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Brain dialysis of neurotransmitters: a commentary
Journal of Neuroscience Methods, 34 (1990) 29-34 Elsevier
29
NSM 01104
Brain dialysis of neurotransmitters: a commentary Gaetano Di Chiara Institute of Experimental Pharmacology and Toxicology, University of Cagliari, Cagliari (Italy) (Received 19 September1989) (Accepted 22 January 1990)
K e y words: Microdialysis; Brain dialysis; Dopamine release; 5-HT release
Brain dialysis has become an important tool for investigatingchanges in the extracellularlevels of neurotransmitters. This paper reviews the experimental variables and criteria that should be considered when interpreting data obtained with the brain dialysis method. Brain dialysis can provide important direct information on the effect of drugs on transmitter release as shown using dopaminergic agonists and antagonists, monoamine uptake inliibitors and drugs of abuse.
Introduction Brain dialysis involves the implantation of a dialysis probe in selected brain areas, perfusing it with a physiological fluid, collecting the fluid and analyzing it for specific substances. The fluid, while flowing inside the probe, extracts from the extracellular brain compartment low-molecularwheight substances which diffuse across the dialytic membrane along their concentration gradient. By this principle substances present in the extraceUular fluid can be monitored, provided that a sensitive method of analysis is available. The advantages of brain dialysis over other techniques for in vivo monitoring of chemical changes in the brain (cup technique, push-pull cannula) were clearly realized since the beginning and were indicated in the 'closed' characteristic of the system which avoids direct contact of the superfusion fluid with the tissue thus reducing local tissue damage and providing relatively clean samples for direct chemical analysis.
Although simple in principle, brain dialysis is an invasive technique which intends to monitor the hopefully undisturbed release of transmitter from nerve terminals by literally inserting into the brain a probe several orders of magnitude larger than the biological structures under study. In fact the outer diameter of currently available probes (0.3-0.8 ram) corresponds to about one thousand times the diameter of the synapses from which neurotransmitter release is to be monitored. On the other hand, in spite of the current use of the term 'microdialysis' for brain dialysis, dialysis probes are 10-100 times larger than other 'micro' tools (micro-pipettes, micro-electrodes) currently used by neuroscientists. These considerations point to the fact that brain dialysis with presently available probes is unable to estimate neurotransmitter release at release sites. Moreover local tissue damage, although reduced, is still expected to play a role in the results obtained.
Experimental variables Correspondence: G. Di Chiaxa, Institute of Experimental Pharmacologyand Toxicology,Universityof Cagliari, Viale A. Diaz, 182, 1-09100Cagliari, Italy.
Soon after the introduction of brain dialysis it became clear that methodological differences could result in major differences in the results obtained.
0165-0270/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
30 Thus, in freely-moving rats implanted 24 h apart with transcerebral probes, neuroleptics stimulated DA release in the caudate in a pharmacologically meaningful manner consistent with their DA-receptor blocking potencies (Imperato and Di Chiara, 1985); in contrast, in freelymoving rats implanted 2 h before with U-probes, the same neuroleptics stimulated inconsistently, in a dose-unrelated manner and, eventually, only at high doses, the output of DA in the caudate (Zetterstrom et al., 1984). In fact, a comparative analysis of the literature reveals that the results provided by brain dialysis are critically dependent upon at least 3 variables: type of probe, post-implantation interval and use of anaesthetized versus freely-moving rats. A 20 times difference between transversal and U-shaped probes has been demonstrated in the ability to recover ACh from the caudate (Damsma et al., 1988). Thus, basal ACh could be detected in dialysates obtained from transcerebral probes but not from U-shaped probes unless a cholinesterase inhibitor was added to the perfusion Ringer (Damsma et al., 1988). Direct comparison of the Ca 2÷ dependency and TTX sensitivity of DA release estimated with the two types of probes (transcerebral vs. U-shaped) in rats implanted acutely (3 h) or subchronically (24 h) reveals that with the transcerebral probe DA release is mostly Ca2+-dependent and TTX-sensitive already 3 h after the implant and totally so 24 h thereafter. Instead, with U-shaped probes DA release is Ca2÷-independent and TTX-insensitive 3 h after the implant which becomes partially Ca2÷-dependent and TTX-sensitive 24 h after (Westerink and De Vries, 1989). Therefore in acutely implanted rats some U-shaped probes seem to detect in basal conditions only an overflow of transmitter, independent from neuronal firing activity and from neurosecretion and probably arising from damaged nerve terminals. It appears therefore that post-implantation interval and type of probe can interact so that differences in probe type become critical at early post-implantation intervals. Differences in probe size and in the related tissue damage might account in part for these differences. A clue for explaining the influence of post-im-
