b) Extracellular neuroactive amino acids in the rat striatum during ischaemia: comparison between penumbral conditions and ischaemia with sustained anoxic depolarisation. J. Neurochem., 61: 178–186. Obrenovitch, T.P., Urenjak, J. and Zilkha, E. (1994) Intracerebral microdialysis combined with recording of extracellular field potential: a novel method for investigation of depolarizing drugs in vivo. Br. J. Pharmacol., 113: 1295–1302. Obrenovitch, T.P., Urenjak, J. and Zilkha, E. (1996) Evidence disputing the link between seizure activity and high extracellular glutamate. J. Neurochem., 66: 2446–2454. Obrenovitch, T.P., Wang, M., Urenjak, J., Butler, M.J. and Dreier, J.P. (2004) The role(s) or nitric oxide during cortical spreading depression. In: Krieglstein, J. and Klumpp, S. (Eds.), Pharmacology of Cerebral Ischemia 2004. Medpharm, Stuttgart, pp. 157–166.

Oldford, E. and Castro-Alamancos, M.A. (2003) Input-specific effects of acetylcholine on sensory and intracortical evoked responses in the ‘‘barrel cortex’’ in vivo. Neuroscience, 117: 769–778. Osborne, P.G., Li, X., Li, Y. and Han, H. (2001) Oxygen-sensing microdialysis probe for in vivo use. J. Neurosci. Res., 63: 224–232. Page, M.E. and Abercrombie, E.D. (1999) Discrete local application of corticotropin-releasing factor increases locus coeruleus discharge and extracellular norepinephrine in rat hippocampus. Synapse, 33: 304–313. Parikh, V., Pomerleau, F., Huettl, P., Gerhardt, G.A., Sarter, M. and Bruno, J.P. (2004) Rapid assessment of in vivo cholinergic transmission by amperometric detection of changes in extracellular choline levels. Eur. J. Neurosci., 20: 1545–1554. Pena, F. and Tapia, R. (1999) Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: a microdialysis and electroencephalographic study. J. Neurochem., 72: 2006–2014. Penalva, R.G., Lancel, M., Flachskamm, C., Reul, J.M., Holsboer, F. and Linthorst, A.C. (2003) Effect of sleep and sleep deprivation on serotonergic neurotransmission in the hippocampus: a combined in vivo microdialysis/EEG study in rats. Eur. J. Neurosci., 17: 1896–1906. Perez-Pinzon, M.A., Nilsson, G.E. and Lutz, P.L. (1993) Relationship between ion gradients and neurotransmitter release in the newborn rat striatum during anoxia. Brain Res., 602: 228–233. Petersen, A., Puschban, Z., Lotharius, J., NicNiocaill, B., Wiekop, P., O’Connor, W.T. and Brundin, P. (2002) Evidence for dysfunction of the nigrostriatal pathway in the R6/ 1 line of transgenic Huntington’s disease mice. Neurobiol. Dis., 11: 134–146. Poe, G.R., Nitz, D.A., Rector, D.M., Kristensen, M.P. and Harper, R.M. (1996) Concurrent reflectance imaging and microdialysis in the freely behaving cat. J. Neurosci. Methods, 65: 143–149. Price, M.L. and Lucki, I. (2001) Regulation of serotonin release in the lateral septum and striatum by corticotropin-releasing factor. J. Neurosci., 21: 2833–2841. Reynolds, N.C. Jr., Lin, W., Cameron, C.M. and Roerig, D.L. (1999) Extracellular perfusion of rat brain nuclei using microdialysis: a method for studying differential neurotransmitter release in response to neurotoxins. Brain Res. Brain Res. Protoc., 4: 124–131. Richards, D.A., Morrone, L.A. and Bowery, N.G. (2000) Hippocampal extracellular amino acids and EEG spectral analysis in a genetic rat model of absence epilepsy. Neuropharmacology, 39: 2433–2441. Rogers, R.C. (1985) An inexpensive picoliter-volume pressure ejection system. Brain Res. Bull., 15: 669–671. Rollema, H., Johnson, E.A., Booth, R.G., Caldera, P., Lampen, P., Youngster, S.K., Trevor, A.J., Naiman, N. and Castagnoli, N. Jr. (1990) In vivo intracerebral microdialysis studies in rats of MPP+ analogues and related charged species. J. Med. Chem., 33: 2221–2230.

