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Diffusion of radiolabeled dopamine, its metabolites and mannitol in the rat striatum studied by dual-probe microdialysis
L. E Agnati: K. Fuxe, C. Nicholson and E. Sykov~i (Eds.) Progress in Brain Research, Vol 125 © 2000 Elsevier Science BV. All rights reserved.
CHAPTER
7
Diffusion of radiolabeled dopamine, its metabolites and mannitol in the rat striatum studied by dual-probe microdialysis Jan Kehr*, Malin H6istad, and Kjell Fuxe Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden
Introduction
One of the often studied aspects of volume transmission (VT) involves its spatio-temporal characterization described as a long-distance and a long-lasting spread of chemical signals within the brain microenvironment (Agnati et al., 1986; Fuxe and Agnati, 1991). A number of morphological and physiological techniques have shown that neurotransmitters, traditionally believed to act only within the limited space of a synaptic cleft, can diffuse over distances exceeding the volume of the synaptic space. Thus, immunocytochemical analysis at both light and electron microscopic levels has revealed differences between neuroanatomical positions of release sites and their respective receptors or transporter proteins, as well as, the existence of extra-synaptic release and release from non-junctional varicosities of transmitters such as monoamines or acetylcholine and neuromodulators such as neuropeptides or adenosine (for review, see Fuxe and Agnati, 1991; Descarries and Umbriaco, 1995; Zoli et al., 1998; 1999; Vizi and Kiss, 1998). Similarly, using neurotransplantation techniques it was shown that neurotransmitters such as dopamine (DA) can diffuse over long distances out from the grafted tissue (Str6mberg et al., 1984). *Corresponding author. Tel.: + 46 8 728 7084; Fax: +46 8 30 28 75; e-mail: [email protected]
Another approach to study VT is based on the use of invasive techniques which allow continuous in vitro or in vivo monitoring of substances involved in chemical neuronal signaling and cellular metabolism (for review, see Kehr, 1999). The principle of such measurements is the same for all types of sensing or sampling devices: a sensor/ probe is stereotaxically implanted into the brain parenchyma, fixed and left to stabilize for a certain period of time (hours to days). Once the steadystate conditions are achieved, the device can generate physical (electrical, optical) signals which reflect the extracellular concentrations of a given analyte, first at 'basal' conditions, followed by a period of pharmacological or physiological stimulation. A major advantage of using the invasive techniques to study VT is a possibility to elucidate mechanisms regulating the properties of the extracellular space in health and disease and the role of extracellular molecular transport for design of novel drug therapies. Microdialysis (Ungerstedt, 1984) has been proven to serve as an efficient tool in studies of brain circuitry and mechanisms of drug actions in vivo. Several mathematical models of diffusion and empirical methods were proposed for estimation of absolute concentrations of extracellular substances (for review, see Kehr, 1993). The principle of microdialysis sampling is based on the diffusion of substances across the concentration gradient in the
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extracellular space and as such, the technique indirectly implies the concept of VT. In spite of that fact, microdialysis has only sparsely been applied in the studies of VT, as compared to the techniques based directly on sensing devices such as voltammetry on carbon fiber electrodes or potentiometry using the ion-selective microelectrodes. A relatively large surface of the microdialysis probe makes it difficult to combine microdialysis with pressure-ejection technique, which is often used with the other two methods as a tool to administer diffusable substances. On the other hand, microdialysis offers a unique possibility to measure several compounds at a time using analytical techniques, such as high-performance liquid chromatography (HPLC), radioimmunoassay or mass spectrometry. Today, microdialysis is probably the most efficient in vivo technique to study brain chemical signaling and to correlate neurochemical analysis with behavioral methods (Zetterstr6m et al., 1986; Young, 1993; Ogren et al., 1996).
Concentration gradients within the extracellular space The extracellular space (ECS) under normal circumstances comprises about 20% of the total brain tissue volume. The extracellular fluid (ECF) contains ions, the concentrations of which are very similar to those found in the cerebrospinal fluid (CSF), as well as a number of long-chain glycosaminoglycans, proteoglycans and glycoproteins tethered to membranes. Neurotransmitters and neuromodulators and also neurotrophic factors and other cytokines in glial and neuronal cells, once released into the ECE all have to traverse the extracellular space on their way to the target receptors. However, the extracellular concentrations of these chemical messengers are far away from being homogeneously distributed within the ECS compartment. For example, for neurotransmitters such as DA, there is a steep gradient of concentrations being up to 6-7 orders of magnitude higher at a release site (a synaptic cleft) than in the outer interstitial space. It was estimated that striatal DA vesicles with a radius of 25 nm contain 25 mM DA, while the concentration of DA in the synaptic cleft, which is about 15 nm wide and 300 nm long,
was around 1.6 mM (Garris et al., 1994). However, the actual extracellular DA concentrations measured by in vivo voltammetry using a thin (5 txm O.D.) carbon fiber electrode was not higher than 20 nM in pargyline-treated rats (Gonon and Buda, 1985) or 0.1-2 txM during electrical stimulation (Kawagoe et al., 1992). Using the devices of even larger diameter such as a 200 txm O.D. microdialysis probe the calculated basal extracellular DA concentration was as low as 5 nM (Parsons and Justice, 1992). Thus, implantable sensors can measure only an 'echo' of the actual release event, whether defined as a spill-over from the synaptic cleft (a leaking synapse) or as a release from a nonjunctional bouton, varicosity or as extra-synaptic release. All these definitions are based on measurements of extracellular chemical signals by invasive means and have a common prerequisite: the overflow of the substance diffusing to the sensing device must be higher than its reuptake, sequestration, clearance to blood or CSF, and enzymatic or other inactivation mechanisms.
Invasive techniques for monitoring volume transmission: role of microdialysis The spatial and temporal resolution of any implantable monitoring device is dependent both on its geometry and the applied detection principle. The invasive techniques can be divided into two main groups: (1) intracorporeal biosensors providing immediate measures of a given analyte and (2) continuous sampling devices generating samples in a form of extracts/perfusates which are further analyzed by an appropriate analytical technique. The response time can be as low as 0.1-1 s for directly detecting voltammetric electrodes, while a relatively laborious and time-consuming analysis of neurotransmitters or neuropeptides sampled by microdialysis requires the fractions to be collected in 5-30 min intervals. The antibody-coated microprobes as described by Duggan and Hendry, 1986, provide excellent spatial resolution but only one time point of about 30 min. The sensing devices and related detection techniques within the first group are highly selective towards one analyte, which is detected directly at the surface of the biosensor implanted into the
181 brain tissue. In the simplest case, the selectivity for a detected endogenous substance is assured by its intrinsic chemical properties such as a relatively specific oxidation potential for DA. A more common way is to utilize the highly specific molecular interactions such as those between ions-ionophores, enzymes-substrates and antibodiesantigens, which all lead, either directly or via an intermediate product, to the changes in physical (electrical or optical) signals. Contrary to most of the biosensors, the implantable sampling devices allow relatively easy intracerebral monitoring of conscious freely moving animals. The sampling device, for example a microdialysis probe is continuously perfused at a low flow-rate (0.1-2 Ixl/min) with a physiological solution, typically a Ringer solution or an artificial cerebrospinal fluid (aCSF). In vivo microdialysis on awake animals eliminates the negative effects of general anesthetics on chemical neurotransmission and cell metabolism. A complete recovery of physiological functions occurs at about 5-7 days after the surgery as revealed by telemetry (Drijfhout et al., 1995). A very useful way to study functional neuroanatomy and the role of specific brain circuits in various behaviors is the so-called dual-probe. Here, one probe is implanted at the cell body level, whereas the second probe is implanted in the terminal area. The first probe is used for chemical stimulation by infusing the drugs while the second probe is used to measure the neurotransmitter release (for review, see Westerink et al., 1998). Here, we describe a newly developed method for studies of long-distance diffusion in the extracellular space. The technique combines dual-probe microdialysis sampling which allows simultaneous infusion and recovery of labeled molecules. The collected samples are separated by HPLC and individual fractions measured by a liquid scintillation counter. Inert markers such as 3H-mannitol can be used to study direct changes of extracellular volume characteristics. Endogenous compounds (e.g. 3H-DA) provide measures of brain permeability, as well as on the rate of cellular uptake and metabolism. As already mentioned in the Introduction, microdialysis sampling builds on the ability of molecules
to diffuse through the ECS; the length of the diffusion path for various molecules has been a matter of intensive research. For example, using voltammetric techniques it was calculated that the half-life and diffusion distance of stimulated DA release from its release site is only 25 ms and 7-10 p~m respectively (for review, see Gonon et al., this book). On the contrary, long distance DA diffusion paths were proposed on the basis of immunohistochemical (Fos-like), electrophysiological and microdialysis studies conducted in unilaterally DA denervated rats, following e.g. d-amphetamine treatment (Bjelke et al., 1994; Schneider et al., 1994). A possibility of longdistance diffusion arising from non-stimulated DA in the rat striatum could be elegantly investigated by use of a dual-probe microdialysis approach as schematically depicted in Fig. 1. Briefly, two microdialysis probes ( C M A / l l , cuprophane membrane: cut-off 6000 Da, length 4 mm and 240 Ixm O.D.) were implanted stereotaxically into the lateral and medial striatum (Paxinos and Watson, 1986) of the halothane anaesthetized 2 ~tl/min • 3H-DA + DA 3H-DOPAC + DOPAC 3H-HVA + HVA
Rin.e^3H-DAi
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Fig. 1. Principal schemeof a dual-probemicrodialysisused for in vivo studies of long-distance diffusion and metabolism of radiolabeled molecules. The first (lateral) microdialysisprobe was used for the infusionof radioactivelabel (e.g. 3H-DA or 3Hmannitol), whereas the second probe, implanted 1 mm apart, recovered all the low-molecular weight substances present in the extracellular fluid including the diffused 3H-label or its metabolites. The samples were separated by microbore LCEC and fractionscorrespondingto the peaks of DA and metabolites were collected and measured in a liquid scintillationcounter.