plantation interval on brain dialysis is provided by the fact that, 2 h after the implant of a dialysis probe in the hippocampus, marked changes in glucose metabolism and blood flow are detected in the implanted area (Benveniste et al., 1987); moreover, probably as a result of massive K ÷ release from damaged elements, spreading depression and depolarization inactivation of intact neurons takes place (Benveniste et al., 1989). Recovery from the above changes appears complete 24 h after the implant of the probe (Benveniste et al., 1987). Finally, anaesthesia can drastically influence the effect of drugs which depend on an intact neuronal excitability and firing activity for their effects. Since stimulation of DA release by drugs like neuroleptics is firing-dependent (Imperato and Di Chiara, 1985), it is not unexpected that these drugs fail to consistently stimulate DA release in anaesthetized rats (Di Chiara and Waldmeier, 1984).
Criteria for brain dialysis The above examples indicate the necessity of establishing specific criteria for evaluating the nature of neurotransmitter output as estimated by any given dialysis technique. In particular these criteria should be applied to the characterization of basal neurotransmitter output which is the basic reference for any experimentally induced change. Due to the 'macro' size of the probes and to their spatial relationship with nerve terminals, brain dialysis is not expected to measure neurotransmitter released at synaptic sites but rather neurotransmitter diffused into the extracellular space after its release. Nonetheless, basal output of DA, NA, ACh and 5-HT is Ca2÷-dependent and TTXsensitive to an extent which ranges from 60% (NA) (Consolo et al. 1987), to 85-100% (DA) (Imperato and Di Chiara, 1984; Westerink and De Vries, 1989), ACh (Damsma et al., 1987) and 5-HT (Carboni and Di Chiara, 1989). Thus, basal output of DA, ACh, 5-HT and NA seems to result from the sequence: action potential, terminal depolarization, activation of voltage-dependent fast Na ÷ channels, activation of voltage-dependent Ca 2+ channels, Ca 2+ influx, exocytosis, trans-
31 mitter release. In aggreement with tiffs, "t-butyrolactone, an agent known to block the firing activity of DA neurons, abolishes basal DA release (Imperato and Di Ciffara, 1984) and prevents impulse-dependent stimulation of DA release (Imperato and Di Chiara, 1985; Imperato and Di Chiara, 1986; Carbonl et al., 1989c). Given these premises, experimental conditions which are unable to reproduce these results for the above transmitters cast serious doubts on the possibility of using neurotransmitter output in dialysates as an estimate of in vivo neurotransmitter release. In the case of amino acids (glutamate, aspartate, giycine), basal output appears both TrX-insensitive and Ca2+-independent (Westerink et al., 1987), suggesting that their release is of non-neural origin or independent from physiological neural activity. One should also consider, however, the possibility of a non-terminal (e.g. dendritic) release of transmitter independent from activation of fast Na + channels and therefore TTX-insensitive. A further complication in this issue is the possibility that physiological transmitter release is carrier-mediated and independent from exocytosis or that exocytosis takes place from intracytoplasmic release of Ca 2+- rather than from Ca 2÷ influx. Therefore, although T r x sensitivity and Ca 2+ dependency should be regarded as basic criteria for brain dialysis of neurotransmitters, they should not be rigidly applied but adapte d to the physiological characteristics of the release of the neurotransmitter under study. Another currently used criterion for testing the physiological nature of neurotransmitter output in dialysates is the ability of high potassium concentrations (30-100 mM) to stimulate it (Imperato and Di Ciffara, 1984; Westerink et al., 1987; Kalrn et al., 1988). This criterion, however, simply indicates that depolarization of neural structures (not only neuronal but also glial) is capable of releasing the transmitter but tells us little about the nature of basal transmitter release. Thus, with striatal U-probes, 5-HT release is strongly stimulated by K + in spite of the fact that it is only partially TTX-sensitive (50%) (Kalrn et al.,1988). Vice-versa, basal aminoacid release is stimulated
