Progress in NeurobiologyVol. 35, pp. 195 to 215, 1990 Printed in Great Britain.All rights reserved

0301-0082/90/$0.00 + 0.50 © 1990PergamonPresspie

MICRODIALYSIS--THEORY AND APPLICATION HELENE BENVE~Sa'E* a n d PETER CHRISTIAN H O T I ~ m I E R t *Neurology Research Laboratory, Department of Medicine (Neurology), Duke University Medical Center, Durham, NC 27710, U.S.A. ~fDepartment of Anesthesiology, Duke University Medical Center, Durham, NC 27710, U.S.A.

(Received 26 February 1990)

CONTENTS 1. Introduction 2. Principle of microdiaiysis 3. Practical aspects 3.1. Implantation of the microdialysis probe in brain tissue 3.2. Apphcation in other tissues 3.3. Perfusion fluids 4. Measurements with microdialysis 4.1. Concentration measurements based on/n vitro calibration 5. Factors affecting substance collection with microdialysis 5.1. Perfusion flow rate and time 5.2. Flux 5.3. Temperatures and changes in outer substance concentration 5.4. Dialysis membrane area and composition 5.5. Diffusion coefficient 6. Effect of microdialysis on the brain microenvironment 6.1. Brain compartmentation 6.2. Tissue reactions 6.3. Local cerebral blood flow and 2-deoxyglucose phosphorylation 6.4. Use of tetrodotoxin and calcium depletion for estimating functional damage after implantation 6.5. The possible influence of the disturbed brain mieroenvironment on results obtained by mierodialysis 7. Solutions to the problem of determining brain interstitial concentrations with microdialysis 7.1. Methods without time resolution 7.2. Methods with time resolution 8. Transformation of glutamate and dopamine dialysate concentrations into brain interstitial concentrations 8.1. Normal brain tissue 8. I. 1. Glutamate 8.1.2. Dopamine 8.2. Interpretation of calculated interstitial glutamate and dopamine concentrations obtained by microdialysis 8.3. Glutamate concentrations in isehemic brain tissue 9. Conclusion References

1. INTRODUCTION Accurate and continuous in vivo measurements of brain interstitial substance concentrations provide a major technical challenge. Among the many innovations that have appeared over the last decades i.e. cortical cup (reviewed by Moroni and Pepeu, 1984), push-pull cannula (reviewed by Phillippu, 1984), ion-selective mieroelectrodes (Thomas, 1978; Zeuthen, 1981) and carbon fiber microelectrodes (Marsden et al., 1984), microdialysis is in principle the only technique which can collect virtually any substance from remote brain regions with a limited amount of tissue trauma. Described as early as 1966 by Bito et aL, the method has undergone several technical improvements (Delgado et aL, 1972; Ungerstedt et aL, 1982; Hamberger et al., 1983;

195 196 196 196 197 197 199 200 200 200 201 201 202 202 203 203 203 203 204 204 204 204 205 207 207 207 207 207 208 208 209

Ungerstedt, 1984) and is rapidly becoming a very popular bioanalytical sampling tool in brain research. However, despite the initial excitement surrounding the concept of mierodialysis and its seemingly limitless applications, this technique like any other is obviously not without problems. Some of these were broadly discussed in a previous report that summarized the many different brain substance concentration measurements that had been performed so far (Benveniste, 1989). In reviewing all these data it is clear that certain uniform guidelines and standards are necessary if mierodialysis is to become truly useful in the future. We will attempt to deal with these issues in the following by including a description of some practical and technical aspects, a brief overview of the many applications and an evaluation of the tissue disturbances caused by the implantation procedure.

195

196

H. BENVENISTEand P. C. HOTTEMEIER

The main focus will be on brain microdialysis and solutions to the problem of calculating interstitial substance concentrations from the data obtained by microdialysis. This effort we believe would be of dubious value unless it was put into perspective with some practical examples. Therefore part of this discussion is centered around the measurement of two neurotransmitters, dopamine and glutamate, in normal and ischemic brain.

Serial

Parallel

2. PRINCIPLE O F MICRODIALYSIS The microdialysis technique uses the dialysis principle and consists of a membrane permeable to water and small solutes which is continuously flushed on one side with a solution devoid of the substances of interest, whereas the other side faces the interstitial space (this obviously only applies for substance collection; if a substance is instead to be delivered, it must be present in the perfusion fluid in a higher concentration than that of the outer medium). A concentration gradient is created causing diffusion of substances from the interstitial space into the dialysis membrane (and vice versa) as illustrated in Fig. 1. The continuous flow through the membrane carries substances to the sampling site for further analysis. A dialysis membrane connected with in- and outlet tubes, allowing fluid to enter and leave the dialysis membrane, respectively, is usually referred to as a 'microdialysis probe'. There are two main types of microdialysis probes: (a) dialysis membranes, formed

I

_ _

Loop

Side by slde

I ~

Concentric

FIG. 2. Designs. Radial sections of a serial microdialysis probe and three different mierodialysis probes with in- and outlet tubes positioned in parallel. The outer diameter of the dialysis membrane is usually 300~00 #M. The length of the membrane is adjusted to the brain regions studied.

as cylinders, connected with in- and outlet tubes in a serial arrangement, and (b) those with in- and outlet tubes positioned in parallel as illustrated in Fig. 2.

brain Interstitial fluid

3. PRACTICAL ASPECTS 0

0

3 . 1 . IMPLANTATION OF THE MICRODIALYSIS

000

PROBE XNBRAIN T~ssuE

o ®1 o o

t=O

d~alysla membrane •

o

0 0

D ~)

"¢"

0

0

O

° ,0

•

0

0

0

~

( 0

o.

It

O 'O

•

L 0 10

•

•

t

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O t~O

FIG. !. The dialysis principle. The basic principle is the positioning of a membrane that allows free diffusion of water and small solutes between the solution of interest (brain interstitial space) and a solution lacking the substances concerned. Because the perfusion fluid is constantly renewed, a concentration gradient is created across the membrane. This forms the basis for diffusion of substances from brain interstitial space into the dialysis membrane. For further details see Section 2.

The brain is especially well-suited for microdialysis because the blood-brain barrier prevents a rapid exchange of most hydrophilic substances between the brain interstitial space and the vascular compartment (Fig. 3). When implanting a microdialysis probe into the brain the animal is anesthetized and positioned in a stereotaxic frame. Having assessed the size of the region of interest it is important to coat and seal off those areas of the microdialysis membrane that may come into contact with adjacent brain regions. The positioning of the probe requires--depending on the probe design--one or two craniectomies on either side of the skull over the brain region of interest. The dura is incised prior to insertion because the dialysis membrane is otherwise easily damaged. During implantation (by means of a micromanipulator attached to the stereotaxic apparatus) the microdialysis probe is usually perfused. This is essential for some dialysis membrane types that do not tolerate drying out and is recommended in general as a means of checking membrane integrity. The microdialysis probe is usually perfused 30 min-2 hr before experiments are carried out so as to obtain 'stable' baseline values (for further details see Sections 5 and 6). For short- or long-term measurements in the awake freely moving

MICRODIALYSIS---THEORY ANDAPPLICATION interstitial

~l 1

compartment

AO•

s compartment

•

•

Oo

FIG. 3. Brain compartmentation. The brain comprises three different water spaces: The intracellular, the interstitial and the vascular space. The microdialysis membrane has direct access to the interstitial fluid compartment. For further details see Sections 3 and 5. animal the microdialysis probe is either fixed to the skull after implantation or implanted via a guide which is fixed to the skull (for further details see Lehmann et al., 1983; Delgado et al., 1984; Imperato and Di Chiara, 1984; Ungerstedt, 1984; Korf and Venema, 1985; Hernandez et al., 1986; Collin and Ungerstedt, 1988).

3.2. APPLICATIONIN OTHERTISSUES Although the microdialysis principle was originally intended for use in brain (Bito et al., 1966; Delgado et al., 1972; Ungerstedt and Pycock, 1974) and later used extensively in this organ (cf. Table l) the range of applications appear to be expanding. A variety of other organs and tissues have been studied with microdialysis (cf. Table 2). The true advantage of this remains to be seen because these organs unlike the brain have no tight barriers between the blood stream and the interstitial space. On the other hand microdialysis should be able to provide much needed information on regional metabolic events that cannot be detected by simple downstream blood sampling. Except for microdialysis in eye (Ben-Nun eta[., 1988) and heart muscle (Hamberger, 1989), the implantation procedure in other tissues seems to be straightforward (Bito et al., 1966; Delgado et al., 1972; Meirieu et al., 1986; Pilowsky et al., 1986; Brodin et al., 1987; L6nnroth et al., 1987; Arner et al., 1988; Hurd et al., 1988; Speciale et al., 1988; Lchmann, 1989; Panter et al., 1990). Ben-Nun et al. (1988) have described in detail the surgical procedures for implantation of a microdialysis probe into the vitreous cavity of the eye. Microdialysis in heart muscle is still at a developmental stage (Hamberger, 1989) and likely very problematic due to difficulties in anchoring the membrane to the moving muscle. Microdialysis in blood appears particularly useful, because it represents a way of continuous sampling without drawing any blood (Arner et al., 1988; Hurd et al., 1988; Speciale et al., 1988). This is obviously a great advantage in small animals with limited blood volumes. 3.3. PERFUSIONFLUIDS Table 3 shows the variety of perfusion fluids that are used for microdialysis. It can be seen that these

197

vary widely with respect to composition and p H - they are 'physiological' (as frequently quoted) in the sense that they are isosmotic with plasma and this is important. For instance perfusion with anisosmotic media changes the dialysate concentration of taurine collected from brain (Soils et al., 1988; Wade et al., 1988; Lehmann, 1989) and muscle (Lehmann, 1989), respectively. Another problem may arise when the investigator wants to measure a substance that is already contained in the perfusion medium. This necessitates a substitution of the substance of interest in accordance with the principles of diffusion (cf. Section 2) while at the same time maintaining the osmolarity of the perfusion fluid. The result may be a change of the initial control conditions in the surrounding tissue, e.g. substitution of choline for sodium has recently been shown to evoke a selective increase of interstitial taurine (Lehmann and Sandberg, 1990). On the other hand, isosmolar replacement of NaCl by sucrose does not seem to affect interstitial amino acid levels (Solis et al., 1988). A physiological fluid is defined as one which is isotonic with respect to blood serum, i.e. of the same osmotic concentration (osmolarity of blood serum = 300 mmol) and also composition. Perfusion fluids that are identical to plasma (for example Ringers solutions) have ion concentrations that differ from those of the brain interstitial space. Normally, the brain possesses homeostatic mechanisms that maintain constant ion concentration in the interstitial fluid and cerebrospinal fluid (CSF) despite variations in plasma composition (Bradbury, 1979). The homeostatic mechanisms are especially efficient for ions such as K +, Ca 2+, and Mg 2+. They operate in conjunction with a low ion permeability in brain capillaries and active ion-transport mechanisms in the choroid plexus and brain endothelial cells (Bradbury, 1979; Hansen, 1985). The interstitial fluid and the CSF are thus protected against changes in plasma ion composition. The dialysis membrane on the other hand has direct access to the interstitial fluid compartment (Fig. 3) and if the latter is perfused with Ca2+-free fluids, interstitial Ca 2+ is drained in a wide area around the dialysis membrane (Fig. 4) (Benveniste et al., 1989). Under such circumstances both active regulatory mechanisms and mass transport by diffusion in the interstitial space are apparently not operating sufficiently fast to permit normal interstitial ion-homeostasis. Interstitial calcium depletion causes dialysate concentrations of various transmitters to decrease (Imperato and Di Chiara, 1984; Westerink and De Vries, 1988). Likewise, if the perfusion fluid contains calcium in a higher concentration than that of the interstitial space (i.e. Ringer's solutions) basal dialysate concentrations of dopamine increase by 70% and the response of dopamine to ~:methyltyrosine (an inhibitor of tyrosine hydroxylase) changes when compared to a perfusion fluid containing a Ca 2+ concentration identical to that of the brain interstitial space (Moghaddam and Bunney, 1989). Consequently it appears to be important to keep the ion composition of the perfusion fluid identical with that of the brain interstitial fluid and not with plasma [unless of course a local lowering of interstitial ion concentrations is intended (Imperato and Di Chiara, 1984; Soils et al., 1986; Westerink and De Vries, 1988;