182 rat. The distance between the membrane centers was 1 mm. The lateral probe was used for the infusion of a radioactive label: 3H-DA or D-3Hmannitol diluted in Ringer solution, at a flow-rate of 2 txl/min. The medial probe, perfused with Ringer solution at the same flow-rate was used for recovery of endogenous (cold) DA and its metabolites, as well as the tritiated labels. Samples were collected at 30-min intervals. Since 3H-mannitol was used as an inert marker, which did not metabolize during its passage through the brain, its radioactivity could be measured directly by use of a liquid scintillation counter. On the contrary, infusion of 3H-DA caused its immediate uptake and enzymatic cleavage to its respective acidic metabolites 3H-DOPAC and 3H-HVA. Thus, measuring the total tritium in samples of 3H-DA infused animals could provide an in vivo index of: (a) DA metabolism, (b) clearance by cerebral blood vessels and (c) uptake into the nerve terminals. To investigate the partition of radioactivity between individual substances, the microdialysis samples were first separated by microbore column liquid chromatography with electrochemical detection (LCEC), as described elsewhere (Kehr, 1999). Fractions corresponding to the individual peaks of DA and its metabolites were collected and measured by a scintillation counter. The clearance of tritiated labels by cerebral microcirculation can be studied by measuring the radioactivity in the blood samples. However, in our experiments we could not detect any radioactivity in the blood collected from the tail vein or intracardially, most likely due to a massive dilution of 'brain-derived' blood at this sampling site. Sampling the blood directly from the jugular vein and/or increasing the radioactivity of the infused label could possibly provide detectable levels. Uptake of 3H-DA to the dopaminergic terminals and the overall distribution of radioactivity within the brain tissue can be studied by using autoradiography of respective brain slices. In fact, the concept of tissue pre-loading, well known from in vitro superfusion techniques, was already demonstrated in the first report on dopamine microdialysis by Ungerstedt and Pycock in 1974. A single probe was used to load 3H-DA into the dopaminergic terminals and following a washout period, the effect of amphetamine on the release of
DA-related radioactivity could be observed. Here, the density of 3H-DA labeling is evaluated histologically for the first time. The diffusion profile of 3H-DA infused via the microdialysis probe in the rat striatum and the analysis of relative tritium density as a function of distance from the probe is shown in Fig. 2. As seen, following 5 hours of continuous infusion, the radioactivity profile shows a steep decay within the first 500 Ixm and being less than 5% of the initial level at a distance of 1 mm, i.e. at the site of the second (detector) probe (not seen in the phospho-imager print of the 10 txm section). Interestingly, the infusion of 3H-mannitol at similar tritium activity concentrations and experimental conditions as for 3H-DA resulted in an almost undetectable radioactive trace of mannitol following 5 days' exposure of the brain slice in a phospho-imager (data not shown). Omitting the brain fixation procedure by intracardial perfusion did not increase the tissue radioactivity of infused 3H-mannitol. This led to the assumption that infused 3H-mannitol is a truly extracellular marker of brain microenvironment being rapidly cleared by the cerebral microcirculation lacking any significant incorporation to the intracellular compartments. 3H-mannitol as a marker of extraceilular space
The usefulness of dual-probe microdialysis method to study diffusion of neurotransmitters within the ECS was initially evaluated by use of the inert lowmolecular weight markers such as 3H-mannitol. Such labels should exhibit fast kinetics of diffusion and equilibration within a typical time frame of an acute microdialysis experiment (6-8 hours including the initial stabilization phase of 90 min). Indeed, as shown in Fig. 3, the recovery of 3Hmannitol (1.5 IxM; specific activity 736.3 GBq/mmol, NEN, USA) reached the steady state within 90 min after start of the infusion, with a corresponding half-time (ts0~,) of 42_+ 1.6 min (mean ___SD, n = 6). From the total 3H-mannitol activity of 820 000+_ 13 000 dpm/10 p~l only about 3% was delivered (lost) into the brain tissue. The curves were calculated using linear regression analysis or Boltzmann sigmoidal nonlinear regression algo-
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rithm (Prism TM, GraphPad Software, according to the following formula: 1 + exp[(ts0~ -
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-60-30 0 30 60 90 120150180210240270300 time (min) Fig. 3. Diffusion profiles of 3H-mannitol in vivo (rat striatum) and in vitro (Ringer solution) using the dual-probe microdialysis. Under in vivo conditions, the steady state was achieved within 90 min after start of the infusion, as demonstrated by the regression curve and the calculated time required to reach half of the steady state (ts0~) which was 42 ± 1.6 min. This indicates the presence of an active clearance processes of 3H-mannitol in the ECE No such equilibrium was seen in a quiescent Ringer solution. Values expressed as percentage of maximal recovered 3H-mannitol concentration at steady state (max Co,t), means ± S.E.M., n = 6 animals.
184 corresponding dual-probe in vitro experiment in Ringer solution at + 24°C and 2 pA/min showed a constant linear increase of recovered radioactivity, indicating non-saturable conditions during the study period (5 hours). The fast equilibrium of mannitol diffusion in the brain together with low background radioactivity suggest that the clearance by blood brain capillaries is the major inactivating mechanism for this molecule. This situation is schematically depicted in Fig. 4. The diffusion curve of 3H-mannitol in 6-hydroxydopamine (6-OHDA) lesioned rats 5 weeks after the unilateral lesions was much faster showing a significantly shorter half-time t50% (33_+ 1 min, P < 0.03) and a significant change in curve steepness (rate constant kss 6_+ 2 min, P < 0 . 0 5 ) vs. the control group. The maximal outflow levels at steady state conditions were about the same in both groups (3600 dpm/10 ixl). This is in agreement with histochemical data, showing that the disappearance of dopaminergic terminals has only moderate effects on gliosis as evaluated by GFAP immunostaining at this time-interval (Str6mberg et al., 1986). Our data on facilitated diffusion of 3H-mannitol in DA denervated striatum of a rat suggests that the
neurodegeneration of the dopaminergic system, seen in Parkinson's disease may be associated with a change of extracellular volume fraction and/or a reduction in tortuosity of the basal ganglia. It can be concluded that under given experimental conditions the kinetic profile of recovered 3H-mannitol is dependent only on the volume fraction/tortuosity characteristics of the ECS and with no significant changes in the brain-blood clearance. Finally, the dual-probe microdialysis using 3H-mannitol or a similar marker could be an interesting complement to the existing electrochemical methods used for the studies of volume fraction and tortuosity of the brain extracellular environment (for review, see Nicholson and Sykov~i, 1998; this volume).