by K + but is virtually Tl'X-insensitive and Ca 2+independent (Westerink et al., 1987). Similar considerations apply to the use of electrical or chemical stimulation of specific neural pathways as a criterion for characterizing basal neurotransmitter release in vivo (Imperato and Di Ciffara, 1984). A currently used criterion for in vivo release is the effectiveness of drugs with neurotransmitterreleasing properties [amphetamine (Zetterstrom et al., 1983; Imperato and Di Ciffara, 1984), fenfluramine (Carboni and Di Chiara, 1989), p-chloroamphetamine (Sharp et al., 1986; Kalrn et al., 1988)]. However, the releasing action of these drugs is TTX-insensitive and Ca2+-independent, in aggreement with a direct displacing action on intraneuronal amine pools (Carboni and Di Chiara, 1989; Carboni et al., 1989c). Thus, the effect of these drugs is not a criterion for physiological in vivo transmitter release. Similar considerations apply to criteria utilizing drugs which interfere with the synthesis, metabolism or compartimentalization of the transmitter (Imperato and Di Chiara, 1984). Using the above criteria for evaluating the various dialysis methods it becomes clear that much in this field has still to be done. For example, no information is available about the Ca 2÷ dependency and TTX sensitivity of neurotransmitter release estimated with concentric probes and with commercially available dialysis probes. In contrast, criteria for a physiological in vivo neurotransmitter release are satisfied by acute and chronic transcerebral probes in different laboratories and for different transmitters [DA (Imperato and Di Ciffara, 1984; Westerink and De "Cries, 1989), NA (L'Heureux et al., 1986), ACh (Consolo et al. 1987; Damsma et al., 1987), 5-HT (Carboni and Di Chiara, 1989) and by U-probes only after chronic implantation (Kalrn et al., 1988; Westerink and De Vries, 1989)].
Some applications of brain dialysis Brain dialysis in freely-moving rats has given major contributions to the knowledge of the mechanism of action of centrally acting drugs by
32 providing direct information on the effect of the drugs on the release of specific neurotransmitters.
Receptor agonists and antagonists Brain dialysis in freely-moving, sub-chronically implanted rats has provided the long sought but never provided evidence for a stimulating effect of systemic neuroleptics on DA release (Imperato and Di Chiara, 1985). It should pointed out that the failure to obtain consistent effects by neuroleptics on DA release in anaesthetized, acutely implanted rats (Di Chiara and Waldmeier, 1984) was instrumental for developing transcerebral dialysis in sub-chronically implanted freely-moving rats (Imperato and Di Chiara, 1985). Both D-2 (Imperato and Di Chiara, 1985) and D-1 receptor antagonists (Imperato et al., 1987) stimulate DA release to a similar extent while DA metabolism is known to be more effectively stimulated by D-2 antagonists. Rapid tolerance takes place to the stimulatory effect of neuroleptics on DA release in the caudate since a second dose of neuroleptic given 3-6 h after the first fails to stimulate DA release (Di Chiara and Imperato, 1985). This process develops during the time of action of a single dose of neuroleptic and explains the observation that stimulation of DA release by classic neuroleptics is short-lasting in comparison with stimulation of DA metabolite output and with motor inhibition. Depolarization blockade of a subpopulation of DA neurons has been suggested as the mechanism of this tolerance (Di Chiara and Imperato, 1985). In contrast to neuroleptics, dopamine receptor agonists, active on D-2 receptors, reduce DA release (Imperato et al., 1988) in aggreement with their ability to inhibit dopaminergic firing activity and to stimulate DA autoreceptors. The doses at which DA-receptor agonists and antagonists (neuroleptics) affect DA release are quite low; thus, agonists like LY 171555, pergolide, apomorphine and BHT 920 act at threshold doses around 25 /~g/kg s.c. (Imperato et al., 1988) while antagonists like haloperidol and SCH 23390 act at threshold doses of 12.5-25 # g / k g s.c. (Imperato and Di Chiara, 1985; Imperato et al., 1987). These doses are in the range of those at which these drugs produce effects in the most sensitive in vivo tests
for central activity. This means that the study of DA release with brain dialysis is among the most sensitive in vivo methods for detecting drug actions at central dopaminergic receptors. By correlating drug effects on DA release to behaviour it is possible to characterize the profile of drugs acting on dopaminergic receptors and to distinguish at least 4 classes of DA agonists (Imperato et al., 1988) differing for their intrinsic activity at D-2 receptors.