198

H. BENVENISTEand P. C. Hf]TTEMEIER TABLE1. APPLICATIONS IN BRAIN AND SPINAL CORD

Ischemia-Hypoxla-Anoxia Benveniste et al., 1984; Hagberg et al., 1984; Hagberg et aL, 1985; Drejer et al., 1985; Hagberg et al., 1986; Lindefors et al., 1986; Phebus et al., 1986; Van Wylen et al., 1986; Hagberg et al., 1987a; Hagberg et al., 1987b; Benveniste and Diemer, 1988a; Globus et al., 1988a; Globus et al., 1988b;Kendrick and Leng, 1988; K o r f e t al., 1988;Slivka et al., 1988; Vulto et al., 1988; Benvenisteet aL, 1989b; Busto et al., 1989;Globus et al., 1989; Hillered et al., 1989; Ikeda et al., 1989; Phebus and Clemens, 1989; Shimada et al., 1989. Hypoglycemia Tossman et al., 1985; Sandberg et al., 1986; Butcher et al., 1987a; Butcher et al., 1987b. Seizures-Hyperexcitation-Kainic acid Lehmann et al., 1983; Lehmann et al., 1984; Jacobsen and Hamberger, 1985; Lehmann et al., 1985a;Vezzani et al., 1985; Lazarewicz et al., 1986a; Lehmann et al., 1986; Pilowsky et al., 1986; Butcher et al., 1987c; Vezzani et al., 1988; Young et al., 1988. Injury Faden et al., 1989; Panter et al., 1990. Hepatic encephalopathy Tossman et al., 1983; Hamberger and Nystr6m, 1984; Tossman et al., 1987. Behavior Ungerstedt et al., 1982; Sharp et al., 1987b;Kendrick et al., 1986; Kendrick et al., 1988a; Kendrick, 1988b;Abercrombie et al., 1989. Dopamine Ungerstedt and Pycock, 1974; Hernand6z et al., 1983;Zetterstr6m et al., 1983; Blakely et al., 1984;Clemens and Phebus, 1984; Imperato and Di Chiara, 1984; Sharp et aL, 1984; Kite et al., 1986;Sharp et aL, 1986a; Sharp et al., 1986b; Bettini et al., 1987; Wood et al., 1987; Alexander et aL, 1988; Damsma et al., 1988b; Carter et al., 1988; Reid et al., 1988; Robinson and Whishaw, 1988; Rollema et aL, 1988; Hurd et al., 1988; Ton et aL, 1988; Wood et aL, 1988; Zetterstr6m et at., 1988; Bean et al., 1989; Hurd and Ungerstedt, 1989a; Hurd and Ungerstedt, 1989b; Hurd and Ungerstedt, 1989e; Hurd and Ungerstedt, 1989d; O'Connor et al., 1989; Westerink and de Vries, 1989; Westerink et al., 1989a; Westerink et al., 1989b. Noradrenaline L'Heureux et al., 1986; Routledge and Marsden, 1987; Abercrombie et al., 1988; Egawa et al., 1988;Kal6n et al., 1988b; Vathy and Etgen, 1988. AcetylchoHne Ajima and Kate, 1987;Console et al., 1987; Damsma et al., 1987a; Damsma et at., 1987b;Damsma et at., 1988a; Damsma et al., 1988c; Herreras et al., 1988; Westerink et al., 1989b. Serotonin Kate et al., 1986; Kal6n et al., 1988a; Sharp et al., 1989. Adenosine Ballarin et al., 1987; Brodie et al., 1987. Amino acids Bite et al., 1986; Delgado et al., 1972; Lehmann and Hamberger, 1983; Sandberg and Lindstr6m, t983; Jacobsen and Hamberger, 1984; Lehmann and Hamberger, 1984; Lerma et al., 1984; Lehmann et al., 1985b; Lerma et al., 1985; Sandberg et al., 1985; Tossman and Ungerstedt, 1985; Jacobsen et al., 1986; Solis et al., 1986; Tossman et al., 1986a; Tossman et al., 1986b; Young and Bradford, 1986; Bradford et al., 1987; Lehmann, 1987; Mufioz et al., 1987; Brodin et al., 1988; Wade et al., 1988; Men6ndez et al., 1989. Peptides Brodin et al., 1983; Lindefors et al., 1985; Lindefors et al., 1986; Brodin et al., 1987; Maidment et al., 1989. Metabolism Zetterstr6m et al., 1982;Hutson et al., 1985;Kuhr et al., 1988; Kuhr and Korf, 1988; Schasfoort et al., 1988; Korf, 1989. Microdialysis methodology Johnson and Justice, 1983; Justice et al., 1983; Delgado et al., 1984; Ungerstedt, 1984; Jacobsen et al., 1985; Korf and Venema, 1985; Hernandez et al., 1986; Lerma et al., 1986;Tossman and Ungerstedt, 1986;Wages et al., 1986;Westerink and Tuinte, 1986; Benvenisteet aL, 1987; Church and Justice, 1987; L6nnroth et al., 1987; Sandberg et al., 1987; Sharp et al., 1987a; Ungerstedt and Hallstr6m, 1987; Alexander et al., 1988a; Benveniste and Diemer, 1988b; Kendrick, 1988; Sells et al., 1988; Westerink and De Vries, 1988; Amberg and Lindefors, 1989; Benveniste, 1989; Benvenisteet al., 1989a; Donzanti and Yamamoto, 1989; Lehmann, 1989; Lindefors et al., 1989; Moghaddam and Bunney, 1989; Reiriz et al., 1989; Lehmann and Sandberg, 1990. r

MICRODIALYSIS----THEORYAND APPLICATION

199

TABLE 2.

Gastrointestinal tract Heart Eye Adrenal gland Blood Subeutis Skeletal muscle

Meirieu et al., 1986 Hamberger, 1989 Ben-Nun et al., 1988 Medvedev et al., 1989 Collin and Ungerstedt, 1988; Hurd et al., 1988; Speciale et al., 1988; Arner et aL, 1989 Bito et al., 1966; Delgado et al., 1972; L6nnroth et aL, 1987; Arner et aL, 1988 Lehmann, 1989

Vezzani et al., 1988; Benveniste et al., 1989; Hurd and Ungerstedt, 1989)]. Interstitial fluids are generally characterized by a very low protein content (see review by Aukland and Nicolaysen, 1981) and therefore never added to perfusion fluids. Furthermore, adding proteins to peffusion fluids would defeat one of the main advantages of microdialysis which is the ability to analyze dialysis substance concentrations directly by high performance liquid chromatography (HPLC) without prior deproteinization. However, in some studies 0.2-0.8% bovine albumin is added to the perfusion medium (Brodin et al., 1983, 1987; Kendrick et aL, 1986, 1988) to avoid peptide 'sticking' to the membrane, plastic- and stainless steel

tubing and thus suboptimal recovery values (see also Section 5.4). In summary, it seems prudent--for microdialysis experiments in brain tissue--to use fluids that contain an identical ionic composition as that of the brain interstitial space and further if one of these ions is under investigation and a substitution is required then to ascertain whether or not this affects control conditions,

4. MEASUREMENTS WITH MICRODIALYSIS Bito et aL (1966) were the first who tried to measure substance concentrations in the brain interstitial

TABLE 3. PERFUSIONFLUID COMPOSITIONS(IN mM UNLESSOTHERWISEINDICATED)

Distilled water Saline: 0.9% NaC1 Saline: 0.9% NaC1; 1.85% CaC12 (pH 7.2) Saline: 0.9% NaC1; 0.5% bovine serum albumin Ringers solution 134 NaC1; 5.9 147 NaCl; 2.4 147 NaCl; 3.4 145 NaC1; 1.3 155 NaCl; 5.5 189 NaC1; 3.9

KC1; 1.3 CaC15 1.2 MgC12 (O2-sat.) CaC12; 4 KC1 (pH 6.0) CaCl2; 4 KC1 (pH 6.1) CaCl2; 4 KC1 (pH 7.2) KCI; 2.3 CaC12 KCl; 3.4 CaC12 (pH 7.2)

Modified Ringers solution 145 NaC1; 2.7 KCl; l MgCI2; 1.2 CaCl:; 0.2 ascorbate (pH 7.4) Buffered Ringers solution 147.2 d NaCl; 3.4 CaCl,; 2.76 KC1; 1.16 MgCl2; 0.62 K2HPO4; 113.5/aM ascorbic acid (pH 6.9) Krelm Ringer solution

147.2 NaC1; 4.0 KCI; 3.4 CaCI2 (pH 6.1) 138 NaCl; II NaHCO3; 5 KCI; I NaH2PO4; 1 CaClz; l MgC12; II glucose (pH7.5) Krebs Ringer bicarbonate

122 NaCI; 3 KC1; 1.2 MgSO4; 0.4 KHzPO4; 25 NaHCO3; 1.2 CaCI2 (pH 7.4) Krebs--Henseleit bicarbonate buffer

118.4 NaC1; 4.7 KC1; 0.6 MgSO4; 2.5 CaC12; 1.2 NaH2PO4; 25 NaHCO3; 11 glucose Mock-¢erebrospinui fluids

120 NaCI; 15 NaHCOa; 5 KC1; 1.5 CaCl:; 1.0 MgSO4; 6 glucose (pH 7.4) 126.5 NaC1; 27.5 NaHCO3; 2.4 KCI; 0.5 KH2PO4; 1.1 CaCl2; 0.85 MgC12; 0.5 Na2SO4; 5.9 glucose (pH 7.5) 127 NaC1; 2,5 KCI; 1.3 CaC12; 0.9 MgCl: (pH 6.0) 127 NaC1; 2.5 KC1; 1.3 CaCI2; 2 MgC12 132.8 NaCl; 3.0 KCI; 24.6 NaHCO3; 6.7 urea; 3.7 glucose 135 NaC1; 3.0 KC1; 1.2 CaCl2; 1.0 MgCl2; 2.0 phosphate (pH 7.4) 140 NaCI; 3.0 KC1; 2.5 CaCl2; l MgCl:; 1.2 Na2HPO4; 0.27 NaH2PO4; 7.2 glucose (pH 7.4) 150 NaCI; 3 KC1; 1.7 CaCI2; 0.9 MgC12 119.5 NaC1; 4.75 KC1; 1.27 CaCl:; 1.19 KH2PO4; 1.19 MgSO4; 1.6 Na, HPO4 (pH 7.2) JPN 35/3---B

200 ],4-

H. BENVENISTEand P. C. Hf2TTEr,mlER mM Ca+* in Interstltlalspace of rat cortex

probe is defined as 'recovery' and expressed either as a ratio or as a percentage. Recovery would normally be determined with the probe in an aqueous solution containing a known concentration of the substance in question. The probe is continuously perfused at a constant flow rate with a fluid devoid of the substance and samples are collected during fixed times intervals. The recovery of the particular substance is then calculated as:

1.2-

1.00.8£

0.4~

Recovery,, ,,,o = Cout/Ci

0.2 t 0

,o'oo

'

20'°0

distance (l~m) between electrode tip and dialysis membrane

FIG. 4. The impact of dialysis with calcium-free fluid on the interstitial milieu in the vicinity of the probe. Perfusion of the probe (2.5/~l/min) was started 1 hr following implantation into the cortex. The interstitial Ca 2+ concentration was measured 30 min after the perfusion start at various distances from the perfused dialysis probe by a Ca2+-sensitive microelectrode. Each point on the curve represents one experiment. Data from Benveniste et al., 1989a. For further details see Sections 3 and 5.

space using dialysis. They collected samples from dialysis membrane bags filled with 6% dextran in saline chronically implanted in dogs (the system was not perfused in any way). Ten weeks later the bag was removed and the fluid analyzed for its contents of amino acids and ions. The K ÷ concentration was found to be 4.71raM. Measurements with ionselective microelectrodes have found brain interstitial K* concentration to be only 3.2raM, sO the bag fluid--or dialysates--apparently did not represent correct interstitial concentrations. In 1972 Delgado and co-workers presented data from a continuously perfused microdialysis probe. Results were given only as dialysate substance concentrations and no attempts were made to transform these into 'true' brain interstitial substance concentrations. Obviously, because the microdialysis probe produces a dialysate of fluid from the interstitial space the substance concentrations measured herein can only be reflections of true brain interstitial substance concentrations. ZetterstrSm et al. (1982) were probably the first who attempted to calculate brain interstitial concentrations from dialysate concentrations using a simple in vitro calibration of the microdialysis probe in water. 4.1.

CONCENTRATION MEASUREMENTS BASED ON IN VITRO CALIBRATION

According to Zetterstr6m et al. (1982) the ratio between the concentration of a substance in the outflow solution and the undisturbed* concentration of the same substance in the solution outside the

* It is important to note that recovery measurements are not evaluated from the substance concentration in the immediate vicinity of the dialysis machine which/s disturbed due to extensive drainage (cf. Section 5.1).

where (?outis the concentration in the outflow solution and Ci the concentration in the medium. Having determined recovery for the substance in question, dialysate substance concentrations are transformed into brain interstitial concentrations using the following formulae: = ~ou~/Recoveryi, vitro"

(1)

Ci is the substance concentration in the brain interstitial space and Cou, is the concentration of the dialysate (Fig. 5). Unfortunately, this simple calculation is not valid because mass transport of substances in brain tissue is different from that of aqueous solutions (see Section 5.5). This means that calculated interstitial concentrations based solely on dialysate concentrations and in vitro recovery--for most substances--underestimate true interstitial concentrations. In the following section we will discuss additional factors that must be taken into account when dialysate concentrations are transformed into brain interstitial concentrations.

5. FACTORS AFFECTING SUBSTANCE COLLECTION WITH MICRODIALYSIS 5.1. PERFUSIONFLOW RATE AND TIME Figure 6 (upper part) illustrates that recovery for a given rnicrodialysis probe increases when perfusion flow rates are kept low and furthermore that recovery changes with time; initially, recovery is high, then rapidly decreases and levels off although it never quite reaches steady state. The slight decrease of recovery after 30--60 min of perfusion is neglected for practical purposes and this part of the curve is used for determinations as described in Section 4.1. It has generally been assumed that the high dialysate substance concentrations immediately after implantation represented a traumatic tissue response [i.e. a local break-down of blood-brain barrier (BBB) and cell membranes]. Although this may play an important role (cf. Section 6) it should be kept in mind that the same time-dependent decrease of recovery is found in aqueous solutions (Delgado et al., 1984; Amberg and Lindefors, 1989; Benveniste et al., 1989; Benveniste, 1989). This is probably due to a steep concentration gradient across the dialysis membrane when the probe is first inserted into the medium which then gradually declines due to intense substance drainage in the immediate vicinity of the membrane (of. Fig. 4). The theory behind both the establishment and timedependency of concentration profiles in the immediate vicinity of a dialysis membrane has recently been explored by Amberg and Lindefors (1989).

MICRODIALYSIS---THEORYAND APPLICATION

I,

201

Com

Re¢°verYln vitro"

Cout C

out

out Rec°verYin

vitro

FIG. 5. Estimation of brain interstitial concentrations from in vitro calibration.

5.2. FLux In contrast to recovery, the flux of substances (the amount harvested by the membrane per unit time) across the dialysis membrane increases with higher flow rates. However, as illustrated in Fig. 6 (lower part), flux is not significantly influenced by perfusion rates above 5-10/~l/min (there is some variability with different probe designs). This is most likely caused by diffusion limitation (the concentration gradient across the dialysis membrane is near maximum at these particular flow rates). Alternatively, a stagnant flux with higher flow rates could also be due to a positive hydrostatic pressure gradient across the membrane which may occur with a rigid tube design and lead to a decrease of mass transport into the probe (Johnson and Justice, 1983). High flow rates with microdialysis are best avoided because the perfusion fluid leaves the probe under pressure and may cause tissue damage---this is exactly the major drawback with the push-pull perfusion technique (see reviews by Ugerstedt (1984) and Benveniste (1989)). The actual flow rates at which significant filtration forces build up across the dialysis membrane are unknown at present and likely influenced by the probe design. Recently, net fluid exchange during microdialysis (in both serial and parallel probe designs) was measured during perfusion flow rates of 2/~l/min and 10/~l/min, respectively. In both instances fluid loss before and after perfusion was less than 0.1% (Lindefors et aL, 1989) suggesting that these flow rates can be used safely.

5.3. TF_~PERArUR~SAND CnANO~ IN OUTER SUBSTANCECONCENTRATION Wages et al. (1986) found that in vitro recovery of 3,4-dihydrophenylacetic acid (DOPAC) at a flow rate of 0.2 #l/min increased approx. 30% when the temperature was increased from 23°C-37°C. These resuits have since been confirmed by others (Benveniste et al., 1989; Lindefors et al., 1989) and are not unexpected because diffusion coefficients are known to increase 1-2%°C (Bard and Faulkner, 1980). Therefore,/n vitro probe calibration should always be performed at temperatures identical to the tissue (Zetterstr6m et aL, 1982; Korf and Venema, 1985; Van Wylen et al., 1986; Wages et al., 1986; Young and Bradford, 1986; Benveniste et al., 1989; Benveniste, 1989). Recovery is independent of changes in the outer (undisturbed) substance concentration (Ungerstedt et al., 1982; Hamberger et aL, 1983; Johnson and Justice, 1983; Sandberg and Lindstr6m, 1983; Ungerstedt, 1984; Tossman and Ungerstedt, 1986; Kendrick, 1988). This is fortunate, because if the opposite were true as pointed out by Johnson and Justice (1983) microdialysis would obviously not be able to measure interstitial concentration changes correctly. When the outer substance concentration is suddenly increased or decreased, the concentration gradient across the dialysis membrane changes correpondingly thus keeping recovery constant. The fact that recovery is independent on changes in the outer substance concentration should not be confused with

H. BENVENIST~andP. C. HOTTEMEm_R

202

ments (Kendrick, 1988). It was found that recovery for certain substances varied as much as 20% between different probes (Kendrick, 1988). Recently, the 'adhesiveness' of the dialysis membrane was quantified by a 'capture probability constant', which according to the authors should be used cautiously, because the 'capture' may be a time-dependent, saturable phenomenon (Lindefors et al., 1989).