Diffusion and metabolism of 3H-dopamine Infusion of 3H-DA (specific activity 2187.1 GBq/ mmol, NEN, USA) at a concentration of 500 nM (corresponds to the mean value of 710 0 0 0 _ 20 000 dpm/10 Ixl) through the microdialysis probe resulted in an average delivery of about 6% which corresponds to in vivo delivery of 30 nM DA. This is only 5-6 times higher than the basal extracellular DA levels estimated by zero-flow and no-net-flux
Fig. 4. A scheme of the diffusion path of 3H-mannitol, in the rat striatum. At steady state, 3H-mannitoldiffusing between the two probes, was distributed only within the ECS compartmentand cleared by cerebral microvesselcircuitry.Signifcantlyfaster diffusion of 3H-mannitolwas observed in rats with loss of DA terminals induced by 6-OHDA injections into the substantia nigra. Thus, 3nmannitol may serve as an inert marker of extracellularvolume fraction and tortuosity.
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The initial hypothesis of long-distance diffusion of striatal DA at basal conditions was not confirmed in this experimental model. The levels of recovered 3H-DA peak at steady-state were only slightly elevated to 45.2_+1.4 dpm/101xl (mean _+SEM, range 42-48 dpm/10 txl) over the threshold values which were 31.3_+1.3 dpm/10txl (range 21-41 dpm/10 Ixl). On the contrary, the basal levels of endogenous (cold) DA in control animals were easily detectable with a mean value of 6.2 nM in 10 txl injected onto the HPLC column. Neither, could 3H-DA be detected in 6-OHDA lesioned animals where the intraneuronal pools of DA terminal monoamine oxidase (MAO) and the dopamine uptake transporter were reduced to a minimum due to an almost complete disappearance of DA-ergic striatal terminals. Here, the counts in fractions of DA peaks were in a similar range as in the non-lesioned animals, i.e. about 35 and 43 dpm/ 10 I~1 in pre-infused and 3H-DA infused periods. Surprisingly, the overall recovered 3H-DA-derived radioactivity in the lesioned group was significantly reduced to 53% of the control group, from 4919_+530 dpm/10 ixl (n=6) to 2609_+205 dpm/ 10txl (n=5) respectively. The distribution of DOPAC, HVA and the unidentified front peak in control and 6-OHDA lesioned groups is schematically depicted in Fig. 6A. The absolute level of radioactivity in the front peak was unaffected by
microdialysis techniques (Parsons and Justice, 1992). In addition, this concentration of DA should still lie within the physiological range of stimulated DA release and as such should not affect the normal function of DA release, autoreceptor feedback and uptake machinery. Under these conditions, the total tritium radioactivity recovered by the second, detector probe was only about 5000 dpm/10 txl in control animals, which means that only 12.5% of delivered 3H-DA was recovered by the second probe. However, this value is not corrected for the in vivo recovery of the second microdialysis probe. Calculating the ts0~ and k~s parameters for the total recovered radioactivity gave the average values of 100_+4 min and 64_+3 min, respectively, i.e. the levels are about twice higher than those obtained with 3H-mannitol. Separation of microdialysis samples by microbore LCEC and subsequent counting of fractions representing DA, DOPAC, HVA and an unidentified compound eluted in the front peak revealed that DOPAC was the fastest diffusing compound (ts0% 85 _+4 min, kss 20 _+1 min; means + S.E.M., n = 6) and contained the largest portion of radioactivity (1634 _+234 dpm/peak) followed by HVA (1343 _+159 dpm/peak) and the unidentified front peak (850_+ 115 dpm/peak), as shown in Fig. 5. Again, a relatively fast steady state could be achieved for all the substances within 120-180 min. 2500-
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186
the 6-OHDA lesion, similar to the case of the inert marker 3H-mannitol as discussed above. These data indicate that the in vivo delivery and recovery of
the two probes was not affected by the 6-OHDA lesion. However, the steady-state levels of metabolites 3H-DOPAC and 3H-HVA were significantly
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Fig. 6. (A) Maximal levels (Cm~x)of tritiated molecules recovered by a second microdialysis probe at steady state in control (n = 6) and 6-OHDA lesioned (n = 5) rats. (B) Calculated half-time values (ts0~) of tritiated DA metabolites and 3H-mannitol in control and 6-OHDA lesioned rats. (C) Calculated diffusion slope values (ts0~) of tritiated DA metabolites and 3H-mannitol in control and 6-OHDA lesioned rats. Statistical analysis was done by Studentg t-test, comparing control vs. lesioned groups, values are expressed as means_+ S.E.M., *: P < 0.05; **: P<0.001; ***: P<0.0003.
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reduced in the 6-OHDA lesioned animals, with maximal reduction for DOPAC down to 14% of control, followed by HVA (28% of controls). This indicates that the striatal MAO activity which is localized predominantly in the dopaminergic terminals (Oreland, 1991) was considerably reduced as a consequence of the 6-OHDA lesion. On the other hand, the slightly less pronounced reduction of HVA suggests that this reduction was caused mostly as a consequence of a diminished pool of DOPAC rather than from reduced activity of extracellular COMT. The calculated kinetic data t50~ and kss for 3HDOPAC, 3H-HVA, the unidentified 3H-DA-derived front peak and 3H-mannitol in control and 6-OHDA lesioned groups are shown in Fig. 6B, C. As seen in Fig. 6B the diffusion half times were significantly reduced by about 40% for all 3H-DArelated peaks, whereas for 3H-mannitol, the corresponding reduction was only 23%. It is notable that 3H-mannitol is cleared much faster under normal conditions than any of the 3H-DArelated compounds in the 6-OHDA lesioned animals. Similarly, the diffusion curves were steeper in the lesioned animals as expressed
mathematically by calculating the diffusion rate kss values using the Boltzmanng non-linear regression (Fig. 6C). Here, again the kss levels were about 40% lower in the 6-OHDA lesioned group, although only the values for 3H-mannitol (see discussion to Figs. 3, 4) and 3H-HVA were significantly different (controls: 25.7_+2.5 min, n=5, 6-OHDA: 16.1_+2.6 min, n=6, P<0.05). A relatively large variation of the mean levels could be explained by poor temporal resolution, i.e. too long (30 min) sampling intervals. Interestingly, there was no difference in maximal radioactivity counts between the control and lesioned animals for 3H-mannitol and the 3H-front peak, whereas 3H-DOPAC and 3HHVA levels were strongly reduced in the lesioned group (see Fig. 6A). This suggests that in the lesioned animals, infused 3H-DA was, in spite of the diminished MAO and DA reuptake activity, unable to diffuse over the longer distances, most likely due to the activation of compensatory mechanisms or the increased brain-blood clearance. Also, it should be considered that 3H-DA itself may exhibit an increased clearance over the brain-blood barrier after the DA denervation. Limited amounts of data are available on the effects
188
of 6-OHDA lesions on the cerebral blood flow (CBF) and on the cerebromicrovascular permeability. Earlier studies have shown a two fold increase of CBF in DA denervated rats following stimulation with apomorphine (Ingvar et al., 1983) probably as a consequence of dopamine receptor supersensitivity. The existence of both D1 and D2 receptors was demonstrated on rat cerebral blood vessels (Amenta et al., 1991). These receptors mediate DA-induced vasodilatation associated with increases in CBF (Sharkey and McCulloch, 1986). In agreement with these data, a reduction of basal CBF was observed in DA denervated caudateputamen (Mraovitch et al., 1993). Recently, it was reported that DA may act as a vasoconstrictor on cortical microvessels (Krimer et al., 1998). However, this effect is probably mediated via agonist activity of DA at adrenergic and serotonergic receptors as discussed by Iadecola, 1998. Also DA terminals contact cerebral endothelial cells indicating a modulation of brain-blood clearance processes of extracellular chemical signals through DA receptors (Krimer et al., 1998). In view of these reports, we hypothesize that the infusion of 3H-DA into the DA denervated rat striatum may cause an immediate increase of local
CBF and capillary permeability augmenting the clearance processes of extacellular 3H-DA and its metabolites as manifested by their reduced extracellular levels and the inability to measure the long-distance diffusion of 3H-DA. Infusion of 3Hmannitol, on the other hand, may not affect CBF nor capillary permeability and consequently no differences at steady state were observed in control and lesioned animals. These results provide new insights into DA VT in the DA denervated striatum and thus in the striatum of Parkinson~ disease patients. D u a l - p r o b e m i c r o d i a l y s i s - a n e w tool for diffusion studies
A scheme shown in Fig. 7 summarizes the proposed diffusion path and metabolism of 3H-DA as studied by the dual-probe microdialysis technique. In control non-lesioned rats, infused 3H-DA is removed from the ECS compartment by three major mechanisms: (a) uptake into the DA-ergic terminals, (b) brain-blood clearance, and (c) enzymatic cleavage by extracellular COMT and mainly DA terminal MAO. Hence, the appearance of
Fig. 7. Scheme of the diffusion path of 3H-DA and formation of its metabolites 3H-DOPAC and 3H-HVA in control rat striatum. At steady state, 3H-DA was cleared by blood capillaries, uptake into the dopaminergic terminals and metabolism by both intracellular and extracellular enzymes. In DA denervated animals, the formation of 3H-DOPAC and 3H-HVA was strongly attenuated due to the absence of DA terminals being the major source of MAO activity for DA metabolism. Consequently, the portion of recovered 3H-HVA formed through extracellular COMT was now higher than 3H-DOPAC (see Fig. 6A).