214 Sanchez-Pernaute, R., Harvey-White, J., Cunningham, J. and Bankiewicz, K.S. (2001) Functional effect of adeno-associated virus mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum of 6-OHDA-lesioned rats. Mol. Ther., 4: 324–330. Santiago, M., Granero, L., Machado, A. and Cano, J. (1995) Complex I inhibitor effect on the nigral and striatal release of dopamine in the presence and absence of nomifensine. Eur. J. Pharmacol., 280: 251–256. Scheller, D.K. (2000) Extracellular taurine as a parameter to monitor cerebral insults ‘on-line’: time courses and mechanisms as studied in vivo. Adv. Exp. Med. Biol., 483: 265–272. Schwarz, A.J., Zocchi, A., Reese, T., Gozzi, A., Garzotti, M., Varnier, G., Curcuruto, O., Sartori, I., Girlanda, E., Biscaro, B., Crestan, V., Bertani, S., Heidbreder, C. and Bifone, A. (2004) Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration. Neuroimage, 23: 296–304. Shimizu-Sasamata, M., Bosque-Hamilton, P., Huang, P.L., Moskowitz, M.A. and Lo, E.H. (1998) Attenuated neurotransmitter release and spreading depression-like depolarizations after focal ischemia in mutant mice with disrupted type I nitric oxide synthase gene. J. Neurosci., 18: 9564–9571. Sierra-Paredes, G. and Sierra-Marcuno, G. (1996) Microperfusion of picrotoxin in the hippocampus of chronic freely moving rats through microdialysis probes: a new method of induce partial and secondary generalized seizures. J. Neurosci. Methods, 67: 113–120. Slezia, A., Kekesi, A.K., Szikra, T., Papp, A.M., Nagy, K., Szente, M., Magloczky, Z., Freund, T.F. and Juhasz, G. (2004) Uridine release during aminopyridine-induced epilepsy. Neurobiol. Dis., 16: 490–499. Smith, T.S. and Bennett, J.P. Jr. (1997) Mitochondrial toxins in models of neurodegenerative diseases. I: in vivo brain hydroxyl radical production during systemic MPTP treatment or following microdialysis infusion of methylpyridinium or azide ions. Brain Res., 765: 183–188. Sotty, F., Souliere, F., Brun, P., Chouvet, G., Steinberg, R., Soubrie, P., Renaud, B. and Suaud-Chagny, M.F. (1998) Differential effects of neurotensin on dopamine release in the caudal and rostral nucleus accumbens: a combined in vivo electrochemical and electrophysiological study. Neuroscience, 85: 1173–1182. Staal, R.G. and Sonsalla, P.K. (2000) Inhibition of brain vesicular monoamine transporter (VMAT2) enhances 1-methyl-4-phenylpyridinium neurotoxicity in vivo in rat striata. J. Pharmacol. Exp. Ther., 293: 336–342. Strong, A.J., Fabricius, M., Boutelle, M.G., Hibbins, S.J., Hopwood, S.E., Jones, R., Parkin, M.C. and Lauritzen, M. (2002) Spreading and synchronous depressions of cortical activity in acutely injured human brain. Stroke, 33: 2738–2743. Swanson, C.J., Perry, K.W. and Schoepp, D.D. (2004) The mGlu2/3 receptor agonist, LY354740, blocks immobilization-induced increases in noradrenaline and dopamine release in the rat medial prefrontal cortex. J. Neurochem., 88: 194–202.