Monoamine reuptake blockers and releasers Blockade of amine reuptake increases its extracellular concentrations as estimated by brain dialysis; thus, cocaine, nomifensine, methylphenidate and phencyclidine increase DA output (Church et al., 1987; Carboni et al., 1989c), desipramine increases NA output (L'Heureux et al., 1986) and chlorimipramine and indalpine increase 5-HT output in dialysates (Kal~n et al., 1988; Carboni and Di Chiara, 1989). The time-course of cocaine concentrations in the extracellular fluid sampled by dialysis appear to match quite nicely that of the increase in DA output (Hurd et al., 1988; Nicolaysen et al., 1988). Brain dialysis has provided clear-cut in vivo evidence for differentiating monoamine reuptake blockers from releasers. Thus, the effect of cocaine, phencyclidine, nomifensine on DA output (Carboni et al., 1989) and of chlorimipramine (Carboni and Di Chiara, 1989) on 5-HT output is blocked by T I X and by lack of Ca 2÷. In contrast, the effect of amphetamine (Carboni et al., 1989c), which releases DA, and of fenfluramine (Carboni and Di Chiara, 1989), which releases 5-HT, is TTX-insensitive and Ca2+-independent. The reason for this difference is readily apparent if one considers that reuptake blockers do not interfere with the release mechanism but simply prevent removal from the extracellular space of the transmitter released by nerve impulses; in contrast, amphetamine and fenfluramine, by displacing amines from vesicular pools into the cytoplasm, make them available for active transport out of the terminal by the reuptake carrier. Transmitter-precursors Administration of the precursor aminoacids (tryptophan for serotonin and tyrosine for dopa-
33 mine) is known to result in a stimulation of the synthesis of the respective transmitters because of the lack of saturation of the rate-limiting enzymes (tryptophan-hydroxylase and tyrosine-hydroxylase) by the in vivo concentration of the precursor aminoacids. Brain dialysis studies have revealed that administration of tryptophan increases extraceUular concentrations of serotonin (Carboni et al., 1989). This effect does not seem the result of an overflow of transmitter as, at least in the case of tryptophan (Carboni et al., 1989), retains the physiological characteristics of basal output, namely, calcium dependency and TITK sensitivity. These results indicate that administration of amino-acid precursors might provide an efficient means for stimulating serotonergic or dopaminergic neurotransmission in experimental or clinical conditions.
Serotonin via excitatory 5-HT3 receptors located in the VTA, might exert a permissive role for the ability of drug stimuli to activate the firing activity of DA neurons. Thus, 5-HT3 antagonists like M D L 72222 and ICS 205-930 prevent the stimulation of DA release by drugs, like morphine and nicotine, which stimulate the firing activity of mesolimbic DA neurons, but not of amphetamine, which stimulates DA release independently from neuronal firing (Carboni et al., 1989a). Correlated with this is the property of 5-HT3 antagonists to block the rewarding properties of morphine and nicotine but not of amphetamine (Carboni et al., 1989b). These results have opened the possibility of utilizing 5-HT3 antagonists in clinical conditions characterized by disorders of motivation and affection such as anxiety, drug dependence, schizo-affective syndromes, etc. etc.
Motivational properties of drugs
References
Various centrally acting drugs are provided with motivational properties which can be positive (rewarding) or negative (aversive). Drugs abused by humans show positive reinforcing properties while drugs like naloxone, picrotoxin, K-opioid agonist are aversive. Brain dialysis has provided seminal evidence of the neurochemical basis of these properties. Drugs abused by humans such a s narcotic analgetics (Di Chiara a n d Imperato,'1988); central stimulants [amphetamine, cocaine, phencyclidine (Carboni et al.,1989c)], ethanol (impetato argd Di Chiara, 1986) and nicotine (Imperato et~ ak, 1986) have in common the property of increasing extracellular DA concentrations preferentially in the limbic n. accumbens as compared to the dorsal caudate (Di Chiara and Imperato, 1988). Therefore, a property of drugs of abuse might be the ability to stimulate DA release preferentially in the mesolimbic DA system. This property is probably related to the psychomotor stimulant and, eventually, to the rewarding properties of these drugs: in fact, blockade of DA receptors (particularly of D-1 receptors) (Leone and Di Chiara, 1987) prevents the motor-stimulant and rewarding effects of drugs of abuse (Wise and Bozarth, 1987).
Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H. (1987) Regionalcerebral glucosephosphorylationand blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat, J. Neurochem.,49: 729-734. Carboni, E. and Di Chiara, G. (1989) Serotonin release estimated by transcortical dialysis in freely moving rats, Neuroscience, 32: 637-645. Carboni, E., Acquas, E., Frau, R. and Di Chiara, G. (1989a) Differential inhibitory effects of a 5-HT3 antagonist on drug-induced stimulation of dopamine release, Eur. J. Pharmacol., 164: 515-519. Carboni, E., Acquas, E., Leone, P. and Di Chiara, G. (1989b) 5HT3 receptor antagonists block morphine- and nicotinebut not amphetamine-induced reward, Psychopharmacology, 97: 175-178. Carboni, E., Imperato, A., Perezzani,L. and Di Chiara, (1989c) Amphetamine, cocaine, phencyclidineand nomifensine increase extracellulardopamine concentrations preferentially in the nucleus accumbens of freely moving rats, Neuroscience, 28: 653-661. Consolo, S., Wu, C.F., Fiorentini, F., Ladinsky, H. and Vezzani, A. (1987) Determination of endogenous acetylcholine release in freely moving rats by transtriatal dialysis coupled to a radioenzymaticassay: effect of drugs, J. Neurochem., 48: 1459-1465. Church, W.H., Justice, J.B. and Byrd, L.D. (1987) Extracellular. dopamine in rat striatum following uptake inhibition by cocaine, nomifensine and benztropine, Europ. J. Pharmacol., 139: 345-348. Damsma, G., Westerink, B.H.C., Imperato, A., Rollema, H., De Vries, J.B. and Horn, A.S. (1987) Automated brain
34 dialysis of acetylcholine in freely moving rats: detection of basal acetylcholine, Life SCI., 41: 873-876. Damsma, G., Westerink, B.H.C., De Boer, P., De Vries, J.B. and Horn, A.S. (1988) Basal acetyleholine release in freely moving rats detected by on-line trans-striatal dialysis: pharmacological aspects, Life SCI., 43: 1161-1168. Di Chiara, G. and Imperato, A. (1985) Rapid tolerance to neuroleptic-induced stimulation of dopamine release in freely moving rats, J. Pharmacol. Exp. Ther., 235: 487-494. Di Chiara, G. and Imperato, A. (1988a) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats, Proc. Natl. Acad. Sci. U.S.A., 85: 5274-5278. Di Chiara, G. and Imperato, A. (1988b) Opposite effects of #and K-opiate agonists on dopamine-release in the nucleus accumbens and in the dorsal caudate of freely moving rats, J. Pharmacol. Exp. Ther., 244: 1067-1080. Di Chiara, G. and Waldmeier, P. (1984) Catecholamine release and metabolism, in: Progress in Catecholamine Research: Basic and Peripheral Mechanisms, Alan R. Liss, Inc., New York, pp. 167-168. Hurd, Y.L., Kehr, J. and Ungerstedt, U. (1988) In vivo microdialysis as a technique to monitor drug transport: correlation of extracellular cocaine levels and dopamine overflow in the rat brain, J. Neurocbem., 51: 1314-1316. Imperato, A. and Di Chiara, 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. Neurosei., 4: 966-977. Imperato, A. and Di Chiara, G. (1985) Dopamine release and metabolism in awake rats after systemic neuroleptics as studied by trans-striatal dialysis, J. Neurosci., 5: 297-306. Imperato, A. and Di Chiara, G. (1986) Preferential stimulation of dopamine-release in the aecumbens of freely moving rats by ethanol, J. Pharmacol. Exp. Ther., 239: 219-228. Imperato, A., Mulas, A. and Di Chiara, G. (1986) Nicotine preferentially stimulates dopamine release in the limbic system of freely moving rats, Eur. J. Pharmacol., 132: 337-338. lmperato, A., Mulas, A. and Di Chiara, G. (1987) The D-1 antagonist SCH 23390 stimulates while the D-1 agonist SKF 38393 fails to affect dopamine release in the dorsal caudate of freely moving rats, Eur. J. Pharmacol., 142: 177-181.