189 3H-DOPAC and 3H-HVA metabolites and ultimately of 3H-DA in the perfusates sampled by the second microdialysis probe should reflect the ratio between these three factors. This combined metabolic-diffusion process is much slower than the diffusion of a labeled inert marker shown in Fig. 4, as evidenced by significantly longer diffusion half times of 3H-DOPAC and 3H-HVA compared to 3Hmannitol. It is suggested that for the DA metabolites there exist in addition to the diffusion characteristics through the ECS compartment also contributions of trans-cellular transport involving, e.g. the brain-blood barrier and the activity of the corresponding metabolizing enzymes. Removal of the DA terminals by lesions with 6-OHDA resulted in a dramatic reduction of the formation of 3H-DA metabolites, but only marginally affected their diffusion speed with no detectable facilitation of 3H-DA diffusion. On the contrary, the inert marker 3H-mannitol diffused significantly faster in the lesioned animals than in the controls. In summary, the dual-probe microdialysis approach shows a clear difference in diffusion properties of the neutral molecules used only as markers of ECS and those endogenously synthesized or metabolized by the brain tissue. In a pathological state, the marker molecules seem to mirror only the 'geometrical' changes of the brain microenvironment (tortuosity, volume fraction), whereas diffusion of the endogenously-derived molecules is affected by all the compensatory mechanisms occurring in the damaged tissue including the brain-blood barrier and cerebral blood flow.
Conclusions The intracerebral dual-probe microdialysis technique allows the study of diffusion of labeled molecules in vivo in anaesthetized or awake animals. The labeled markers can be biologically inert molecules or endogenous substances delivered and sampled without any volume changes or other disturbances to brain homeostasis. Diffusion kinetics and steady-state levels of DA metabolites could be studied over long (> 1 mm) inter-probe distances, whereas studies of DA diffusion should be conducted at shorter ( - 0.1 mm) distances. The dual-probe microdialysis technique
can provide an in vivo index of a complex interplay of clearance and metabolic processes of extracellular labels at basal, non-stimulated conditions and as such, the technique can be a complement to the existing voltammetric and potentiometric methods. Finally, the dual-probe technique can be used to study molecular transport within the brain microenvironment in various neuropathological models of brain diseases or following pharmacological stimuli, helping to understand the role of VT at such conditions.
References Amenta, E, Ricci, A. and Vega, J.A. (1991)Autoradiographic localization of dopamine receptors in rat cerebral blood vessels. Eur.J. Pharmacol., 192: 123-132. Agnati, L.E, Fuxe, K., Zoli, M., Zini, I., Toffano, G. and Ferraguti, A. (1986) A correlation analysis of the regional distribution of central enkephalin and beta-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. Acta Physiol. Scand., 128: 201-207. Bjelke, B., Strrmberg, I., O'Connor W.T., Andbjer, B., Agnati, L.E and Fuxe, K. (1994) Evidence for volume transmission in the dopamine denervated neostriatum of the rat after a unilateral nigral 6-OHDA microinjection. Studies with systemic d-amphetamine treatment. Brain Res., 662:11-24. Descarries, L., Beaudet, A. and Watkins, K.C. (1975) Serotonin nerve terminals in adult rat neocortex. Brain Res., 100: 563-588. Descarries, L. and Umbriaco, D. (1995) Ultrastructural basis of monoamine and acetylcholine function in CNS. Sere. Neurosei., 7: 309-318. Drijfhout, W.J., Kemper, R.H., Meerlo, P., Koolhaas, J.M., Grol, CJ. and Westerink, B.H. (1995) A telemetry study on the chronic effects of microdialysis probe implantation on the activity pattern and temperature rhythm of the rat. J. Neurosci. Meth., 61: 191-196. Duggan, A.W. and Hendry, I.A. (1986) Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody coated microelectrodes. Neurosci. Lett., 68: 134-140. Fuxe, K. and Agnati, L.F. (1991) Two principal modes of electro-chemical communicationin the brain: volume versus wiring transmission. In: K. Fuxe and L.F. Agnati (Eds), Volume Transmission in the Brain. Novel Mechanisms for Neural Transmission. Advances in Neuroscience, Vol. 1,
Raven Press, New York, pp. 1-11. Garris, P.A., Ciolkowski, E.L., Pastore, P. and Wightman, R.M. (1994) Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain. J. Neurosci., 14: 6084-6093.
190 Gonon, E and Buda, M. (1985) Regulation of dopamine release by impulse flow and by autoreceptors as studied by in vivo voltammetry in the rat striatum. Neuroscience, 14: 765-774. Iadecola, C. (1998) Neurogenic control of the cerebral microcirculation: is dopamine minding the store? Nature, 1: 263-265. Ingvar, M., Lindvall, O. and Stenevi, U. (1983) Apomorphineinduced changes in local cerebral blood flow in normal rats and after lesions of the dopaminergic nigrostriatal bundle. Brain Res., 262: 259-265. Jansson, A., O'Connor, W.T., StrOmberg, I., Ftrander, P., Rimondini, R., Zoli, M., Agnati, L.E and Fuxe, K. (1999) Actions of cerebroventricular dopamine injections on the unilaterally dopamine denervated striatum. Focus on periventricular striatum. Manuscript. Kawagoe, K.T., Garris, P.A. and Wiedemann, D.J. and Wightman, R.M. (1992) Regulation of transient dopamine concentration gradients in the microenvironment surrounding nerve terminals in the rat striatum. Neuroscience, 51: 55-64. Kehr, J. (1993) A survey on quantitative microdialysis: theoretical models and practical implications. J. Neurosci. Meth., 48: 251-261. Kehr, J. (1999) Monitoring chemistry of brain microenvironment: biosensors, microdialysis and related techniques. In: U. Windhorst and H. Johansson (Eds), Modern Techniques in Neuroscience Research. Springer-Verlag, Heidelberg, pp. 1149-1198.