A I= is t o

10

E 411 2O

0i

. 4

8

12 recovery %

16

0

5 . 5 . DIFFUSION COEFFICIENT

0

Recovery % 10 8

6

~

4 2 0

5

10

15

20

Flux (dpm/rnin)

FtG. 6. Upper part: Recovery of 14C-mannitol. A typical curve of recovery as a function of perfusion flow rate (11) and time (@), respectively. Recovery is inversely related to perfusion flow rate and time, respectively. When perfused at a constant flow rate, relative recovery is initially high but decreases rapidly. The recovery at individual perfusion flow rates was measured after 90 min of perfusion. Data from Benveniste et al., 1989a. Lower part: In contrast to recovery values, the net flux across the dialysis membrane is only slightly influenced by perfusion rates above 5pl/min10#l/min (varies with microdialysis probe designs). Data from Benveniste eI al., 198%. For further details see Section 5. recovery and time-dependency (cf. Section 5.1). The latter phenomenon is also dependent on concentration gradient changes but here the 'undisturbed' outer concentration does not change correspondingly and recovery therefore decreases. 5.4. DIALYSIS MEMBRANE A R E A AND COMPOSITION

Recovery is directly proportional to the size of the dialysis membrane area (Hamberger et al., 1983; Sandberg and Lindstr6m, 1983; Ungerstedt, 1984; Tossman and Ungerstedt, 1986). This is advantageous when one wishes to detect brain interstitial substances that are present in very low concentrations. By increasing the membrane area low concentrations can be detected with reasonably high perfusion flow rates while maintaining an adequate time resolution. Dialysis membrane materials are known to interact with transported substances via unknown mechanisms and affect mass transport. For instance, recovery of acidic amino acids is lower than that of the neutral ones (Sandberg and Lindstr6m, 1983). Another extensive study examined how different dialysis membrane materials affected in vitro recovery measure-

It is known that the diffusion coefficient is inversely proportional to the size of the substance in question, i.e. solute radius (for futher details see Friedman, 1986). Consequently, diffusion coefficients will be lower for substances with higher molecular weights which may help explain the inverse relationship between recovery and molecular weight (Kendrick, 1988; Benveniste et al., 1989). Because this is found even with dialysis membranes characterized by high cut-offs (50,000) (Kendrick, 1988) diffusion (for molecular weights up to 5000 daltons) is not likely to be limited by membrane pore size. Recovery~,~t,o is known only for a small number of substances and is--with few exceptions--less than that found in simple, aqueous media* (L6nnroth et al., 1987; Arner et al., 1988; Benveniste et aL, 1989; Lindefors et al., 1989). This is because diffusion coefficients in complex media (i.e. the brain is a complex medium comprising the interstitial-, the intracellular- and the vascular compartment) are lower when compared to those in aqueous solutions. Diffusion of most hydrophilic substances is impeded by impermeable cell membranes (Nicholson et al., t979; Nicholson and Rice, 1986) and mass transport is confined to the interstitial space (ct) which comprises only 20% of the total brain volume (Levin et al., 1970; Nicholson et al., 1979; Nichoison and Phillips, 1981). Several studies illustrate the importance of taking these factors into account. Lazarewicz et al. (1986) measured the Ca 2÷ concentration in dialysates collected from a horizontal tube implanted in rabbit hippocampus during perfusion with a Ca 2+free medium. With 20% in vitro recovery, the brain extracellular Ca 2÷ level was calculated to be approx. 0.75 mM. This is considerably below the known extracellular Ca 2+ concentration in brain, i.e. 1.2-1.3 mM (see also Vezzani et al., 1988). In another study in vitro recovery of adenosine changed from 20.3% at a flow rate of 2/~l/min to nearly 100% when the flow rate was decreased to 0.1/~l/min (see also Section 5.1). When repeated in rive, the respective adenosine concentrations recovered in dialysates increased from 0.12-1.26 #M. The calculated 'interstitial' adenosine concentration based on in vitro recovery, amounted to 0.12/0.20, or 0.60 #M, but should in fact have been twice as high (i.e. approx. 1.26 with 100% recovery) * For substances like K ÷ and glycerol the in vivo recovery equals that of in vitro (Arner et al., 1988; Benveniste et al., 1989; Lindefors et al., 1989). This is in accordance with previous investigations showing that K + in brain rapidly traverses the cell membrane due to rapid exchange mechanisms (Nicholson et al., 1979; Nicholson and Phillips, 1981). Furthermore, glycerol--a lipophilic molecule---is not very likely to be impeded by the presence of cell membranes.

MICRODIALYSIS---THEoRYAND APPLICATION

(Van Wylen et al., 1986). Recently, Alexander et al. (1988) demonstrated the significant difference between in vitro and /n vivo recovery measurements using tritiated water (THO). For a given microdialysis probe and perfusion flow rate (above 0.2/zl/min) in vitro THO recovery was always higher than that in vivo. 6. EFFECT OF MICRODIALYSIS ON THE BRAIN MICROENVIRONMENT 6.1. BRAIN COMPARTMENTATION

The brain comprises three different water spaces: the intracellular, the interstitial and the vascular space (Fig. 3). The determination of substances in the brain interstitial space with microdialysis obviously requires that the implantation procedure leaves the boundaries between these spaces intact. Blood-brain barrier integrity has been measured after implantation of a microdialysis probe with BBB-impermeable substances such as ~-amino-isobutyrate and sodium technitate (Benveniste et al., 1984; Tossman and Ungerstedt, 1986). Despite some degree of brain tissue trauma with the insertion procedure (see below) it was nevertheless found that the BBB around the microdialysis probe was intact shortly (30 min-2 hr) after implantation. Previously, Hamberger and co-workers (1983) and Hamberger and Nystr6m (1984) compared amino acid concentrations in blood, CSF, and brain tissue (homogenates) with concentrations in dialysates 24 hr after probe implantation. 'Interstitial' amino acid concentrations (calculated according to Eqn. 1) were distinctly lower than those of blood and tissue but correlated remarkably well with CSF values. These results suggested that the BBB was intact. There are as yet no reports on BBB integrity in chronic preparations > 1 day). 6.2. TISSUEREACTIONS It is important to know the time-course of tissue changes after implantation of the probe, because the trauma itself, the inflammatory changes and subsequent gliosis and fibrosis may inflict severe changes of local brain metabolism and blood flow. In addition, tissue diffusion characteristics may also change. Tissue reactions were studied after implantation of a serial microdialysis probe in rat hippocampus (Benveniste and Diemer, 1988). Within two days the tissue adjacent to the membrane (a 50#m border zone) exhibited edema, minor hemorrhages and accumulations of polymorphonuclear leukocytes. Eosinophilic neurons, indicating cell death, were occasionally present 100-150#m from the implant. Otherwise, normal neuropil surrounded the microdialysis membrane. After three days there was astrocyte hypertrophy followed by connective tissue displacement after 2 weeks. The latter was still present after 60 days (see also Hamberger and NystrSm, 1984). These results were largely confirmed by Shuaib et aL (1990) using the silver degeneration staining method. They further demonstrated that degenerating axons were present both adjacent to the implantation site and also in remote brain areas

203

such as the corpus callosum and contralateral hippocampus. Tissue reactions are classic and unavoidable in the vicinity of both implants and lesions (Del Rio Hotega and Penfield, 1927; Stensaas and Stensaas, 1976; Collias and Manuelidis, 1957). In view of this, the reliability of chronic microdialysis measurements becomes questionable (to be discussed further in Section 6.5). 6.3. LOCALCEREBRALBLOODFLOW AND 2-DEOXYGLUCOSEPHOSPHORYLATION 4-Iodo[N -methyl- ~4C]-antipyrine autoradiographs --illustrating regional cerebral blood flow--from rat brains with a borizontally placed microdialysis probe in hippocampus demonstrate that immediately (within I hr) after implantation hippocampal blood flow is reduced by 40% (Benveniste et al., 1987). At the same time local cerebral blood flow (LCBF) is decreased by 60% in remote brain regions such as striatum, lateral septum and thalamus. 2-Deoxy-D[14C]glucose (2DG) autoradiographs reveal two kinds of changes within 3 hr after implantation: (a) Distinct regions with increased 2-DG uptake in close proximity to the dialysis membrane, and (b) a general reduction in 2-DG uptake in both hippocampus and remote brain regions. LCBF and local cerebral 2DG phosphorylation (LCMR~) were near normal in all brain regions 24 hr after implantation. All these experiments have since been repeated using a vertical probe design (Sandberg and Benveniste, 1989). Hippocampal CBF was unchanged 2 hr after insertion whereas LCMR~ was moderately increased (approx. 50% of the values obtained with the horizontal probe). Importantly, there were no remote changes of either blood flow or glucose consumption. It is well-known that brain lesions cause a metabolic deactivation of those brain areas anatomically connected with the lesioned region (Feeney and Baron, 1984). This phenomenon is termed 'diaschisis'. However, the changes of LCMR~ and LCBF found in remote brain areas after implantation of the dialysis membrane were widespread and transient and therefore uncharacteristic of diaschisis (Feeney and Baron, 1984). Alternatively, these findings could be explained by the induction of a spreading depression (SD) (Leao, 1944) which is known to cause widespread transient changes of LCBF and LCMRac (Shinohara et al., 1979; Lauritzen, 1987). In experiments where a microdialysis probe was combined with a calcium-sensitive microelectrode there were transient changes of the DC-potential and the interstitial Ca 2÷ concentration characteristic of a SD (Fig. 7) (Benveniste et al., 1989). In cortex, a SD is characterized by depression of evoked and spontaneous EEG activity and a negative shift of the DC-potential (Nicholson and Kraig, 1981; Hansen, 1985). This in turn is associated with changes in the distribution of ions between the intra- and extracellular compartments: Interstitial potassium concentration increases and those of calcium, sodium and chloride decrease (Hansen, 1985). Most ion concentrations spontaneously revert to normal after 30 sec-1 min.

204

H. BENVENISTEand P. C. H(YrrEMEER m M Ca +'~

12 05

~

O1 3 min

FIG. 7. Elicitation of a spreading depression. The arrow indicates the time of insertion of the microdialysis probe. Data from Benveniste et al., 1989a. For further information see Section 6. 6.4. USE OF TETRODOTOXIN AND CALCIUM

DEPLETIONFORESTIMATINGFUNCTIONAL DAMAGEAFTERIMPLANTATION

Westerink and De Vries (1988) evaluated functional tissue damage after probe implantation using tetrodotoxin (TTX) and calcium-free perfusion fluids. In normal brain tissue both maneuvers would abolish transmitter release and for instance dopamine should not appear in the dialysates*. Acutely and several hours after implantation of a parallel designed probe (o.d. 800/~m), dopamine release was not inhibited by calcium depletion and only moderately so by TTX. When a transtriatal dialysis probe (with a smaller outer diameter) was used acutely, dopamine output displayed some calcium and TTX dependency. When tested after 24 hr dopamine output was completely abolished with both probe designs suggesting a return to normal tissue function--this is in accord with the normalization of cerebral blood flow and 2-DG phosphorylation that also occurs at this time. 6.5. THE POSSIBLEINFLUENCEOF THE DISTURBED BRAIN MICROENV1RONMENTON RESULTS OBTAINED BY MICRODIALYSIS Based on histological, functional, metabolic and blood flow changes caused by the probe insertion it appears that the optimal time for commencing microdialysis is 8-48 hr after implantation. At this time local blood flow and glucose metabolism is minimally * It is well-known that calcium ions play a fundamental role in the process of 'excitation-secretion coupling' in neurochemical transmission. Release of transmitter occurs as a result of the arrival of an action potential at the nerve terminal. The depolarization phase of the action potential, results in an opening of voltage-dependent Ca2+ channels in the presynaptic membrane. The influx of Ca 2+ down its electrochemical gradient through calcium channels leads to a transient rise in intracellular Ca2+ which triggers a transient release of the transmitter. The Ca2+ entry into the presynaptic terminal can be divided into two phases, i.e. the early phase which is abolished by tetrodotoxin (TTX) (which blocks sodium-dependent voltage-operated channels) and the late phase which is unaffected by TTX but can be inhibited by Mn 2+ and Co2+. Consequently, TTX, inorganic Ca 2+ blockers or calcium depletion inhibit transmitter release (cf. review by T6r6k, 1989).

disturbed, the tissue releases transmitter(s) (dopamine) in a voltage-dependent manner and chronic tissue reactions have not yet developed. However, this optimal time is often either impractical or unobtainable. This is particularly so in experiments where microdialysis measurements are combined with electrophysiological recordings. Futhermore, there is still some controversy as to whether experiments carried out with acute or chronical implanted probes give similar substance concentration measurements. Some data but not all show that control levels of amino acids and biogenic amines and changes of these induced by various stimuli differ between acute and chronic preparations. Korf and Venema (1985) found a steady increase in the resting release of several amino acids over a period of 9 days. The same authors also found that responses to concentrated potassium infusions and electroconvulsive shock differed with time. A similar trend has been observed by Westerink and Tuinte (1986) who found that potassium-induced dopamine release disappeared 3 days after implantation whereas amphetamineinduced dopamine release was still present but diminished 8 days after implantation (see also Roltema et aL, 1988). In contrast, Delgado et al. (1984) found near-constant concentrations of aspartate, threonine and glutamate over a period of 10 months. Additionally, Strecker et al. (1986) using dialysis membrane of smaller dimensions than those of Westerink and De Vries (1986), demonstrated an almost complete inhibition of dopamine release after apomorphine in an acute implantation situation.

7. SOLUTIONS TO THE PROBLEM OF DETERMINING BRAIN INTERSTITIAL CONCENTRATIONS W I T H MICRODIALYSIS We previously discussed that the calculation of 'true' brain interstitial substance concentrations based exclusively on dialysate concentrations and in vitro recovery leads to significant underestimations due to the fact that mass transport of substances in vivo differs from that in vitro (of. Sections 4.1 and 5.5). Other approaches are therefore required and those that have been used so far can be divided into two categories: (a) Methods that operate without time resolution, i.e. perfusion flows are either very low (or non-existent), or the calculation requires repetitive measurements which means that substance concentration fluctuations during some short experimental intervention cannot be detected, and (b) methods that allow a continuous transformation of dialysate concentrations without a loss of time resolution. 7.1.