Takata, K., Takeda, Y., Sato, T., Nakatsuka, H., Yokoyama, M. and Morita, K. (2005) Effects of hypothermia for a short period on histologic outcome and extracellular glutamate concentration during and after cardiac arrest in rats. Crit. Care Med., 33: 1340–1345. Taylor, D.L., Davies, S.E., Obrenovitch, T.P., Doheny, M.H., Patsalos, P.N., Clark, J.B. and Symon, L. (1995) Investigation into the role of N-acetylaspartate in cerebral osmoregulation. J. Neurochem., 65: 275–281. Thakkar, M.M., Winston, S. and McCarley, R.W. (2003) A1 receptor and adenosinergic homeostatic regulation of sleep-wakefulness: effects of antisense to the A1 receptor in the cholinergic basal forebrain. J. Neurosci., 23: 4278–4287. Tian, G.F., Azmi, H., Takano, T., Xu, Q., Peng, W., Lin, J., Oberheim, N., Lou, N., Wang, X., Zielke, H.R., Kang, J. and Nedergaard, M. (2005) An astrocytic basis of epilepsy. Nat. Med., 11: 973–981. Tovar-Y-Romo, L.B. and Tapia, R. (2006) Cerebral neurons of transgenic ALS mice are vulnerable to glutamate release stimulation but not to increased extracellular glutamate due to transport blockade. Exp. Neurol. Tsukada, H., Nishiyama, S., Fukumoto, D., Ohba, H., Sato, K. and Kakiuchi, T. (2004) Effects of acute acetylcholinesterase inhibition on the cerebral cholinergic neuronal system and cognitive function: functional imaging of the conscious monkey brain using animal PET in combination with microdialysis. Synapse, 52: 1–10. Urenjak, J., Zilkha, E., Gotoh, M. and Obrenovitch, T.P. (1997) Effect of acidotic challenges on local depolarizations evoked by N-methyl-D-aspartate in the rat striatum. Life Sci., 61: 523–535. Vespa, P., Bergsneider, M., Hattori, N., Wu, H.M., Huang, S.C., Martin, N.A., Glenn, T.C., McArthur, D.L. and Hovda, D.A. (2005) Metabolic crisis without brain ischemia is common after traumatic brain injury: a combined microdialysis and positron emission tomography study. J. Cereb. Blood Flow Metab., 25: 763–774. Wahl, F., Obrenovitch, T.P., Hardy, A.M., Plotkine, M., Boulu, R. and Symon, L. (1994) Extracellular glutamate during focal cerebral ischaemia in rats: time course and calcium dependency. J. Neurochem., 63: 1003–1011. Wang, M., Urenjak, J., Fedele, E. and Obrenovitch, T.P. (2004) Effects of phosphodiesterase inhibition on cortical spreading depression and associated changes in extracellular cyclic GMP. Biochem. Pharmacol., 67: 1619–1627. Waterfall, A.H., Singh, G., Fry, J.R. and Marsden, C.A. (1996) Acute acidosis elevates malonaldehyde in rat brain in vivo. Brain Res., 712: 102–106. West, A.R. and Grace, A.A. (2004) The nitric oxide-guanylyl cyclase signaling pathway modulates membrane activity States and electrophysiological properties of striatal medium spiny neurons recorded in vivo. J. Neurosci., 24: 1924–1935. Wu, W.R., Zhu, Z.T. and Zhu, X.Z. (2000) Differential effects of L-deprenyl on MPP+- and MPTP-induced dopamine overflow in microdialysates of striatum and nucleus accumbens. Life Sci., 67: 241–250.

215 Zackheim, J. and Abercrombie, E.D. (2005) Thalamic regulation of striatal acetylcholine efflux is both direct and indirect and qualitatively altered in the dopamine-depleted striatum. Neuroscience, 131: 423–436. Zhang, deX., Williamson, J.M., Wu, H.Q., Schwarcz, R. and Bertram, E.H. (2005) In situ-produced 7-chlorokynurenate has different effects on evoked responses in rats with limbic

epilepsy in comparison to naive controls. Epilepsia, 46: 1708–1715. Zimmer, L., Mauger, G., Le Bars, D., Bonmarchand, G., Luxen, A. and Pujol, J.F. (2002) Effect of endogenous serotonin on the binding of the 5-HT1A PET ligand 18F-MPPF in the rat hippocampus: kinetic beta measurements combined with microdialysis. J. Neurochem., 80: 278–286.