Krimer, L.S., Mully, III, E.C., Williams, G.V. and GoldmanRakic, P. (1998) Dopaminergic regulation of cerebral cortical microcirculation. Nature, 1: 286-289. Mraovitch, S., Calando, Y., Onteniente, B., Peschanski, M. and Seylaz, J. (1993) Cerebrovascular and metabolic uncoupling in the caudate-putamen following unilateral lesion of the mesencephalic dopaminergic neurons in the rat. Neurosci. Lett., 157: 140-144. Nicholson, C. and Sykov~i, E. (1998) Extracellular space structure revealed by diffusion analysis. Trends Neurosci., 21: 207-215. Ogren, S.O., Kehr, J. and Schttt, P.A. (1996) Effects of ventral hippocampal galanin on spatial learning and on in vivo acetylcholine release in the rat. Neuroscience, 75: 1127-1140.
Oreland, L. (1991) Monoamine oxidase, dopamine and Parkinsong disease. Acta Neurol. Scand., 84: 60-65. Parsons, L.H. and Justice, J.B., Jr. (1992) Extracellular concentration and in vivo recovery of dopamine in the nucleus accumbens using microdialysis. J. Neurochem., 58: 212-218.
Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Schneider, J.S., Rothblat, D.S. and DiStefano, L. (1994) Volume transmission of dopamine over large distances may contribute to recovery from experimental Parkinsonism. Brain Res., 643: 86-91. Sharkey, J. and McCulloch, J. (1986) In: C. Owman and J.E. Hardebo (Eds), Neuronal Regulation of Brain Circulation, Elsevier, New York, pp. 111-127. Strtmberg, I., Bjtrklund, H., Dahl, D., Jonsson, G., Sundstrtm, E. and Olson, L. (1986) Astrocytic responses to dopaminergic denervations by 6-hydroxydopamine and 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine as evidenced by glial fibrillary acidic protein immunohistochemistry. Brain Res. BulL, 17: 225-236. Strtmberg, I., Herrera-Marschitz, M., Hultgren, L., Ungerstedt, U. and Olson L. (1984) Adrenal medullary implants .in the dopamine denervated striatum. I. Acute catecholamine levels in grafts and host caudate as determined by HPLCelectrochemistry and fluorescence histochemical image analysis. Brain Res., 297: 41-51. Ungerstedt, U. (1984) Measurement of neurotransmitter release by intracranial dialysis. In: C.A. Marsden (Ed.), Measurement of Neurotransmitter Release In Vivo. Methods in Neurosciences, Vol. 6. Wiley, New York, pp. 81-105. Ungerstedt, U. and Pycock, C. (1974) Functional correlates of dopamine neurotransmission. Bull. Schweiz. Akad. Med. Wis., 30: 44-55. Vizi, E.S. and Kiss, J.P. (1998) Neurochemistry and pharmacology of the major hippocampal transmitter systems: synaptic and non-synaptic interactions. Hippocampus, 8: 566-607. Westerink, B.H., Drijfhout, W.J., van Galen, M., Kawahara, Y. and Kawahara, H. (1998) The use of dual-probe microdialysis for the study of catecholamine release in the brain and pineal gland. Adv. Pharmacol., 42: 136-140. Young, A.M. (1993) Intracerebral microdialysis in the study of physiology and behaviour. Rev. Neurosci., 4: 373-395. Zetterstrtm, T., Herrera-Marschitz, M. and Ungerstedt, U. (1986) Simultaneous measurement of dopamine release and rotational behaviour in 6-hydroxydopamine denervated rats using intracerebral dialysis. Brain Res., 376: 1-7. Zoli, M., Torri, C., Ferrari, R., Jansson, A., Zini, I., Fuxe, K. and Agnati, L.F. (1998) The emergence of the volume transmission concept. Brain Res. Rev., 26: 136-147. Zoli, M., Jansson, A., Sykov~i, E., Agnati, L.F. and Fuxe, K. (1999) Volume transmission in the brain and its relevance for neuropsychopharmacology. Trends Pharmacol. Sci., 20: 142-150.
CHAPTER
7
Diffusion of radiolabeled dopamine, its metabolites and mannitol in the rat striatum studied by dual-probe microdialysis Jan Kehr*, Malin H6istad, and Kjell Fuxe Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden
Introduction
One of the often studied aspects of volume transmission (VT) involves its spatio-temporal characterization described as a long-distance and a long-lasting spread of chemical signals within the brain microenvironment (Agnati et al., 1986; Fuxe and Agnati, 1991). A number of morphological and physiological techniques have shown that neurotransmitters, traditionally believed to act only within the limited space of a synaptic cleft, can diffuse over distances exceeding the volume of the synaptic space. Thus, immunocytochemical analysis at both light and electron microscopic levels has revealed differences between neuroanatomical positions of release sites and their respective receptors or transporter proteins, as well as, the existence of extra-synaptic release and release from non-junctional varicosities of transmitters such as monoamines or acetylcholine and neuromodulators such as neuropeptides or adenosine (for review, see Fuxe and Agnati, 1991; Descarries and Umbriaco, 1995; Zoli et al., 1998; 1999; Vizi and Kiss, 1998). Similarly, using neurotransplantation techniques it was shown that neurotransmitters such as dopamine (DA) can diffuse over long distances out from the grafted tissue (Str6mberg et al., 1984). *Corresponding author. Tel.: + 46 8 728 7084; Fax: +46 8 30 28 75; e-mail: [email protected]
Another approach to study VT is based on the use of invasive techniques which allow continuous in vitro or in vivo monitoring of substances involved in chemical neuronal signaling and cellular metabolism (for review, see Kehr, 1999). The principle of such measurements is the same for all types of sensing or sampling devices: a sensor/ probe is stereotaxically implanted into the brain parenchyma, fixed and left to stabilize for a certain period of time (hours to days). Once the steadystate conditions are achieved, the device can generate physical (electrical, optical) signals which reflect the extracellular concentrations of a given analyte, first at 'basal' conditions, followed by a period of pharmacological or physiological stimulation. A major advantage of using the invasive techniques to study VT is a possibility to elucidate mechanisms regulating the properties of the extracellular space in health and disease and the role of extracellular molecular transport for design of novel drug therapies. Microdialysis (Ungerstedt, 1984) has been proven to serve as an efficient tool in studies of brain circuitry and mechanisms of drug actions in vivo. Several mathematical models of diffusion and empirical methods were proposed for estimation of absolute concentrations of extracellular substances (for review, see Kehr, 1993). The principle of microdialysis sampling is based on the diffusion of substances across the concentration gradient in the
180
extracellular space and as such, the technique indirectly implies the concept of VT. In spite of that fact, microdialysis has only sparsely been applied in the studies of VT, as compared to the techniques based directly on sensing devices such as voltammetry on carbon fiber electrodes or potentiometry using the ion-selective microelectrodes. A relatively large surface of the microdialysis probe makes it difficult to combine microdialysis with pressure-ejection technique, which is often used with the other two methods as a tool to administer diffusable substances. On the other hand, microdialysis offers a unique possibility to measure several compounds at a time using analytical techniques, such as high-performance liquid chromatography (HPLC), radioimmunoassay or mass spectrometry. Today, microdialysis is probably the most efficient in vivo technique to study brain chemical signaling and to correlate neurochemical analysis with behavioral methods (Zetterstr6m et al., 1986; Young, 1993; Ogren et al., 1996).