METHODSWITHOUTTIMERESOLUTION

According to Fig. 6, major reductions of perfusion flow rates result in recovery values that approach 100%. This would in theory eventually produce dialysate concentrations equal to those of the brain interstitial space. Actually Bito et al. (1966) were the first who accomplished this with an unperfused implanted dialysis bag. The dialysate concentrations they obtained are probably slightly overestimated-for instance resting glutamate concentrations

205

MICRODIALYSIS---THEORYAND APPLICATION

amounted to 450/~i (Sections 4, 8.1 and 8.2). Wages et al. (1986) measured dopamine in dialysates at flow rates of 0.1 and 2/~l/min, respectively, and found that dialysate dopamine concentrations decreased from 9 to 2 ma. Similar results were found for adenosine by Van Wylen et al. (1986). Jacobson et al. (1985) solved diffusion equations and found that recovery depended on perfusion flow rate, membrane area, and an 'average mass transfer coefficient'. From their equation and with the use of regression analysis they could estimate extracellular concentrations. Interstitial concentrations derived from the curve fit were only slightly higher than those based on in vitro recovery measurements. The equation assumed a constant concentration profile outside the probe and furthermore a dialysis membrane that constituted a diffusional resistance. However, both assumptions are probably wrong. First, because the microdialysis probe constantly drains substances from the brain, tissue diffusion gradients change correspondingly (become less steep with time) and lead to a reduction of mass transport (cf. Section 5.1). Secondly, dialysis affects substance concentrations quite far from the probe as shown in Fig. 4, and not only in its near vicinity. This is evidence that the membrane does not constitute a major diffusion barrier and may therefore be regarded as part of the medium. Lerma et al. (1986) measured amino acid concentrations in dialysates collected from a dialysis membrane loop in which the perfusate circulated for several hours. Assuming that compounds crossed the dialysis membrane by simple diffusion, amino acid concentrations were calculated by computerized nonlinear regression analysis of the dialysis data at different circulation times. For instance resting glutamate concentrations were found to be 2.9/~i which is surprisingly low considering the fact that others have measured much higher levels (Young and Bradford, 1986; Globus et al., 1988; Faden et al., 1989) even though dialysates were not recircled. Unfortunately, Lerma et al. (1986) did not test the validity of their method by measurements of ion concentrations in the dialysate as other investigators have done (Bito et al., 1966; Benveniste et al., 1988, 1989) and the results are therefore difficult to evaluate. L6nnroth et al. (1987) presented yet another method that in principle would produce a 'true' interstitial concentration of any compound in the brain----or in any tissue for that matter. The dialysis membrane--implanted in subcutis--was perfused with perfusion media containing glucose in different concentrations. Using regression analysis they determined the exact point at which no net flux of glucose occurred across the membrane. This glucose concentration would be in equilibrium with that of the surrounding tissue and hence equal to it. Lindefors et al. (1989) recently presented a somewhat similar approach as Lfnnroth and co-workers (1987) by infusing the substance of interest via the microdialysis probe and calculating recovery as follows: Recovery =

c~° - C~n_~

c~°

(2)

where Cm is the substance concentration in the perfusion medium, and Ci,-o,t is the concentration after passage through the medium. Having determined recovery /n vivo the interstitial concentration (Ci) could be calculated from Eqn. 1 (Section 4.1). The problem with this approach is, as pointed out by the authors, that recovery determinations using Eqn. 2 cannot discriminate between what is actually transported through the membrane and what may be adhering to it. This appears for some unknown reason to be a more significant problem for recovery measurements determined by Eqn. 2 than with Eqn. 1. Some substances 'stick' to the membrane (cf. Sections 3.3 and 5.4) and there is, for example, a discrepancy between in vitro recovery of sucrose determined from Eqns 1 and 2, respectively (Lindefors et al., 1989).

7.2. METHODSWITHTIME RESOLUTION Benveniste et al. (1989) calculated interstitial substance concentrations assuming that mass transport of substances between the probe and the tissue only occurs by diffusion*. In a simple medium (i.e. without cells), the flux J measured in relation to the total area is given by Ficks law as: t3C J = -O-dr

(3)

where D is the diffusion coefficient, C is the substance concentration in a unit volume and r is the distance measured perpendicular to the area considered. In the case of a tissue with a total volume of V and interstitial space volume V0, the flux J to the probe is: l = - ~ D --OC

t~r

(4)

where ct = V o / V , 15 is the diffusion coefficient in the tissue interstitial space. It is assumed that cells do not exchange the substances of interest with the interstitial space. These theoretical considerations show that mass transport or flux in vivo is always smaller than in vitro because ~t is less than 1 and/~ less than D. The so-called tortuosity factor (As= D/I~) describes the increased diffusion pathway in vivo due to the presence of impermeable cell membranes (Safford and Bassingthwaighte, 1977). Assuming that diffusion around the probe is axisymmetrical and strictly radial (for a more extensive mathematical analysis see Benveniste et al., 1989) Eqn. 4 can be

* It is well-established that mass transport in brain interstitial space occurs both by diffusion and convection, i.e. convection fmass transport caused by fluid motion (Bradbury et al., 1981;Cserr et al., 1981; Szentistv~.nyiet al., 1984). However, we will not consider the latter process because diffusion is usually the dominant mass transport mechanism in the interstitial fluid. Nevertheless, when calibrating the microdialysis probe/n vitro, we have to avoid convection which can easily be introduced by stirring. Convection is avoided if the aqueous solution is solidified with agar (0.2-1.0%).

206

H, BENVENISTEand P. C. HfdTTEMEIER

solved and the undisturbed (cf. Section 4.1) brain interstitial substance is found:

8"

Ci = [K*A21ot] x ~'o~t/Recovery~n ,,,,o.

4

(5)

Equation 5 states that the interstitial concentration can be calculated by inserting in vitro recovery, ~'out and K22/cc The inclusion of the interstitial volume fraction a and the tortuosity factor 2 in the equation is not surprising and was pointed out by Nicholson and Phillips (1981) who described diffusion from a point source in brain tissue. A comparable situation prevails when substances move in the interstitial space towards the microdialysis probe. Diffusion is retarded by the tortuous interstitial space and the amount of substance reaching the probe is reduced by the volume fraction. They determined the value of 22/ct for tetramethylammonium (TMA, an extracellular marker in brain tissue) using iontophoresis and ion-selective microelectrodes (Nicholson et al., 1979; Nicholson and Phillips, 1981) and found an extracellular volume fraction of 0.2 and a tortuosity factor of 1.6 which would give 22/ct value of appoximately 12. A similar value was found for Ca 2+ using microdialysis (Benveniste et al., 1989). The ratio of 12 for Ca 2÷ is consistent with the microelectrode study of Nicholson and Rice (1986), who showed that Ca 2÷ diffusion in brain interstitial space is similar to that of TMA, and with that of Safford and Bassingthwaighte (1977), who showed that 4SCa2+ mainly diffuses in the interstitial space of myocardial tissue. Therefore for these calculations one needs to know both the outflow concentration and the in vitro recovery of the substance at 37°C and also the diffusion characteristics in vivo (i.e. ct, 2 and K). It is worth emphasizing that the use of Eqn. 5 is only valid if the surrounding microenvironment is inactive. The effects of cellular uptake, exchange mechanisms and metabolism of a substance on ~t, ). and K are not fully understood at present and only a few transmitters and ions have been characterized in this respect (Rice et aL, 1985; Nicholson and Rice, 1986). Considering all these theoretical aspects and the many uncertainties involved it is obvious that the determination of *When determining brain interstitial concentrations according to Eqn. 5 with microdialysis it is also necessary to incorporate an additional factor, K. The factor K, given by:

iL expresses the differences between substance concentrations on the inside of the dialysis membrane, when dialysis is performed in simple, Cb, and complex, ~'b media, respectively. If mass transport in brain tissue equaled that of an aqueous solution, then Cb = Cb, or K = 1. However, measurements of TMA concentration profiles in red blood cell suspensions (RBCs) and aqueous solutions, respectively, have shown that Cb > ~'b (Fig. 8), which indicates that diffusion in water is driven by a smaller concentration gradient across the dialysis membrane compared with diffusion complex media. We may interpretate this as if recovery in water is likely to be underestimated because diffusion in this particular medium is sufficiently fast to counteract the sink action of the microdialysis probe (Benveniste et al., 1989). t Diffusion coefficient of calcium.

3= 3

i. 0 0 lime/min

FIG. 8. The time-course of glutamate concentration changes in dialysates collected in CAI of hippoeampus before, during and after 10 min of ischemia. The dark and white arrow indicate the onset of ischemia and recirculation, respectively. Data from Benveniste et aL, 1989b. For further information see Section 8.

interstitial concentration with microdialysis using Eqn. 5 can only be approximated. Lindefors and co-workers (1989) presented a solution where the interstitial concentration (~'i) was found to be related to the dialysate concentration (Cout) in the following way: Cou~q Ci = 4zcLa/3 h(t/3/R 2)

(6)

where q is the perfusion flow rate, L the dialysis membrane length, ,t the interstitial volume fraction, /3 the diffusion coefficient in brain tissue. The term h(t) is a function of the scaled, non-dimensional time, t = t t / 3 / R 2, R is the radius of the dialysis probe [for details of the mathematical analysis see (Arner et al., 1989)]. The diffusion coefficient in brain tissue is, as previously discussed, determined by: /3 = D i ). 2.

Inserting this expression into Eqn. 6 we get: _

qCout 22

Ci = 4x-~-~ ~ t ) '

(7)

As in Eqn. 5, the interstitial substance concentration Ci is directly proportional to 22 and inversely proportional to cc Therefore, knowledge of the in vivo diffusion characteristics is also required for this equation. This is partly circumvented by calculating the diffusion coefficient in vivo using the following formula: /3 = Recovery/, vivo x D (8) Recoveryi, vitro X O~ where Recoveryi, vwo is determined from Eqn. 2. To test the validity of Eqn. 6 we can calculate the interstitial Ca 2+ concentration using data from Benveniste et al. (1989): q = 5/~i/min = 8.3 x 10-" m3/sec; Cout=0.0078 mM; L = 2 x 10 -3m; /3 = D/22 = 0.79 x 10-9t/1.62 = 3.08 x 10 -1° m2/sec; ct = 0.2 (Nicholson et al., 1979); h(t) = h ( t t D / R 2) = h(5400 sec x 3.08 x 10 -l° mZ/sec/(260 x 10 -6 m ) 2) = h(24.6) = 0.23 (Amberg and Lindefors, 1989; Lindefors et al., 1989), the diffusion coefficient of calcium in vitro is 0.79 x 10-gm2/sec (Hille, 1984)

207

M1CRODIALYSIS~THEORY AND APPLICATION

and inserting these parameters into Eqn. 6 the interstitial Ca 2+ concentration amounts to: 8.3 x 10 -H m3/sec x 0.0078 mM ~i =

4 n x 2 x 10 -3 m x 0 . 2

= 1.81 raM.

x 3.08 x 10-1°m2/sec x 0.23 which is somewhat higher than that found by ion-selective microelectrodes (1.2-1.3 mM) (Hansen, 1985). It appears that the use of Eqn. 6 tends to overestimate brain interstitial substance concentrations (see Sections 8.1 and 8.3). In the next section we will transform glutamate and dopamine dialysate concentrations into interstitial concentrations using both Eqns 5 and 6 for comparison.

daltons). Rice et al. (1985) determined the diffusion coefficient for dopamine in brain tissue to be 0.68 x 10-t°m2/sec. According to Eqn. 6 the interstitial glutamate concentration is: 0.441~M X 8.3 x 10 -H m3/sec ~'~i 4 x it x 2 x 10 -3 m x 0.2 x 0.68 345/~M. X 10-1°m2/sec x 0.31 where Co,t = 0.44/~M, q = 5 #1/rain = 8.3 x 10 -11 m3/ see, L = 2 x 10 -3 m (Co,t, q and L originate from data by Benveniste et al., 1989), D = 0.68 x 10 -~° m2/ sec (Rice et al., 1985), h ( t ) = 0.31 (Lindefors et al., 1989) and g = 0.2 (Nicholson et al., 1979). 8.1.2. Dopamine

8. T R A N S F O R M A T I O N O F GLUTAMATE AND D O P A M I N E DIALYSATE CONCENTRATIONS INTO BRAIN INTERSTITIAL CONCENTRATIONS 8. I. NORMAL BRAIN TISSUE 8.1.1. Glutamate

Equation 5 transforms dialysate concentrations into brain interstitial concentrations but does n o t - as previously stressed---consider transmitter uptake and/or metabolism. Bradford et al. (1987) demonstrated that glutamate concentrations in dialysates increased by 75% when a glutamate uptake inhibitor was added to the perfusion medium. Therefore, unless this uptake is corrected fo r prior to using Eqn. 5, interstitial (synaptic) glutamate concentrations would probably be underestimated: Corrected dialysate glutamate concentration= dialysate glutamate concentration × correction factor ~out = 0.44 ]IM X 1.75 = 0.77 #M.

[0.44gM is the average dialysate concentration of glutamate in rat CAI hippocampus at a perfusion flow rate of 5 #l/rain (Benveniste et al., 1989).] Inserting this value in Eqn. 5:

In order to calculate interstitial dopamine concentrations with Eqn. 5 we will use data from Hurd and Ungerstedt (1989d). They measured basal striatal concentrations of dopamine in rats before and after systemic administration of cocaine which inhibits dopamine high affinity uptake (see review by Hurd, 1989). Dialysate concentrations of dopamine increased from 10.6 to 45 nM during cocaine administration. Thus, having corrected for uptake we can now use Eqn. 5: Ci = [(K x 22)/~] x (Co,t/Recovery~, vii,o) and we get Ci =

Ci = [(0.7 x 1.62)/0.2] x (0.77 #M/0.05) = 138 #M where K =0.7 (Benveniste et al., 1989), 2 = 1.6 (Nicholson and Philips, 1981), ct = 0 . 2 (Nicholson and Philips, 1981) and the recovery~, v~,ro of glutamate at 37°C is 5% with a perfusion flow rate of 5/d/rain (Tossman and Ungerstedt, 1986; Benveniste, 1989). It is important to note, that our use of 2 and ~t found for TMA, assumes that diffusion characteristics for glutamate and TMA, respectively, are similar. Unlike Eqn. 5, Eqn. 6 by Lindefors et al. (1989) apparently does not require a correction for transmitter uptake (see Lindefors et aL, 1989). Unfortunately, the diffusion coefficient for glutamate in brain tissue is unknown and no attempts have been made to calculate this value according to Eqn. 8. In the following, therefore, we will assume that the diffusion coefficient for glutamate is similar to that of dopamine which is known (the molecular weights of dopamine and glutamate are quite similar, 156 vs 169

0.2 x 0.24

= 2.2 ]2M

where Co,t = 45 nm, ,2. = 1.6, K =0.7, ~t = 0.2 and 0.24 represents the recovery~ vi,rofor dopamine, when a 4 mm CMA/10 dialysis membrane is perfused at 2 #1/min (Collin and Ungerstedt, 1989; Hurd and Ungerstedt, 1989d). Once again it is important to note that our use of 2 and ~t found for TMA, assumes that diffusion characteristics for dopamine and TMA, respectively, are similar. Interstitial dopamine concentrations calculated from Eqn. 6 yields

Ci = [(K x 22)Ict)] x (Coot/Recoveryi, vi~,o), we arrive at an interstitial concentration of:

45nMX 1.62x0.7

•i =

10.6riM x 3.3 x 10 -ll 4xTrx4xl0_3mx0.2

=3.3gM

X 0.68 x 10-1°m:/sec x 0.31 where ~'o,t is 10.6nM, q = 3.3 x 10 -llm3/sec, L = 4 x 10 -3 m (~'o,t, q and L originate from data by Hurd and Ungerstedt, 1989),/) = 0.68 x 10 -l° m2/sec (Rice et al., 1985), h(t) = 0.31 (Lindefors et al., 1989) and ~t = 0.2. 8.2. INTERPRETATIONOF CALCULATEDINTERSTITIAL GLUTAMATEAND DOPAMINECONCENTRATIONS OBTAINED BY MICRODIALYSIS Results from the previous section suggest that calculations of glutamate and dopamine interstitial concentrations from Eqns 5 and 6 correlate reasonably well. As anticipated both methods provide concentrations that are several-fold higher than values obtained by using the simple/n vitro recovery formula (Eqn. 1). How do we assess the accuracy of these determinations? This is not possible for glutamate

208

H. BENVENISTEand P. C. HOTTEMEmR

because there are no standards to compare with. For dopamine there appears to be a good correlation between microdialysis calculations and measurements performed with carbon fiber microelectrodes (Moghaddam et al., 1987). On the other hand is it at all relevant from a biological standpoint to be concerned with the accuracy of these microdialysis determinations? Neurotransmitter concentrations are obviously not constant but fluctuate within a few hundred microseconds as a result of release and uptake (Alberts et al., 1983). Therefore microdialysis with its limited time resolution cannot measure actual transmitter concentration changes in the synaptic cleft during neurotransmission but only an average of these. This is important to keep in mind when evaluating experimental data. A good example is the apparent paradox that the rather high resting glutamate concentrations we find in vivo are potentially neurotaxic in vitro (Choi et al., 1987; Frandsen and Schousboe, 1987; Frandsen et al., 1989; Rosenberg and Aizenman, 1989). However, with reference to our previous arguments we may assume that neurons are not constantly exposed to these 'high' average glutamate concentrations and therefore not damaged. In the following section we will determine interstitial (synaptic) glutamate concentrations during transient global ischemia. In contrast to normal tissue this situation is characterized by electrical silence, no transmitter uptake and therefore a more stabile transmitter concentration which can be calculated and evaluated by Eqns 5 and 6, respectively. 8.3. GLUTAMATE CONCENTRATIONS IN ISCHEMIC BRAIN TISSUE

Glutamate may play an important role in the pathogenesis of selective neuronal injury and death following transient cerebral ischemia (Jargensen and Diemer, 1982; Wieloch, 1985a; Rothman and Olney, 1986; Greenamyre, 1986; Auer and Siesj6, 1988; Choi, 1989). In short the excitotoxin hypothesis explains selective neuronal death induced by transient global ischemia as a result of an abnormally high concentration of glutamate present in the synaptic cleft during ischemia. This triggers excessive and uncontrolled Ca 2÷ influx in the postsynaptic neurons that ultimately causes cell death. Microdialysis has been crucial for the verification of this hypothesis. Figure 8 demonstrates the time-course of glutamate concentration changes in dialysates collected from the rat CA1 hippocampal region before, during 10 min of ischemia and upon recirculation (Benveniste et al., 1989b). Glutamate concentrations increase in the dialysates during ischemia in all animals. These results have largely been confirmed by several other investigators. Hagberg et al. (1985) demonstrated a 3.5-fold increase of glutamate during 10min of ischemia in the rabbit hippocampus, Globus et al. (1988a) and Korf et al. (1988) found a 7-fold release of glutamate during 20 min of ischemia in rat striatum, Shimada et al. (1989) demonstrated a 5- to 10-fold increase of glutamate in cat cortex and finally Hillered et al. (1989) measured (>40.fold increase) of glutamate in striatum after permanent occlusion of the middle cerebral artery.

The calculation of 'true' glutamate concentrations during ischemia requires as previously discussed that glutamate dialysate levels are transformed into brain interstitial concentrations. This is first done using Eqn. 5. High affinity glutamate uptake is nonfunctional during ischemia (Barbour et aL, 1988) and therefore any corrections for this effect is unnecessary. The mean glutamate concentration measured in dialysates during 5-10rain of ischemia is 2.7/~M (Benveniste et al., 1989). The Recoveryinoi,,o of glutamate is 5% (Tossman and Ungerstedt, 1986) and the interstitial space size is known to shrink during ischemia by 50% to 0.1 (Hansen and Olsen, 1980). The tortuosity factor has not been determined during ischemia and we must therefore use the value of 1.6 obtained in normal brain tissue (Nicholson et al., 1979; Nicholson and Rice, 1986). Inserting all these parameters into Eqn. 5 we arrive at an interstitial glutamate concentration of: Ci = 0.7 x 0.62/0.1) × 2.7#u/0.05 = 968/~M which is still somewhat underestimated because the tortuosity factor may increase during ischemia (Nicholson and Rice, 1986). Glutamate concentrations evaluated by Eqn. 6 yield: •i = 2.7/ZM x 8.3 x 10-11 m3/sec =4.2ram 4 x n x 2 x 10-3m× 100.1 x 0.68 x 10-1°m:/sec x 0.31 where ~'out= 2.7/~M, q = 8.3 x 10 -H m3/sec, L = 2 x 10 -3 m (Benveniste et al., 1989), ~ = 0.1 (Hansen and Olsen, 1980), D =0.68 × 10-1°m2/sec (Rice et al., 1985) and h(t)=0.31 (Lindefors et al., 1989). It is noteworthy that the simple recovery formula (Eqn. 1), would only have given a glutamate concentration of 2.7/,u/0.05 = 54 # u which appears to represent a 20- to 80-fold underestimation of the 'true' interstitial (synaptic) glutamate concentration calculated from Eqns 5 and 6. This is important for the verification of the excitotoxin hypothesis because glutamate concentrations of 1--4mM are definitely toxic during a brief exposure whereas concentrations in the low micromolar range require much longer time to produce neuronal injury (Choi et al., 1987; Frandsen and Schousboe, 1987; Frandsen et al., 1989; Rosenberg and Aizenman, 1989). 9. CONCLUSION The microdialysis technique is an important breakthrough in the neurobioiogical sciences and has provided much needed information on the complex functions of the intact brain. The real benefit of expanding the applications to include other organs and tissues remains to be seen but preliminary results seem promising. However, despite its increasing popularity, microdialysis is still far from being the routine method it was perhaps intended to be. In the past, much work has necessarily gone into improving the technology involved whereas less attention was focused on the many methodological problems and limitations that could lead to erroneous data interpretations and conflicting results. Hopefully, the preceding chapters have clearly illustrated that this trend is

MICRODIALYSIS---THEORYAND APPLICATION changing and that an increasing effort is directed towards finding solutions to the many data analytical problems that are still partially unresolved.

REFERENCES ABERCROMBm,E. D., KELLER,R. W. JR and ZIGMOND,M. J. (1988) Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: Pharmacological and behavioral studies. Neuroscience 27, 897-904. ABERCRO~mm,E. D., KEmm, K. A., DxFmscmA, D. S. and ZmMOND, M. J. (1989) Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J. Neurochem. 52, 1655-1658. AJXMA,A. and KATO,T. (1987) Brain dialysis: detection of acetylcholine in the striatum of unrestrained and unanesthetized rats. Neurosci. Left. 81, 129-132. ALnERTS,B., BRAY,D., LEWIS,J., RAm, M., ROBERTS,K. and WATSON, J. D. (1983) Molecular Biology o f the Cell. Garland Publishing Inc.: New York. ALEXANDER,G. M., GROTHUSEN,J. R. and SCh'WARTZMAN, R. J. (1988a) Flow dependent changes in the effective surface area of microdialysis probes. Life Sci. 43, 595-601. ALEXANDER,N., NAKAHARA,D., OZAKI, N., KANEDA,N., SASAOKA,T., IWATA,N. and NAGATSU,T. (1988b) Striatai dopamine release and metabolism in sinoaorticdenervated rats by in vivo microdialysis. Am. J. Physiol. 254, R396-R399. AMBERG,G. and LINDEFORS,N. (1989) Intracerebral microdialysis: II. Mathematical studies of diffusion kinetics. J. Pharmac. Meth. 22, 157-183. ARNER, P., BOLINDER,J., ELIASSON,A., LUNDIN, A. and UNGERSTEDT,U. (1988) Microdialysis of adipose tissue and blood for/n vivo lipolysis studies. Am. J. Physiol. 255, E737-E742. AUER, R. N., and SmsJ6, B. K. (1988) Biological differences between ischemia, hypoglycemia and epilepsy. Ann. Neurol. 24, 699-707. AUKLAND,K. and NICOLAYSEN,G. (1981) Interstitial fluid volume: Local regulatory mechanisms. Physiol. Rev. 61, 556~44. BALLARIN, M., HERRERA-MARSCHITZ, M., CASAS, i . and UNGERSTEDT,U. (1987) Striatai adenosine levels measured 'in vivo' by microdiaiysis in rats with unilateral dopamine denervation. Neurosci. Lett. 83, 338-344. BARBOUR,B., BREW, H. and ATTWELL,D. (1988) Electrogenic glutamate uptake in gliai cells is activated by intracellular potassium. Nature 335, 433-435. BEAN, A. J., DURING,M. J. and ROTH, R. H. (1989) Stimulation-induced release of coexistent transmitters in the prefrontal cortex: An in vivo microdialysis study of dopamine and neurotensin release. J. Neurochem. 53, 655-657. BARD,A. G. and FAULKN.ER,L. R. (1980) Electrical Methods, p. 153. Wiley: New York. BEN-NUN,J., COOPER,R. L., CRINGLE,S. J. and CONSTABLE, I. J. (1988) A new technique for in vivo intraocular pharmacokinetic measurements. Archs Ophthal. N.Y. 106, 254-259. BENVENISTE, H., DREJER, J., SCHOUSBOE,A. and DIEMER, N. H. (I 984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracellular microdialysis. J. Neurochem. 43, 1369-1374. BENVENISTE, H., DREJER, J., SCHOUSBOE,A. and DIEMER, N. H. (1987) Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. J. Neurochem. 49, 729-734.

209

I~NVL~TS, H. and ~ , N. H. (1988a) Early postiscbemic 45Ca accumulation in rat dentate hilus. J. Cereb. Blood flow Metab. 8, 713-719. I~NV~'IST~, H. and ~ , N. H. (1988b) Cellular reactions to implantation of a microdialysis tube in the rat hippocampus. Acta neuropath. Bar/. 74, 234-238. BEI~,'VEmSTE,H. (1989) Brain microdialysis. J. Neurochem. 52, 1667-1679. BEUVEN.tSTE,H., HANSON.,A. J. and OTrOSEN,N. S. (1989a) Determination of brain interstitial concentrations by microdialysis. J. Neurochem. 52, 1741-1750. BE~XSTE, H., JfltRGENSEN, M. B., S^m3B~G, M., CHRISTEN'SEN,T., HAGBERG,H. and Dm~m~, N. H. (1989b) Isehemic damage in hippocampal CAI is dependent on glutamate release and intact innervation from CA3. J. Cereb. Blood flow Metab. 9, 626-639. BETTINI, E., CECl, A., SPINELLI,R. and SAMARIN.,R. (1987) Neuroleptic-like effects of the I-isomer of fenfluramine on striatal dopamine release in freely moving rats. Biochem. Pharmac. 36, 2387-2391. BITO, L., DAVSON,H., LEVIN,E., MURRAY,M. and SNIDER, N. (1966) The concentrations of free amino acids and other electrolytes in cerebraispinal fluid, in vivo dialysate of brain, and blood plasma of the dog. J. Neurochem. 13, 1057-1067. BLAKELY,R. D., WAGES,S. A., JUSTICE,J. B., HERDON,J. G. and NEILL, D. B. (1984) Neuroleptics increase striatal catecholamine metabolites but no ascorbic acid in dialyzed perfusate. Brain Res. 308, 1-8. BRADSURY,M. W. B. (1979) The Concept o f a Blood-Brain Barrier. Wiley: London. BRADBURY, i . W. B., CSERR, S. F. and WESTROP, R. J. (1981) Drainage of cerebral interstitial fluid into deep cervical lymph of the rabbit. Am. J. Physiol. 240, F329-F336. BRADFORD, H. F., YOUNG, A. M. J. and CROWDER,J. M. (1987) Continuous glutamate leakage from brain cells is balanced by compensatory high-affinity reuptake transport. Neurosci. Lett. 81, 296-302. BRODm, M. S., LEE, K., FREDHOLM, B. B., STAHLE, L. and DUNWlDDIE,T. V. (1987) Central versus peripheral mediation of responses to adenosine receptor agonists: evidence against a central mode of action. Brain Res. 415, 323-330. BRODIN, E., LINDEVORS,N. and UNGERSTEDT,U. (1983) Potassium evoked in vivo release of substance P in rat caudate nucleus measured using a new technique of brain dialysis and an improved substance P-radioimmunoassay. Acta physiol, scand. (suppl. 515), 17-20. BRODIN,E., LINDEROTH,B., GAZELIUS,B. and UNGERSTEDT, U. (1987) In vivo release of substance P in cat dorsal horn studied with microdialysis. Neurosci. Lett. 76, 357-362. BROOIN, L., TOSSMAN,U., OHTA, Y., UNGERSTEDT,U. and GmLLNER, S. (1988) The effect of an uptake inhibitor (dihydrokainate) on endogenous excitatory amino acids in the lamprey spinal cord as revealed by microdialysis. Brain Res. 458, 166-169. BUSTO, R., GtOBUS, M. Y. T., DmrRICH, W. D., MARTINEZ, E., VALD~, I. and GINSBERG,M. D. (1989) Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in the rat brain. Stroke 20, 904-910. BUTCHER, S. P., SANDBERG, M., HAGnFJtG, H. and HAMBERGER,A. (1987a) Cellular origin of endogenous amino acids released into the extracellular fluid of the rat striatum during severe insuiin-produced hypoglycemia. J. Neurochem. 48, 722-728. BUTCHER,S. P., JACOBSON,I., SANDBERG,M., HAGBERG,H. and HAMBERGER,A. (1987b) 2-Amino-5-phosphonovalerate attenuates the severe hypoglycemia-induced loss of perforant path-evoked field potentials in the rat hippocampus. Neurosci. Lett. 76, 296-300.