Concentration gradients within the extracellular space The extracellular space (ECS) under normal circumstances comprises about 20% of the total brain tissue volume. The extracellular fluid (ECF) contains ions, the concentrations of which are very similar to those found in the cerebrospinal fluid (CSF), as well as a number of long-chain glycosaminoglycans, proteoglycans and glycoproteins tethered to membranes. Neurotransmitters and neuromodulators and also neurotrophic factors and other cytokines in glial and neuronal cells, once released into the ECE all have to traverse the extracellular space on their way to the target receptors. However, the extracellular concentrations of these chemical messengers are far away from being homogeneously distributed within the ECS compartment. For example, for neurotransmitters such as DA, there is a steep gradient of concentrations being up to 6-7 orders of magnitude higher at a release site (a synaptic cleft) than in the outer interstitial space. It was estimated that striatal DA vesicles with a radius of 25 nm contain 25 mM DA, while the concentration of DA in the synaptic cleft, which is about 15 nm wide and 300 nm long,
was around 1.6 mM (Garris et al., 1994). However, the actual extracellular DA concentrations measured by in vivo voltammetry using a thin (5 txm O.D.) carbon fiber electrode was not higher than 20 nM in pargyline-treated rats (Gonon and Buda, 1985) or 0.1-2 txM during electrical stimulation (Kawagoe et al., 1992). Using the devices of even larger diameter such as a 200 txm O.D. microdialysis probe the calculated basal extracellular DA concentration was as low as 5 nM (Parsons and Justice, 1992). Thus, implantable sensors can measure only an 'echo' of the actual release event, whether defined as a spill-over from the synaptic cleft (a leaking synapse) or as a release from a nonjunctional bouton, varicosity or as extra-synaptic release. All these definitions are based on measurements of extracellular chemical signals by invasive means and have a common prerequisite: the overflow of the substance diffusing to the sensing device must be higher than its reuptake, sequestration, clearance to blood or CSF, and enzymatic or other inactivation mechanisms.
Invasive techniques for monitoring volume transmission: role of microdialysis The spatial and temporal resolution of any implantable monitoring device is dependent both on its geometry and the applied detection principle. The invasive techniques can be divided into two main groups: (1) intracorporeal biosensors providing immediate measures of a given analyte and (2) continuous sampling devices generating samples in a form of extracts/perfusates which are further analyzed by an appropriate analytical technique. The response time can be as low as 0.1-1 s for directly detecting voltammetric electrodes, while a relatively laborious and time-consuming analysis of neurotransmitters or neuropeptides sampled by microdialysis requires the fractions to be collected in 5-30 min intervals. The antibody-coated microprobes as described by Duggan and Hendry, 1986, provide excellent spatial resolution but only one time point of about 30 min. The sensing devices and related detection techniques within the first group are highly selective towards one analyte, which is detected directly at the surface of the biosensor implanted into the
181 brain tissue. In the simplest case, the selectivity for a detected endogenous substance is assured by its intrinsic chemical properties such as a relatively specific oxidation potential for DA. A more common way is to utilize the highly specific molecular interactions such as those between ions-ionophores, enzymes-substrates and antibodiesantigens, which all lead, either directly or via an intermediate product, to the changes in physical (electrical or optical) signals. Contrary to most of the biosensors, the implantable sampling devices allow relatively easy intracerebral monitoring of conscious freely moving animals. The sampling device, for example a microdialysis probe is continuously perfused at a low flow-rate (0.1-2 Ixl/min) with a physiological solution, typically a Ringer solution or an artificial cerebrospinal fluid (aCSF). In vivo microdialysis on awake animals eliminates the negative effects of general anesthetics on chemical neurotransmission and cell metabolism. A complete recovery of physiological functions occurs at about 5-7 days after the surgery as revealed by telemetry (Drijfhout et al., 1995). A very useful way to study functional neuroanatomy and the role of specific brain circuits in various behaviors is the so-called dual-probe. Here, one probe is implanted at the cell body level, whereas the second probe is implanted in the terminal area. The first probe is used for chemical stimulation by infusing the drugs while the second probe is used to measure the neurotransmitter release (for review, see Westerink et al., 1998). Here, we describe a newly developed method for studies of long-distance diffusion in the extracellular space. The technique combines dual-probe microdialysis sampling which allows simultaneous infusion and recovery of labeled molecules. The collected samples are separated by HPLC and individual fractions measured by a liquid scintillation counter. Inert markers such as 3H-mannitol can be used to study direct changes of extracellular volume characteristics. Endogenous compounds (e.g. 3H-DA) provide measures of brain permeability, as well as on the rate of cellular uptake and metabolism. As already mentioned in the Introduction, microdialysis sampling builds on the ability of molecules
to diffuse through the ECS; the length of the diffusion path for various molecules has been a matter of intensive research. For example, using voltammetric techniques it was calculated that the half-life and diffusion distance of stimulated DA release from its release site is only 25 ms and 7-10 p~m respectively (for review, see Gonon et al., this book). On the contrary, long distance DA diffusion paths were proposed on the basis of immunohistochemical (Fos-like), electrophysiological and microdialysis studies conducted in unilaterally DA denervated rats, following e.g. d-amphetamine treatment (Bjelke et al., 1994; Schneider et al., 1994). A possibility of longdistance diffusion arising from non-stimulated DA in the rat striatum could be elegantly investigated by use of a dual-probe microdialysis approach as schematically depicted in Fig. 1. Briefly, two microdialysis probes ( C M A / l l , cuprophane membrane: cut-off 6000 Da, length 4 mm and 240 Ixm O.D.) were implanted stereotaxically into the lateral and medial striatum (Paxinos and Watson, 1986) of the halothane anaesthetized 2 ~tl/min • 3H-DA + DA 3H-DOPAC + DOPAC 3H-HVA + HVA
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Fig. 1. Principal schemeof a dual-probemicrodialysisused for in vivo studies of long-distance diffusion and metabolism of radiolabeled molecules. The first (lateral) microdialysisprobe was used for the infusionof radioactivelabel (e.g. 3H-DA or 3Hmannitol), whereas the second probe, implanted 1 mm apart, recovered all the low-molecular weight substances present in the extracellular fluid including the diffused 3H-label or its metabolites. The samples were separated by microbore LCEC and fractionscorrespondingto the peaks of DA and metabolites were collected and measured in a liquid scintillationcounter.