210

H. BENVEhqSTEand P. C. HOTTo~mR

BUTCHER, S. P., LAZAREWICZ,J. W. and HA~mr~GER, A. (1987c) In vivo mierodialysis studies on the effects of decortication and excitotoxic lesion on kalnic acidinduced calcium fluxes, and endogenous amino acid release, in the rat striatum. J. Neurochem. 49, 1355-1360. CARTER, C. J., L'HEuREux, R. L. and SCATTON,B. (1988) Differential control by N-methyI-D-aspartate and kalnate of striatal dopamine release in vivo: A trans-striatal dialysis study. J. Neurochem. 51, 462-468. CHOI, D. (1989) Toward a new pharmacology of isehemic neuronal death. In: Collins RC, moderator. Selective vulnerability of the brain: New insights into the pathophysiology of stroke. Ann. intern. Med. 110, 992-1000. CHOI, D. W., MAULUCCI-GEDDE,M. and KRIEGSTEIN,A. R. (1987) Glutamate neurotoxicity in cortical cell culture. J. Neurosci. 7, 357-368. CHURCH,W. H. and JUSTICE,J. B. JR (1987) Rapid sampling of extracellular dopamine in vivo. Analyt. Chem. 59, 712-716. CLEMENS,J. A. and PHEBUS,L. A. (1984) Brain dialysis in conscious rats confirms in vivo electrochemical evidence that dopaminergic stimulation releases ascorbate. Life Sci. 35, 671~577. COLLIAS, J. C. and MANUEUDIS,E. E. (1957) Histopathological changes produced by implanted electrodes in cat brains. Comparison with histopathological changes in human and experimental puncture wounds. J. Neurosurg. 14, 302-328. COLLIN, A.-K. and UNGERSTEDT,U. (1988) Microdialysis, User's guide 4th edition. Carnegie Medicin. CONSOLO, S., Wu, C. F., FIORENT~Ni,F., L~INSK'¢, H. and VEZZANI,A. (1987) Determination of endogenous acetylcholine release in freely moving rats by transstriatal dialysis coupled to a radioenzymatic assay: Effect of drugs. J. Neurochem. 48, 1459-1465. CSERR, H. F., COOLER,D. N., SUm, P. K. and PATLAK,C. S. (1981) Efflux of radiolabeled polyethylene glycols and albumin from rat brain. Am. J. Physiol. 240, F319-F328. DAMSMA,G., WESTERINK,B. H. C., IMPERATO,A., ROLLEMA, H., DE VRIES,J. B. and HORN, A. S. (1987a) Automated brain dialysis of acetylcholine in freely moving rats: Detection of basal acetylcholine. Life Sci. 41, 873-876. DAMSMA,G., WESTERINK,B. H. C., DE VRIES,J. B., VAN DEN BERG, C. J. and HORN, A. S. (1987b) Measurement of acetylcholine release in freely moving rats by means of automated intracerebral dialysis. J. Neurochem. 48, 1523-1528. DAMSMA, G., BIESSELS,P. T. M., WESTERINK,B. H, C., DE VRIES,J. B. and HORN,A. S. (1988a) Differential effects of 4-aminopyridine and 2,4-diaminopyridine on the in vivo release of acetylcholine and dopamine in freely moving rats measured by intrastriatal dialysis. Eur. J. Pharmac. 145, 15-20. DAMSMA,G., WESTER1NK,B. H. C., DE BOER, P., DE VR1ES, J. B. and HORN,A. S. (1988b) The effect of systematically applied cholinergic drugs on the striatal release of dopamine and its metabolites, as determined by automated brain dialysis in conscious rats. Neurosci. Lett. 89, 349-354. DAMSMA,G., WESTERINK,B. H. C., DE BOER, P., DE VRIES, J. B. and HORN,A. S. (1988c) Basal acetylcholine release in freely moving rats detected by on-line trans-striatal dialysis: Pharmacological aspects. Life Sci. 43, 1161-1168. DELGADO,J. M. R., DEFEUDIS,F. V., ROTH, R. H., RYUGO, D. K. and MITRUKA, B. M. (1972) Dialytrode for long term intercerebral perfusion in awake monkeys. Archs int. Pharmaeodyn. 198, 9-21. DELGADO, J. M. R., LERMA,J., MARTINDEL RIO, R. and SoLIS, J. M. (1984) Dialytrode technology and local profiles of amino acids in the awake cat brain. J. Neurochem. 42, 1218-1228.

DEL RIO HOTEGA, P. and PENFIELO, W. (1927) Cerebral cicatrix: the reaction of neuroglia and microglia to brain wounds. Bull. Johns Hopkins Hosp. 41, 278-303. DONZ~qXa, B. A. and YAra~d~OTO,B. K. (1988) An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates. Life Sci. 43, 913-922. DREIER, J., BENVENISTE,H., DmMER,N. H. and SCHOUSBOE, A. (1985) Cellular origin of ischemia-induced glutamate release from brain tissue in vivo and in vitro. J. Neurochem. 45, 145-151. EGAWA,M., HOEBEL,B. G. and STONE,E. A. (1988) Use of microdialysis to measure brain noradrenerglc receptor function in vivo. Brain Res. 458, 303-308. FADEN, A. I., DEMEDIUK,P., PANTER,S. S. and V1NK, R. (1989) The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244, 798-800. FRANDSEN,AA. and SCHOUSBOE,A. (1987) Time and concentration dependency of the toxicity of excitatory amino acids on cerebral neurones in primary culture. Neurochem. Int. 10, 583-591. FRANOSEN,AA., DREJER,J. and SCHOUSBOE,A. (1989) Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-d-aspartate (NMDA) as well as non-NMDA receptors. J. Neurochem. 53, 297-299. FRIEDMAN,M. H. (I 986) Principles and Models of Biological Transport. Springer: Berlin. FEEHEY,D. M., and BARON,J.-C. (1986) Diaschisis. Stroke 17, 817-829. GLOBUS,M. Y. T., BUSTO,R., DIETRICH,D. W., MARTINEZ, E., VALDES, I. and GINSBERG,M. D. (1988a) Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and y-aminobutyric acid studied by intracerebral microdialysis. 3". Neurochem. 51, 1455-1464. GLOBUS,M. Y. T., BUSTO,R., DIETRICH,W. D., MARTINI~Z, E., VALDES,I. and GINSBERG,M. D. (1988b) Intra-ischemic extracellular release of dopamine and glutamate is associated with striatal vulnerability to ischemia. Neurosci. Lett. 91, 36-40. GLO~US, M. Y. T., Busro, R., DIETRICH,W. D., MARTINEZ, VALDI~S,I. and G1NSBERG,M. D. (1989) Direct evidence for acute and massive norepinephrine release in the hippocampus during transient ischemia. J. Cereb. Blood flow Metab. 9, 892-896. GREENAMYRE,J. T. (1986) The role of glutamate in Neurotransmission and in Neurologlc disease. Archs NeuroL 43, 1058-1063. HAGBERG, H., LEHMANN, A. and HAMBERGER,A. (1984) Inhibition by verapamil of ischemic Ca 2+ uptake in the rabbit hippocampus. J. Cereb. Blood flow Metab. 4, 297-300. HAGBERG, H., LEHMANN,A., SANDBERG,M., NYSTR~}M,B., JACOBSON,I. and HAMBERGER,A. (1985) Ischemia-induced shift of inhibitory and excitatory amino acids from intrato extracellular compartments. J. Cereb. Blood flow Metab. 5, 413-419. HAGBERG,H., ANOERSSON,P., BUTCHER,S., SANDBERG,M., LEHMANN, A. and HAMSERGERA. (1986) Blockade of N-methyl-D-aspartate-sensitive acidic amino acid receptors inhibits ischemia-induced accumulation of purine catabolites in the rat striatum. Neurosci. Lett. 68, 311-316. HAGBERG,H., ANDERSSON,P., KJELLMER,I., THIRINGER,K. and THORDSTEIN,M. (1987a) Extracellular overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia. Neurosci. Lett. 78, 311-317. HAGBERG,H., ANDERSSON,P., LAZAREWICZ,J., J ACOBSON,1., BUTCHER, S. and SANDBERG,M. (1987b) Extracellular adenosine, inosine, hypoxanthine and xanthine in relation to tissue nucleotides and purines in rat striatum during transient ischemia. J. Neurochem. 49, 227-231.

MICROD1ALY~--THI~RYAND APPLICATION HAMBERGER,A., JACOBSON,I., MOLXN,S.-O., NYSTR6M, B., SANDBERG,M. and UNOERstevr, U. (1982) Metabolic and transmitter compartments for glutamate. In: Neurotransmitter Interaction and Compartmentation, pp. 359-378. Ed. H. F. BRADFORD. Plenum. HAMBEROER,A., BER~OLD, C.-H., KARLSSON,B., LEmVXANN, A. and NVSTg6M, B. (1983) Extracellular GABA, glutamate and glutamine in vivo perfusion dialysis of the rabbit hippooampus. In: Glutamine, Glutamate and GABA in the Central Nervous System, pp. 473-492. Alan R. Liss Inc.: New York. HAMnERGER,A. and NYSTR6M, B, (1984) Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy. Neurochem. Res. 9, 1181-1192. HAMBEROER,A. (1989) Microdialysis in clinical diagnosis: amino acid patterns in the temporal cortex and the myocardium. Current Separations 9, 119. HANSEN, A. J. and OLSEN, C. E. (1980) Brain extraceUular space during spreading depression and ischemia. Acta physiol, scand. 168, 355-365. HANSEN,A. J. (1985) Effect of anoxia on ion distribution in the brain. Physiol. Rev. 65, 101-148. HERNANDEZ, L., PAEZ, X. and HAMLIN,C. (1983) Neurotransmitters by local intracerebral dialysis in anesthetized rats. Pharmac. Biochem. Behav. 18, 159-162. HERNANDEZ,L., STANLEY,B. G. and HOEBEL,B. G. (1986) A small, removable microdialysis probe. Life Sci. 39, 2629-2637. HERRERAS,O., SOLfS,A. S., MARTINDEL Rio, R. and LERMA, J. (1988) Sensory modulation of hippocampal transmission. II. Evidence for a cholinergic locus of inhibition in the Sehaffer-CA1 synapse. Brain Res. 461, 303-313. HILLE, B. (1984) lonic Channels o f Excitable Membranes. Sinauer Associates: Sunderland, Massachusetts. HILLERED,L., HALLSTROM,A., SEGERSV)~RD,S., PERSSON,L. and UNGERSTEDT,U. (1989) Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. J. Cereb. BIoodflow Metab. 9, 607-616. HURD, Y. L., KEHR, J. and UNOERSTEDT,U. (1988) In vivo microdialysis as a technique to monitor drug transport: Correlation of extracelhilar cocaine levels and dopamine overflow in the rat brain. J. Neurochem. 51, 1314-1316. HURD, Y. (1989) In vivo brain pharmacology of cocaine, amphetamine and related drugs: A microdialysis study. Dissertation, Department of Pharmacology, Karolinska Institute, Stockholm. HURD, Y. and UNGERSTEDT,U. (1989a) In vivo neurochemical profile of dopamine uptake inhibitors and releasers in rat caudate-putamen. Eur. J. Pharmac. 166, 251-260. HURD,Y. and UNGERSTEDT,U. (1989b) Ca2+-dependence on the amphetamine, nomifensine, and Lu 19-005 effect on in vivo dopamine transmission. Fur. J. Pharmac. 166, 261-269. HURD, Y. L. and UNGERSTEDT,U. (1989c) Influence of a carrier transport process on in vivo release and metabolism of dopamine: Dependence on extracellular Na +. Life Sci. 45, 283-293. HURD, Y. L. and UNOERSTEDT, U. (1989d) Cocaine: An in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum. Synapse 3, 48-54. HUTSON, P. H., SARNA, G. 8., KANTAMANENI,B. D. and CURZON,G. (1985) Monitoring the effect of a tryptophan load on brain indole metabolism in freely moving rats by simultaneous cerebrospinal fluid sampling and brain dialysis. J. Neurochem. 44, 1266-1273. IKEDA, M., NAKAZAWA, T., ABe-, K., KAZ,mKO, T. and YAMATSU,K. (1989) Extracellular accumulation of glutamate in the hippocampus induced by ischemia is not calcium dependent--in vitro and in vivo evidence. Neurosci. Lett. 96, 202-206.

211

IMPERATO,A. and DI C'msa~, G. (1984) Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: A new method for the study of the in vivo release of endogenous dopamine and metabolites. J. Neurosci. 4, 966-977. JACOmON,I. and H ~ o ~ , A. (1984) Veratridine-induced release in vivo and in vitro of amino acids in the rabbit olfactory bulb. Brain Res. 299, 103-112. JACOSSON,I. and HAt,mEROER,A. (1985) Kainic acid-induced changes of extracellular amino acid levels, evoked potentials and EEG activity in the rabbit olfactory bulb. Brain Res. 348, 289-296. JACOBSON, I., SANDeERO, M. and HAMeEROER,A. (1985) Mass transfer in brain dialysis devices--a new method for the estimation of extracellular amino acids concentration. J. Neurosci. M e t e 15, 263-268. JACOBSON,I., HAGBERG,H., SANDnERG,M. and HAMBERGER, A. (I 986) Ouabain-indoced changes in extracellular aspartate, glutamate and GABA levels in the rabbit olfactory bulb in vivo. Neurosci. Lett. 64, 211-215. JOHNSON,R. D. and JUSTICE,J. B. (1983) Model studies for brain dialysis. Brain Res. Bull. 10, 567-571. JUSTICE, J. B., WAGES, S. A. and MICHAEL,A. C. (1983) Interpretations of voltammetry in the striatum based on chromatography of striatal dialysate. J. Liq. Chromat. 6, 1873-1896. J~OENS~N, M. B. and DmMER,N. H. (1982) Selective neuron loss after cerebral ischemia in the rat: Possible role of transmitter glutamate. Acta neurol, scand. 66, 536-546. KAI~N, P., STRECKF.R,R. E., ROSENOREN,E. and BJORKLUND, A. (1988a) Endogenous release of neuronal serotonin and 5-hydroxyindoleacetic acid in the caudate-putamen of the rat as revealed by intracerebal dialysis coupled to highperformance liquid chromatography with fluorimetric detection. J. Neurochem. 51, 1422-1435. KALI~N,R., KOKAIA,M., LINDVALL,O. and BJ6RKLUND,A. (1988b) Basic characteristics of noradrenaline release in the hiplxx~mpus of intact and 6-hydroxydopaminelesioned rats as studied by in vivo microdialysis. Brain Res. 474, 374-379. KATO, T., DONG, B., IsmL K. and KINEMUCHI,H. (1986) Brain dialysis: In vivo metabolism of dopamine and serotonin by monoamine oxidase A but not B in the striatum of unrestrained rats. J. Neurochem. 46, 1277-1282. KENT)RICK, K. M., BALDWIN, B. A., COOPER, T. R. and St~RMAN, D. F. (1986) Uric acid is released in the zona incerta of the subthalamic region of the sheep during rumination and in response to feeding and drinking stimuli. Neurosci. Left. 70~ 272-277. KENDRICK, K. M., KEVERNE, E. B., CHAPMAN, C. and BALDWIN,B. A. (1988a) Microdialysis measurement of oxytocin, aspartate, g-aminobutyric acid and glutamate release from the olfactory bulb of the sheep during vaginocervical stimulation. Brain Res. 442, 171-174. KENDRICK, K. M., KEVERNE, E. B., CHAPMAN, C. and BALDWIN,B. A. (1988b) Intereranial dialysis measurement of oxytocin, monoamine and uric acid release from the olfactory bulb and substantia nigra of sheep during parturition, suckling, separation from lambs and eating. Brain Res. 439, 1-10. KENDRICK, K. M. and LENO, G. (1988) Haemorrhageinduced release of noradrenaline, 5-hydroxytryptamine and uric acid in the supraoptic nucleus of the rat, measured by microdialysis. Brain Res. 440, 402--406. KENDRICK, K. M. (1988) Use of microdialysis in neuroendocrinology. Meth. Enzymol. 168, 182-205. KITO,S., SmMOYAMA,M. and ARAKAWA,R. (1986) Effects of neurotrausmitters or drugs on the in vivo release of dopamine and its metabolites. Jap. J. Pharmac. 40, 57-67. KORF, J. and VENEMA,K. (1985) Amino acids in rat striatal dialysate: Methodological aspects and changes after eleetroconvulsive shock. J. Neurochem. 45, 1341-1348.

212

H. BENVEmSTEand P. C. H0"rlEMEER

KoP,v, J., KLEIN, H. C. VENEU.A,K. and POSTEMA,F. (1988) Increases in striatal and hippocampal impedance and extracellular levels of amino acids by cardiac arrest in freely moving rats. J. Neurochem. 50, 1087-1096. KORF, J. (1989) Lactography: A novel technique to monitor brain energy metabolism in physiology and pathology. Neurosci. Res. Commun. 4, 129-138. KUHR, W. G. and Kol~, J. (1988) Extracellular lactic acid as an indicator of brain metabolism: Continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis. J. Cereb. Blood flow Metab. 8, 130-137. KUHR, W. G., VAN DEN BERG, C. J. and KORF, J. (1988) In vivo identification and quantitative evaluation of carriermediated transport of lactate at the cellular level in the striatum of conscious, freely moving rats. J. Cereb. Blood flow Metab. 8, 848-856. LAURITZEN,M. (1987) Cerebral blood flow in migraine and cortical spreading depression. Acta neurol, scand. 76(! 13s), 1--40. LAZAREWICZ, J. W., LEHMANN, A., HAGBERG, H. and HAMBERGER,A. (1986a) Effects of kainic acid on brain calcium fluxes studied in vivo and in vitro. J. Neurochem. 46, 494-498. LAZAREWlCZ, J. W., LEH~NN, A. and HAMBERGER,A. (1986b) Effects of Ca 2+ entry blockers on kainate-induced changes in extraceUular amino acids and Ca 2+ in vivo. J. Neurosci. Res. 18, 341-344. LEAO, A. P. P. (1944) Spreading depression of activity in cerebral cortex. J. Neurophysiol. 7, 359-390. LEHMANN, A., ISACSSON,H. and HAMBVa~GER,A. (1983) Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus. J. Neurochem. 40, 1314-1320. LEHMANN,A. and HAMB~GER,A. (1983) Dihydrokainic acid affects extracellular taurine and phosphoethanolamine levels in the hippocampus. Neurosci. Lett. 38, 67-72. LEHMANN,A. and HAMBERGER,A. (1984) A possible interrelationship between extracellular taurine and phosphoethanolamine in the hippocampus. J. Neurochem. 42, 1286-1290. LEHMANN,A., HAGBERG,H. and HAMBERGER,A. (1984) A role for taurine in the maintenance of homeostasis in the central nervous system during hyperexcitation? Neurosci. Lett. 52, 341-346. LEHMANN,A., HAGBERG,H., JACOBSON,I. and HAMBERGER, A. (1985a) Effects of status epilepticus on extracellular amino acids in the hippocampus. Brain Res. 359, 147-151. LEHMANN, A., LAZAREWlCZ,J. W. and ZEISE, M. (1985b) N-Methylaspartate-evoked liberation of taurine and phosphoethanolamine in vivo : Site of release. J. Neurochem. 45, 1172-1177. LEHMANN, A., SANDBERG,M. and HUXTABLE,R. J. (1986) In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizuresusceptible rats. Neurochem. Int. 8, 513-520. LEHMANN, A. (1987) Evidence for a direct action of Nmethylaspartate on non-neuronal cells. Brain Res. 411, 95-101. LEHMANN,A. (1989) Effects of microdialysis-perfusion with anisoosmotic media on extracellular amino acids in the rat hippocampus and skeletal muscle. J. Neurochem. 53, 525-535. LEHMANN,A. and SANOBERG,M. (1990) In vivo substitution of choline for sodium evokes a selective osmoinsensitive increase of extracellular taurine in the rat hippocampus. J. Neurochem. 54, 126-129. LERMA, J., HERRERAS,0., HERRANZ, A. S., MU~OZ, D. and MARTINDELR~O,R. (1984) In vivo effects of nipecotic acid on levels of extracellular GABA and taurine, and hippocampal excitability. Neuropharmacology 23, 595-598. LERMA,J., HERRANZ,A. S., HERRERm,O., MU~OZ, D., SOLiS, J. M., MARTINDEE Rio, R. and DELGADO,J. M. R. (1985)

~-Aminobutyric acid greatly increases the/n vivo extracellular taurine in the rat hippocampus. J. Neurochem. 44, 983-986. LERMA,J., HERRANZ,A. S., HERRF.Rm,O., AnRAIRA,V. and MARTIN DEL Rio, R. (1986) In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis. Brain Res. 384, 145-155. LEVlN,V. A., FENSTERMACHER,J. D. and PATLAK,C. S. (1970) Sucrose and inulin space measurements of cerebral cortex in four mammalian species. Am. J. Physiol. 219, 1528-1533. L'HEUREUX, R., DENNIS, T., CURET, O. and SCATTON,B. (1986) Measurement of endogenous noradrenaline release in the rat cerebral cortex in vivo by transcortical dialysis: Effects of drugs affecting noradrenergic transmission. J. Neurochem. 46, 1794-1801. LINDEFORS,N., BRODIN,E., THEODO~N-NORHEIM, E. and UNGERSTEDT,O. (1985) Regional distribution and in vivo release of tachykinin-like immunoreactives in rat brain: Evidence for regional differences in relative proportions of tachykinins. Regul. Peptides 10, 217-230. LINDEFORS, N., YAMAMOTO, Y., PANTALEO, T., LAGERCRANTZ, H., BRODIN, E. and UNGERSTEDT,U. (1986) In vivo release of substance P in the nucleus tractus solitarii increases during hypoxia. Neurosci. Left. 69, 94-97. LINDEFORS, N., AMBERG, G. and UNGERSTEDT,U. (1989) Intracerebral microdialysis: I. Experimental studies of diffusion kinetics. J. Pharmac. Meth. 22, 141-156. L6NNROTH,P., JANSSON,P.-A. and SMITH,U. (1987) A microdialysis method allowing characterization of intercellular water space in humans. Am. J. Physiol. 253, E228-E231. MA1DMENT, N. T., BRUMBAUGH, D. R., ERDELYI, E. and EVANS,D. J. (1989) Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo. Neuroscience 33, 549-55% MARSDEN, C. A., BRAZELL, M. P. and MAIDMENT,N. T. (1984) An introduction to in vivo electrochemistry. In: Measurement of Neurotransmitter Release In Vivo, pp. 127-153. Ed. C. A. MARSDEN.Wiley: New York. MEDVEDEV, O. S., KUZMIN, A. I., SELIVANOV,V. N. and SYSOEV,A. B. (1989) Pharmacological and physiological analysis of catecholamine secretion by the adrenal gland using in vivo microdialysis in the awake rat. Current Separations 9, 89. MEIRIEU,O., PAIRET,M., SUTRA,J. E. and RUCKEBUSCH,M. (1986) Local release of monoamines in the gastrointerstinal tract: An in vivo study in rabbits. Life Sci. 38, 827-834. MENI~NDEZ,N., HERRERAS,O., SOLiS,J. M., HERRANZ,A. S. and MARTIN DEE RiO, R. (1989) Extracellular taurine increase in rat hippocampus evoked by specific glutamate receptor activation is related to the excitatory potency of glutamate agonists. Neurosci. Lett. 102, 64-69. MOGHADDAM, B., SCHENK, J. O., STEWART, W. B. and HANSEN, A. J. 0987) Temporal relationship between neurotransmitter release and ion flux during spreading depression and anoxia. Can. J. Physiol. Pharmac. 65, If05-1110. MOGHADDAM,B. and BUNNEY,B. S. (1989) Ionic composition of microdialysis perfusion solution alters the pharmacological responsiveness and basal outflow of striatal dopamine. J. Neurochem. 53, 652-654. MORONI,F. and PEPEU,G. (1984) The cortical cup technique. In: Measurement of Neurotransmitter Release In Vivo, pp. 63-81. Ed. C. A. MARSDEN.Wiley: New York. MuI~OZ, M. D., HERRERAS,O., HERRANZ,A. S., SOLtS,J. M., MARTIN DEL RiO, R. and LERMA,J. (1987) Effects of dihydrokainic acid of extracellular amino acids and neuronal excitability in the in vivo rat hippocampus. Neuropharmacology 26, 1-8. NICHOLSON, C., PHILLIPS, J. M. and GARDNER-MEDWlN, A. R. (1979) Diffusion from an iontophoretic point source

MICRODIALY$1&---THEORYANDAPPLICATION

213

adrenaline and noradrenaline measured/n vivo. Brain Res. 426, 103-I 11. SAFFOm3,R. E. and BmSINGTh'W~GI-rrE,J. B. (1977) Calcium diffusion in transient and steady states in muscle. J. Biophys. 20, 113-136. Application of Ion-selective Microelectrodes. Ed. T. SANDBFaG,M. and LINDSTROM,S. (1983) Amino acids in the ZEUTHZN.Elsevier: Amsterdam. NICtIOLSON, C. and PmLLIPS, J. M. (1981) Ion diffusion dorsal lateral geniculate nucleus of the cat--collection in modified by tortuosity and volume fraction in extravivo. J. Neurosci. Meth. 9, 65-74. cellular microenvironment of rat cerebellum. J. Physiol., SANDBERG, M., NYSTRtM, B. and HAMnERGER,A. (1985) Lond. 321, 225-257. Metabolically derived aspartate---elevated extracellular NICHOLSON, C. and RICE, M. E. (1986) The migration of levels/n vivo in iodoacetate poisoning. J. Neurosci. Res. substances in the neuronal microenvironment. Ann. N.Y. 13, 489-495. Acad. Sci. 481, 55-71. SANDBERG,M., BUTCHER, S. P. and HAGBERG,H. (1986) O'CONNOR, W. T., DREW,K. L. and UNGVaS~DT, U. (1989) Extracellular overflow of neuroactive amino acids during Differences in dopamine release and metabolism in rat severe insulin-induced hypoglycemia: In vivo dialysis of striatal subregions following acute clozapine using in vivo the rat hippocampus. J. Neurochem. 47, 178-184. microdialysis. Neurosci. Lett. 98, 211-216. SANDBERG,M., HAGBERG,H., JACOBSON,I., KARLSSON,B., PANTER,S. S., YUM, S. W. and FADEN,A. I. (1990) Alteration LEHMANN, A. and HAMeERGER,A. (1987) Analysis of in extracellular amino acids after traumatic spinal cord amino acids: Neurochemical application. Life Sci. 41, injury. Ann. Neurol. 27, 96-99. 829-832. PHEBus, L. A., PERRY, K. W., CLE~,~NS,J. A. and FULLER, SANDBERG,M. and BENVENISTE,H. (1988) Blood flow and R. W. (1986) Brain anoxia releases striatal dopamine in glucose phosphorylation in the rat hippocampus after rats. Life Sci. 38, 2447-2453. implantation of a dialysis probe. 7th ESN, June 12-17. Ptw~us, L. A. and CLEMENS,J. A. (1989) Effects of transient, Gothenburg. global, cerebral ischemia on striatal extracellular dopaSCHmFOORT,E. C. M., DE BRUIN,L. A. and KoRv, J. (1988) mine, serotonin and their metabolites. Life Sci. 44, Mild stress stimulates rat hippocampal glucose utilization 1335-1342. transiently via NMDA receptors, as assessed by lactograPmLLIPPU,A. (1984) Use of push-pull cannulae to determine phy. Brain Res. 475, 58-63. the release of endogenous neurotransmitters in distinct SHARP, T., MAID~NT, N. T., BRAZELL, M. P. and brain areas of anesthetized and freely moving animals. Z~T~RSTR6M, T. (1984) Changes in monoamine metabIn: Measurement of Neurotransmitter Release In Vivo, olites measured by simultaneous/n vivo differential pulse pp. 3-39. Ed. C. A. MARSDEN.Wiley: New York. voltammetry and intracerebral dialysis. Neuroscience 12, PILOWSKY,P. M., KAPOOR,V., MINSON,J. B., WEST, M. J. 1213-1221. and CHALMERS,J. P. (1986) Spinal cord serotonin release SHARP, T., ZETTERSTR6M, T., CmUS~NSON, L. and and raised blood pressure after brainstem kainic acid UNGERS~DT, U. (1986a) p-Chloroamphetamine releases injection. Brain Res. 366, 345-357. both serotonin and dopamine into rat brain dialysate in RADHAKISHUN, F. S., VAN REE, J. M. and WESTERINK, vivo. Neurosci. Lett. 72, 320-324. B. H. C. (1988) Scheduled eating increases dopamine SHARP, T., ZETTERSTR6M,T. and UNGERSTEDT,U. (1986b) release in the nucleus accumbens of food-deprived rats as An in vivo study of dopamine release and metabolism in assessed with on-line dialysis. Neurosci. Lett. 85, 351-356. rat brain regions using intracerebral dialysis. J. NeuroREID, M., HERRERA-MARSCHITZ,M., HOKFELT,T., TERENIUS, chem. 47, 113-122. L. and UNGERSTEDT,U. (1988) Differential modulation of SHARP, T., ZETTERSTR()M,T., SERIES,H. G., CARLSSON,A., striatal dopamine release by intranigral injection of ?GRAHAME-SM1TH,D. G. and UNGERSTEDT,U. (1987a) aminobutyric acid (GABA), dynorphin A and substance HPLC-EC analysis of catechols and indoles in rat brain P. Eur. J. Pharmac. 147, 411-420. dialysates. Life Sci. 41, 869-872. REIRIZ,J., MENA,M. A., BAZAN,E., MURADAS,V., LERMA,J., SHARP, T., ZETTERSTR6M, T., LJUNGBERG, T. and DELGADO,J. M. R. and DE Yf~nENES,J. G. (1989) Temporal UNGERSTEOT,U. (1987b) A direct comparison of amphetprofiles of levels of monoamines and their metabolites in amine-induced behaviours and regional brain dopamine striata of rats implanted with dialysis tubes, d. Neurorelease in the rat using intracerebral dialysis. Brain Res. chem. 53, 789-792. 401, 332-330. RICE, M. E., GERHARDT,G. A., HIERL,P. M., NAGY, G. and SHARP,T. and FOSTER,G. A. (1989) In vivo measurement ADAMS,R. N. (1985) Diffusion coefficients of neurotransusing microdialysis of the release and metabolism of mitters and their metabolites in brain extracellular fluid 5-hydroxytryptamine in raphe neurones grafted to the rat space. Neuroscience 15, 891-902. hippocampus. J. Neurochem. 53, 303-306. ROBINSON,T. E. and WmSHAW, I. Q. (1988) Normalization SHARP, T., BRAMWELL,S. R., CLARK,D. and GRAHAMEof extraceUular dopamine in striatum following recovery SMrm, D. G. (1989) In vivo measurement of extraceUular from a partial unilateral 6-OHDA lesion of the substantia 5-hydroxytryptamine in hippocampus of the anaesnigra: A microdialysis study in freely moving rats. Brain thetized rat using microdialysis: Changes in relation to Res. 450, 209-224. 5-hydroxytryptaminergic neuronal activity. J. NeuroROLLEMA,H., KUHR, W. G., KRANENBORG,G., DE VRIES,J. chem. 53, 234-240. and VAN DEN BERG, C. (1988) MPP+-induced effiux of SHIMADA, N., GRAF, R., ROSNER, G., WAKAYAMA,A., dopamine and lactate from rat striatum have similar time GEORGE, C. P. and HEISS, W.-D. (1989) Ischemic flow courses as shown by in vivo brain dialysis, d. Pharmac. threshold for extracellular glutamate increase in cat exp. Ther. 245, 858-866. cortex. J. Cereb. Blood flow Metab. 9, 603-606. ROSENBERG,P. A. and AIZENMAN,R. (1989) Hundred-fold SmNOH~,A, M., DOLLINGER,B., BROWN,G., RAPOPORT,S. increase in neuronal vulnerability to glutamate toxicity in and SOKOLOFF,L. (1979) Cerebral glucose utilization: astrocyte-poor cultures of rat cerebral cortex. Neurasci. local changes during and after recovery from spreading Lett. 103, 162-168. cortical depression. Science 203, 188-190. ROTtIMAN,S. M. and OLNEY,J. W. (1986) Glutamate and the SHUAm,A., Xu, K., CRAIN,B., SIRE~,A.-L., FEUF.gSTEIN,G., pathophysiology of hypoxic-ischemic brain damage. Ann. HALLENBECK,J. and DAVIS,J. (1990) Assessment of damNeurol. 19, 105-111. age from implantation of microdialysis probes in the rat RotrrLECX3E, C. and MARSDEN,C. A. (1987) Comparison of hippocampus with silver degeneration staining. Neurosci. the effects of selected drugs on the release of hypothalamic Lett., in press. in the brain: role of tortuosity and volume fraction. Brain Res. 169, 580-584. NICHOLSON, C. and KRAm, R. P. (1981) The behavior of extracellular ions during spreading depression. In: The