182 rat. The distance between the membrane centers was 1 mm. The lateral probe was used for the infusion of a radioactive label: 3H-DA or D-3Hmannitol diluted in Ringer solution, at a flow-rate of 2 txl/min. The medial probe, perfused with Ringer solution at the same flow-rate was used for recovery of endogenous (cold) DA and its metabolites, as well as the tritiated labels. Samples were collected at 30-min intervals. Since 3H-mannitol was used as an inert marker, which did not metabolize during its passage through the brain, its radioactivity could be measured directly by use of a liquid scintillation counter. On the contrary, infusion of 3H-DA caused its immediate uptake and enzymatic cleavage to its respective acidic metabolites 3H-DOPAC and 3H-HVA. Thus, measuring the total tritium in samples of 3H-DA infused animals could provide an in vivo index of: (a) DA metabolism, (b) clearance by cerebral blood vessels and (c) uptake into the nerve terminals. To investigate the partition of radioactivity between individual substances, the microdialysis samples were first separated by microbore column liquid chromatography with electrochemical detection (LCEC), as described elsewhere (Kehr, 1999). Fractions corresponding to the individual peaks of DA and its metabolites were collected and measured by a scintillation counter. The clearance of tritiated labels by cerebral microcirculation can be studied by measuring the radioactivity in the blood samples. However, in our experiments we could not detect any radioactivity in the blood collected from the tail vein or intracardially, most likely due to a massive dilution of 'brain-derived' blood at this sampling site. Sampling the blood directly from the jugular vein and/or increasing the radioactivity of the infused label could possibly provide detectable levels. Uptake of 3H-DA to the dopaminergic terminals and the overall distribution of radioactivity within the brain tissue can be studied by using autoradiography of respective brain slices. In fact, the concept of tissue pre-loading, well known from in vitro superfusion techniques, was already demonstrated in the first report on dopamine microdialysis by Ungerstedt and Pycock in 1974. A single probe was used to load 3H-DA into the dopaminergic terminals and following a washout period, the effect of amphetamine on the release of
DA-related radioactivity could be observed. Here, the density of 3H-DA labeling is evaluated histologically for the first time. The diffusion profile of 3H-DA infused via the microdialysis probe in the rat striatum and the analysis of relative tritium density as a function of distance from the probe is shown in Fig. 2. As seen, following 5 hours of continuous infusion, the radioactivity profile shows a steep decay within the first 500 Ixm and being less than 5% of the initial level at a distance of 1 mm, i.e. at the site of the second (detector) probe (not seen in the phospho-imager print of the 10 txm section). Interestingly, the infusion of 3H-mannitol at similar tritium activity concentrations and experimental conditions as for 3H-DA resulted in an almost undetectable radioactive trace of mannitol following 5 days' exposure of the brain slice in a phospho-imager (data not shown). Omitting the brain fixation procedure by intracardial perfusion did not increase the tissue radioactivity of infused 3H-mannitol. This led to the assumption that infused 3H-mannitol is a truly extracellular marker of brain microenvironment being rapidly cleared by the cerebral microcirculation lacking any significant incorporation to the intracellular compartments. 3H-mannitol as a marker of extraceilular space
The usefulness of dual-probe microdialysis method to study diffusion of neurotransmitters within the ECS was initially evaluated by use of the inert lowmolecular weight markers such as 3H-mannitol. Such labels should exhibit fast kinetics of diffusion and equilibration within a typical time frame of an acute microdialysis experiment (6-8 hours including the initial stabilization phase of 90 min). Indeed, as shown in Fig. 3, the recovery of 3Hmannitol (1.5 IxM; specific activity 736.3 GBq/mmol, NEN, USA) reached the steady state within 90 min after start of the infusion, with a corresponding half-time (ts0~,) of 42_+ 1.6 min (mean ___SD, n = 6). From the total 3H-mannitol activity of 820 000+_ 13 000 dpm/10 p~l only about 3% was delivered (lost) into the brain tissue. The curves were calculated using linear regression analysis or Boltzmann sigmoidal nonlinear regression algo-
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-60-30 0 30 60 90 120150180210240270300 time (min) Fig. 3. Diffusion profiles of 3H-mannitol in vivo (rat striatum) and in vitro (Ringer solution) using the dual-probe microdialysis. Under in vivo conditions, the steady state was achieved within 90 min after start of the infusion, as demonstrated by the regression curve and the calculated time required to reach half of the steady state (ts0~) which was 42 ± 1.6 min. This indicates the presence of an active clearance processes of 3H-mannitol in the ECE No such equilibrium was seen in a quiescent Ringer solution. Values expressed as percentage of maximal recovered 3H-mannitol concentration at steady state (max Co,t), means ± S.E.M., n = 6 animals.
184 corresponding dual-probe in vitro experiment in Ringer solution at + 24°C and 2 pA/min showed a constant linear increase of recovered radioactivity, indicating non-saturable conditions during the study period (5 hours). The fast equilibrium of mannitol diffusion in the brain together with low background radioactivity suggest that the clearance by blood brain capillaries is the major inactivating mechanism for this molecule. This situation is schematically depicted in Fig. 4. The diffusion curve of 3H-mannitol in 6-hydroxydopamine (6-OHDA) lesioned rats 5 weeks after the unilateral lesions was much faster showing a significantly shorter half-time t50% (33_+ 1 min, P < 0.03) and a significant change in curve steepness (rate constant kss 6_+ 2 min, P < 0 . 0 5 ) vs. the control group. The maximal outflow levels at steady state conditions were about the same in both groups (3600 dpm/10 ixl). This is in agreement with histochemical data, showing that the disappearance of dopaminergic terminals has only moderate effects on gliosis as evaluated by GFAP immunostaining at this time-interval (Str6mberg et al., 1986). Our data on facilitated diffusion of 3H-mannitol in DA denervated striatum of a rat suggests that the
neurodegeneration of the dopaminergic system, seen in Parkinson's disease may be associated with a change of extracellular volume fraction and/or a reduction in tortuosity of the basal ganglia. It can be concluded that under given experimental conditions the kinetic profile of recovered 3H-mannitol is dependent only on the volume fraction/tortuosity characteristics of the ECS and with no significant changes in the brain-blood clearance. Finally, the dual-probe microdialysis using 3H-mannitol or a similar marker could be an interesting complement to the existing electrochemical methods used for the studies of volume fraction and tortuosity of the brain extracellular environment (for review, see Nicholson and Sykov~i, 1998; this volume).
Diffusion and metabolism of 3H-dopamine Infusion of 3H-DA (specific activity 2187.1 GBq/ mmol, NEN, USA) at a concentration of 500 nM (corresponds to the mean value of 710 0 0 0 _ 20 000 dpm/10 Ixl) through the microdialysis probe resulted in an average delivery of about 6% which corresponds to in vivo delivery of 30 nM DA. This is only 5-6 times higher than the basal extracellular DA levels estimated by zero-flow and no-net-flux
Fig. 4. A scheme of the diffusion path of 3H-mannitol, in the rat striatum. At steady state, 3H-mannitoldiffusing between the two probes, was distributed only within the ECS compartmentand cleared by cerebral microvesselcircuitry.Signifcantlyfaster diffusion of 3H-mannitolwas observed in rats with loss of DA terminals induced by 6-OHDA injections into the substantia nigra. Thus, 3nmannitol may serve as an inert marker of extracellularvolume fraction and tortuosity.
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The initial hypothesis of long-distance diffusion of striatal DA at basal conditions was not confirmed in this experimental model. The levels of recovered 3H-DA peak at steady-state were only slightly elevated to 45.2_+1.4 dpm/101xl (mean _+SEM, range 42-48 dpm/10 txl) over the threshold values which were 31.3_+1.3 dpm/10txl (range 21-41 dpm/10 Ixl). On the contrary, the basal levels of endogenous (cold) DA in control animals were easily detectable with a mean value of 6.2 nM in 10 txl injected onto the HPLC column. Neither, could 3H-DA be detected in 6-OHDA lesioned animals where the intraneuronal pools of DA terminal monoamine oxidase (MAO) and the dopamine uptake transporter were reduced to a minimum due to an almost complete disappearance of DA-ergic striatal terminals. Here, the counts in fractions of DA peaks were in a similar range as in the non-lesioned animals, i.e. about 35 and 43 dpm/ 10 I~1 in pre-infused and 3H-DA infused periods. Surprisingly, the overall recovered 3H-DA-derived radioactivity in the lesioned group was significantly reduced to 53% of the control group, from 4919_+530 dpm/10 ixl (n=6) to 2609_+205 dpm/ 10txl (n=5) respectively. The distribution of DOPAC, HVA and the unidentified front peak in control and 6-OHDA lesioned groups is schematically depicted in Fig. 6A. The absolute level of radioactivity in the front peak was unaffected by
microdialysis techniques (Parsons and Justice, 1992). In addition, this concentration of DA should still lie within the physiological range of stimulated DA release and as such should not affect the normal function of DA release, autoreceptor feedback and uptake machinery. Under these conditions, the total tritium radioactivity recovered by the second, detector probe was only about 5000 dpm/10 txl in control animals, which means that only 12.5% of delivered 3H-DA was recovered by the second probe. However, this value is not corrected for the in vivo recovery of the second microdialysis probe. Calculating the ts0~ and k~s parameters for the total recovered radioactivity gave the average values of 100_+4 min and 64_+3 min, respectively, i.e. the levels are about twice higher than those obtained with 3H-mannitol. Separation of microdialysis samples by microbore LCEC and subsequent counting of fractions representing DA, DOPAC, HVA and an unidentified compound eluted in the front peak revealed that DOPAC was the fastest diffusing compound (ts0% 85 _+4 min, kss 20 _+1 min; means + S.E.M., n = 6) and contained the largest portion of radioactivity (1634 _+234 dpm/peak) followed by HVA (1343 _+159 dpm/peak) and the unidentified front peak (850_+ 115 dpm/peak), as shown in Fig. 5. Again, a relatively fast steady state could be achieved for all the substances within 120-180 min. 2500-
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3 H-DOPAC
3 H-HVA
3 H-mannitol
Fig. 6. (A) Maximal levels (Cm~x)of tritiated molecules recovered by a second microdialysis probe at steady state in control (n = 6) and 6-OHDA lesioned (n = 5) rats. (B) Calculated half-time values (ts0~) of tritiated DA metabolites and 3H-mannitol in control and 6-OHDA lesioned rats. (C) Calculated diffusion slope values (ts0~) of tritiated DA metabolites and 3H-mannitol in control and 6-OHDA lesioned rats. Statistical analysis was done by Studentg t-test, comparing control vs. lesioned groups, values are expressed as means_+ S.E.M., *: P < 0.05; **: P<0.001; ***: P<0.0003.