214

H. BENVENlSTEand P. C. HOTTEMEmR

SLIVKA, A., BRANNAN, T. S., WEINBERGER,J., KNOTT, P. J. and COHEN, G. (1988) Increase in extracellular dopamine in the striatum during cerebral ischernia: A study utilizing cerebral microdialysis. J. Neurochem. 50, 1714-1718. SOLiS, J. M., HERRANZ,A. S., HERRERAS,O., MU~OZ, M. D., MARTIN DEL RiO, R. and LERMA, J. (1986) Variation of potassium ion concentration in the rat hippocampus specifically affects extracellular taurine levels. Neurosci. Lett. 66, 263-268. SOLIS, J. M., HERRANZ, A. S., HERRERAS,O., LERMAN,J. and MARTIN DEL Rio, R. (1988) Does taurine act as an osmoregulatory substance in the rat brain? Neurosci. Lett. 91, 53-58. SPECIALE, C., UNGERSTEDT, U. and SCHWARCZ, R. (1988) Effect of kynurenine loading on quinolinic acid production in the rat: studies in vivo and in vitro. Life Sci. 43, 777-786. STErqSAAS, S. and STENSAAS,L. (1976) The reaction of the cerebral cortex to chronically implanted plastic needles. Acta neuropath. Berl. 35, 187-203. STRECKER, R. E., SHARP, T., BRUNDIN, P., ZETTERSTR(SM,T., UNGERSTEOT, U. and BJORKLUND, A. (1987) Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 22, 169-178. STR(SMBERG,I., HERRERA-MARSCHITZ,M., UNGERSTEDT,U., EBENDAL, T. and OLSON, L. (1985) Chronic implants of chromaffin tissue into the dopamine-denervated striatum. Effects of NGF on graft survival, fiber growth and rotational behavior. Expl Brain Res. 60, 335-349. SZENTISTV~,NYI, I., PATLAK, C. S., ELLIS, R. A. and CSERR, H. F. (1984) Drainage of interstitial fluid from different regions of rat brain. Am. J. Physiol. 246, F835-F844. THOMAS, R. C. (1978) Ion-sensitive lntracellular Microelectrodes. Academic Press: New York. TON, J. M. N. C., GERHARDT,G. A., FRIEDMANN,M., ETGEN, A. M., ROSE, G. M., SHARPLESS,N. S. and GARDNER, E. L. (1988) The effects of A9-tetrahydrocannabinol on potassium-evoked release of dopamine in the rat caudate nucleus: an in vivo electrochemical and in vivo microdialysis study. Brain Res. 451, 59~58. TOSSMAN, U., ERIKSSON, S., DELIN, A., HAGENFELDT, L., LAW, D. and UNGERSTEDT,U. (1983) Brain amino acids measured by intracerebral dialysis in porta-cacal shunted rats. J. Neurochem, 41, 1046-1051. TOSSMAN, U., WIELOCH, T. and UNGERSTEDT, U. (1985) yAminobutyric acid and taurine release in the striatum of the rat during hypoglycemic coma, studied by microdialysis. Neurosci. Lett. 62, 231-235. TOSSMAN, O. and UNGERSTEDT, U. (1985) The effect of apomorphine and pergolide on the potassium-evoked overflow of GABA in rat striatum studied by microdialysis. Eur. J. Pharmac. 123, 295-298. TOSSMAN, U. and UNGERSTEDT, U. (1986) Microdialysis in the study of extracellular levels of amino acids in the rat brain. Acts physiol, scand. 128, 9-14. TOSSMAN, U., JONSSON, G. and UNGERSTEDT, U. (1986a) Regional distribution and extracellular levels of amino acids in rat central nervous system. Acta physiol, scand. 127, 533-545. TOSS.MAN, U., SEGOVIA, J. and UNGERSTEDT, U. (1986b) Extracellular levels of amino acids in striatum and globus pallidus of 6-hydroxydopamine-lesioned rats measured with microdialysis. Acta physiol, scand. 127, 547-531. TOR(SK, T. L. (1989) Neurochemical transmission and the sodium-pump. Prog. Neurobiol. 32, I 1-76. UNGERSTEDT, U. and PYCOCK, C. (1974) Functional correlates of dopamine neurotransmission. Bull. schweiz. Akad. reed. Wiss. 1278, 1-13. UNGERSTEDT, O., HERRERA-MARSCHITZ, M., JUNGNELIUS, U., STAHLE, L., TOSSMAN,U. and ZETTEi~STROM,T. (1982) Dopamine synaptic mechanisms reflected in studies combining behavioural recordings and brain dialysis. In:

Advances in Dopamine Research, pp. 219-231. Ed. M. KOTISAKA. Pergamon Press: New York. UNOERSTEDT, U. (1984) Measurement of neurotransmitter release by intracraniat dialysis. In: Measurement of Neurotransmitter Release In Vivo, pp. 81-105. Ed. C. A. MAgSDEN. Wiley: New York. UNGERSTEDT, U. and HALLSTROM, A. (1987) In vivo microdialysis--A new approach to the analysis of neurotransmitter in the brain. Life Sci. 41, 861-864. VAN WYLEN,D. G. L., PARK, T. S., RUBIO, R. and BERNE, R. M. (1986) Increases in cerebral interstitial fluid adenosine concentration during hypoxia, local potassium infusion, and iscbemia. J. Cereb~ BIood flow Metab. 6, 522-528. VATHY, I. and ETGEN, A. M. (1988) Ovarian steroids and hypothalamic norepinephrine release: Studies using in vivo brain microdialysis. Life Sci. 43, 1493-1499. VEZZANI, A., UNGERSTEDT, U., FRENCH, E. n. and SCrlWARCZ, R. (1985) In vivo brain dialysis of amino acids and simultaneous EEG measurements following intrahippocampal quinolinic acid injection: Evidence for a dissociation between neurochemical changes and seizures. J. Neurochem. 45, 335-344. VEZANNI, A., Wu, H. W., ANGELICO, P., STASI, M. A. and SAMAraS, R. (1988) Quinolic acid-induced seizures, but not nerve cell death, are associated with extracellular Ca 2+ decrease assessed in the hippocampus by brain dialysis. Brain Res. 454, 289-297. VULTO, A. G., SHARP, T., UNGERSTEDT, U. and VERSTEEG, D. H. G. (1988) Rapid postmortem increase in extracellular dopamine in the rat brain as assessed by brain microdialysis. J. Neurochem. 51, 746-749. WADE, J. V., O ~ s , J. P., SAMSON,F. E., NELSON, S. R. and PAZEDERNIK, T. L. (1988) A possible role for taurine in osmoregulation within the brain. J. Neurochem. 51, 740-745. WAGES, S. A., CHURCH, W. H. and JUST1CE,J. B. JR (1986) Sampling considerations for on-line microbore liquid chromatography of brain dialysate. Analyt. Chem. 58, 1649-1655. WESTEmNK, B. H. C. and TUINTE, M. H. J. (1986) Chronic use of intracerebral dialysis for the in vivo measurement of 3,4-dihydroxyphenylethylamine and its metabolite 3,4dihydroxyphenylacetic acid. J. Neurochem. 46, 181-185. WESTERINK,B. H. C. and DE VRIES,J. B. (1988) Characterization of in vivo dopamine release as determined by brain microdialysis after acute and subehronic implantations: Methodological aspects. J. Neurochem. 51, 683~87. WESTERINK, B. H. C. and DE VRIES, J. B. (1989) On the mechanism of neuroleptic induced increase in striatal dopamine release: Brain dialysis provides direct evidence for mediation by autoreceptors localized on nerve terminal. Neurosci. Lett. 99, 197-202. WESTERINK, B. H. C., HOFSTEEDE, R. M., TUNTLER, J. and DE VINES, J. B. (1989a) Use of calcium antagonism for the characterization of drug-evoked dopamine release from the brain of conscious rats determined by microdialysis. J. Neurochem. 52, 722-729. WESTERINK, B. H. C., DAMSMA,G. and DE VRIES,J. B. (1989b) Effect of ouabain applied by intrastriatal microdialysis on the in vivo release of dopamine, acetylcholine, and amino acids in the brain of conscious rats. J. Neurochem 52, 705-712. WmLOCH, T. (1985) Neurochemical correlates to selective neuronal vulnerability. Prog. Brain Res. 63, 69-85. WOOD, P. L., KIM, H. S. and MARIEN, M. R. (1987) Intracerebral dialysis: Direct evidence for the utility of 3-MT measurements as an index of dopamine release. Life Sci. 41, 1-5. WOOD, P. L., KIM, H. S., STOCKLIN, K. and RAo, T. S. (1988) Dynamics of the striatal 3-MT pool in rat and mouse: species differences as assessed by steady-state measurements and intracerebral dialysis. Life Sci. 42, 2275-2281.

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215

YOUNG, A. M. J. and BRADVOP, D, H. F. (1986) Excitatory ZETTEP.STR6M,T., BRUNDIN, P., GAGE, F. H., SHARP, T., ISACSON, O., DU~,~TT, S. B., UNGERffI~T, U. and amino acid neurotransmitters in the corticostriate pathBJGRKLUND, A. (1986) In vivo measurement of sponway: Studies using intracerebral microdialysis in vivo. taneous release and metabolism of dopamine from intraJ. Neurochem. 47, 1399-1404. striatal nigral grafts using intracerebral dialysis. Brain YOUNG, A. M. J., CROWDER,J. M. and BRADFORD,H. F. Res. 362, 344-349. (1988) Potentiation by kainate of excitatory amino acid release in striatum: Complementary in vivo and in vitro ZETTERSTR(~M, T., SHARP, T., COLLIN, A. K. and UNGERSTEDT,U. (1988) In vivo measurement of extraexperiments. J. Neurochem. 50, 337-345. cellular dopamine and DOPAC in rat striatum after ZETTERSTROM,T., VERNET,L., UNGERSTEDT,U., TOSSMAN, various dopamine-releasing drugs; implications for the U., JONZON,B. and FREDHOLM,B. B. (1982) Purine levels origin of extracellular DOPAC. Eur. J. Pharmac. 148, in the intact rat brain. Studies with an implanted perfused 327-334. hollow fibre. Neurosci. Lett. 29, l l l - l l 5 . ZETT~mTR6M, T., SHARP, T., MAr.SDEN, C. A. and ZEUTm~N,T. (1981) The application of ion selective microelectrodes. In" The Application of lon-selective MicroUNGFaSTEDT,U. (1983) In vivo measurement of dopamine electrodes. Ed. T. ZEUTH~N.Elsevier: Amsterdam. and its metabolites by intracerebral dialysis: Changes after d-amphetamine. J. Neurochem. 41, 1769-1773.

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