187
C
A t, w
E al
,ag
3 H-front
3H-DOPAC
3 H-HVA
3 H-mannitol
Fig. 6. Continued
reduced in the 6-OHDA lesioned animals, with maximal reduction for DOPAC down to 14% of control, followed by HVA (28% of controls). This indicates that the striatal MAO activity which is localized predominantly in the dopaminergic terminals (Oreland, 1991) was considerably reduced as a consequence of the 6-OHDA lesion. On the other hand, the slightly less pronounced reduction of HVA suggests that this reduction was caused mostly as a consequence of a diminished pool of DOPAC rather than from reduced activity of extracellular COMT. The calculated kinetic data t50~ and kss for 3HDOPAC, 3H-HVA, the unidentified 3H-DA-derived front peak and 3H-mannitol in control and 6-OHDA lesioned groups are shown in Fig. 6B, C. As seen in Fig. 6B the diffusion half times were significantly reduced by about 40% for all 3H-DArelated peaks, whereas for 3H-mannitol, the corresponding reduction was only 23%. It is notable that 3H-mannitol is cleared much faster under normal conditions than any of the 3H-DArelated compounds in the 6-OHDA lesioned animals. Similarly, the diffusion curves were steeper in the lesioned animals as expressed
mathematically by calculating the diffusion rate kss values using the Boltzmanng non-linear regression (Fig. 6C). Here, again the kss levels were about 40% lower in the 6-OHDA lesioned group, although only the values for 3H-mannitol (see discussion to Figs. 3, 4) and 3H-HVA were significantly different (controls: 25.7_+2.5 min, n=5, 6-OHDA: 16.1_+2.6 min, n=6, P<0.05). A relatively large variation of the mean levels could be explained by poor temporal resolution, i.e. too long (30 min) sampling intervals. Interestingly, there was no difference in maximal radioactivity counts between the control and lesioned animals for 3H-mannitol and the 3H-front peak, whereas 3H-DOPAC and 3HHVA levels were strongly reduced in the lesioned group (see Fig. 6A). This suggests that in the lesioned animals, infused 3H-DA was, in spite of the diminished MAO and DA reuptake activity, unable to diffuse over the longer distances, most likely due to the activation of compensatory mechanisms or the increased brain-blood clearance. Also, it should be considered that 3H-DA itself may exhibit an increased clearance over the brain-blood barrier after the DA denervation. Limited amounts of data are available on the effects
188
of 6-OHDA lesions on the cerebral blood flow (CBF) and on the cerebromicrovascular permeability. Earlier studies have shown a two fold increase of CBF in DA denervated rats following stimulation with apomorphine (Ingvar et al., 1983) probably as a consequence of dopamine receptor supersensitivity. The existence of both D1 and D2 receptors was demonstrated on rat cerebral blood vessels (Amenta et al., 1991). These receptors mediate DA-induced vasodilatation associated with increases in CBF (Sharkey and McCulloch, 1986). In agreement with these data, a reduction of basal CBF was observed in DA denervated caudateputamen (Mraovitch et al., 1993). Recently, it was reported that DA may act as a vasoconstrictor on cortical microvessels (Krimer et al., 1998). However, this effect is probably mediated via agonist activity of DA at adrenergic and serotonergic receptors as discussed by Iadecola, 1998. Also DA terminals contact cerebral endothelial cells indicating a modulation of brain-blood clearance processes of extracellular chemical signals through DA receptors (Krimer et al., 1998). In view of these reports, we hypothesize that the infusion of 3H-DA into the DA denervated rat striatum may cause an immediate increase of local
CBF and capillary permeability augmenting the clearance processes of extacellular 3H-DA and its metabolites as manifested by their reduced extracellular levels and the inability to measure the long-distance diffusion of 3H-DA. Infusion of 3Hmannitol, on the other hand, may not affect CBF nor capillary permeability and consequently no differences at steady state were observed in control and lesioned animals. These results provide new insights into DA VT in the DA denervated striatum and thus in the striatum of Parkinson~ disease patients. D u a l - p r o b e m i c r o d i a l y s i s - a n e w tool for diffusion studies
A scheme shown in Fig. 7 summarizes the proposed diffusion path and metabolism of 3H-DA as studied by the dual-probe microdialysis technique. In control non-lesioned rats, infused 3H-DA is removed from the ECS compartment by three major mechanisms: (a) uptake into the DA-ergic terminals, (b) brain-blood clearance, and (c) enzymatic cleavage by extracellular COMT and mainly DA terminal MAO. Hence, the appearance of
Fig. 7. Scheme of the diffusion path of 3H-DA and formation of its metabolites 3H-DOPAC and 3H-HVA in control rat striatum. At steady state, 3H-DA was cleared by blood capillaries, uptake into the dopaminergic terminals and metabolism by both intracellular and extracellular enzymes. In DA denervated animals, the formation of 3H-DOPAC and 3H-HVA was strongly attenuated due to the absence of DA terminals being the major source of MAO activity for DA metabolism. Consequently, the portion of recovered 3H-HVA formed through extracellular COMT was now higher than 3H-DOPAC (see Fig. 6A).
189 3H-DOPAC and 3H-HVA metabolites and ultimately of 3H-DA in the perfusates sampled by the second microdialysis probe should reflect the ratio between these three factors. This combined metabolic-diffusion process is much slower than the diffusion of a labeled inert marker shown in Fig. 4, as evidenced by significantly longer diffusion half times of 3H-DOPAC and 3H-HVA compared to 3Hmannitol. It is suggested that for the DA metabolites there exist in addition to the diffusion characteristics through the ECS compartment also contributions of trans-cellular transport involving, e.g. the brain-blood barrier and the activity of the corresponding metabolizing enzymes. Removal of the DA terminals by lesions with 6-OHDA resulted in a dramatic reduction of the formation of 3H-DA metabolites, but only marginally affected their diffusion speed with no detectable facilitation of 3H-DA diffusion. On the contrary, the inert marker 3H-mannitol diffused significantly faster in the lesioned animals than in the controls. In summary, the dual-probe microdialysis approach shows a clear difference in diffusion properties of the neutral molecules used only as markers of ECS and those endogenously synthesized or metabolized by the brain tissue. In a pathological state, the marker molecules seem to mirror only the 'geometrical' changes of the brain microenvironment (tortuosity, volume fraction), whereas diffusion of the endogenously-derived molecules is affected by all the compensatory mechanisms occurring in the damaged tissue including the brain-blood barrier and cerebral blood flow.
Conclusions The intracerebral dual-probe microdialysis technique allows the study of diffusion of labeled molecules in vivo in anaesthetized or awake animals. The labeled markers can be biologically inert molecules or endogenous substances delivered and sampled without any volume changes or other disturbances to brain homeostasis. Diffusion kinetics and steady-state levels of DA metabolites could be studied over long (> 1 mm) inter-probe distances, whereas studies of DA diffusion should be conducted at shorter ( - 0.1 mm) distances. The dual-probe microdialysis technique
can provide an in vivo index of a complex interplay of clearance and metabolic processes of extracellular labels at basal, non-stimulated conditions and as such, the technique can be a complement to the existing voltammetric and potentiometric methods. Finally, the dual-probe technique can be used to study molecular transport within the brain microenvironment in various neuropathological models of brain diseases or following pharmacological stimuli, helping to understand the role of VT at such conditions.
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