Imperato, A., Tanda, G., Frau, R. and Di Chiara, G. (1988), Pharmacological profile of dopamine receptor agonists as studied by brain dialysis in behaving rats, J. Pharmacol. Exp. Ther., 245: 257-264. Kal6n, P., Streeker, R.E., Rosengren, E. and Bj6rklund, A. (1988) Endogenous release of neuronal serotonin and 5-hydroxyindoleacetic acid in the caudate-putamen of the rat as revealed by intracerebral dialysis coupled to high performance liquid chromatography with fluorimetric detection, J. Neurochem., 51: 1422-1435. Leone, P. and Di Chiara, G. (1987) Blockade of D-1 receptors by SCH 23390 antagonizes morphine- and amphetamineinduced place preference conditioning, Eur. J. Pharmacol., 135: 251-254. 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. Nicolaysen, L.C., Pan, H.-T. and Justice, Jr., J.B. (1988) Extracellular cocaine and dopamine concentrations are linearly related in rat striatum, Brain Res., 456: 317-323. Sharp, T., Zetterstrom, T., Christmanson, L. and Ungerstedt, U. (1986) p-Chloroampbetamine releases both serotonin and dopamine into rat brain dialysates 'in vivo', Neurosci. Lett., 72: 320-324. Westerink, B.H.C. and De "Cries, J.B. (1987) Characterization of "in vivo" dopamine release as determined by brain microdialysis after acute and subchronic implantations: methodological aspects, J. Neurocbem., 51: 683-687. Westerink, B.H.C., Damsma, G., Rollema, H., De Vries, J.B. and Horn, A.S. (1987) Scope and limitations of "in vivo" brain dialysis: a comparison of its application to various transmitter systems, Life Sci., 41: 1763-1776. Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, U. (1983) "In vivo" measurement of dopamine and its metabolites by intracerebral dialysis: changes after damphetamine, J. Neurochem., 41: 1769-1773. Zetterstrom, T., Sharp, T. and Ungerstedt, U. (1984) Effect of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat studied by intracerebral dialysis, Eur. J. Pharmacol., 106: 27-37.
29
NSM 01104
Brain dialysis of neurotransmitters: a commentary Gaetano Di Chiara Institute of Experimental Pharmacology and Toxicology, University of Cagliari, Cagliari (Italy) (Received 19 September1989) (Accepted 22 January 1990)
K e y words: Microdialysis; Brain dialysis; Dopamine release; 5-HT release
Brain dialysis has become an important tool for investigatingchanges in the extracellularlevels of neurotransmitters. This paper reviews the experimental variables and criteria that should be considered when interpreting data obtained with the brain dialysis method. Brain dialysis can provide important direct information on the effect of drugs on transmitter release as shown using dopaminergic agonists and antagonists, monoamine uptake inliibitors and drugs of abuse.
Introduction Brain dialysis involves the implantation of a dialysis probe in selected brain areas, perfusing it with a physiological fluid, collecting the fluid and analyzing it for specific substances. The fluid, while flowing inside the probe, extracts from the extracellular brain compartment low-molecularwheight substances which diffuse across the dialytic membrane along their concentration gradient. By this principle substances present in the extraceUular fluid can be monitored, provided that a sensitive method of analysis is available. The advantages of brain dialysis over other techniques for in vivo monitoring of chemical changes in the brain (cup technique, push-pull cannula) were clearly realized since the beginning and were indicated in the 'closed' characteristic of the system which avoids direct contact of the superfusion fluid with the tissue thus reducing local tissue damage and providing relatively clean samples for direct chemical analysis.
Although simple in principle, brain dialysis is an invasive technique which intends to monitor the hopefully undisturbed release of transmitter from nerve terminals by literally inserting into the brain a probe several orders of magnitude larger than the biological structures under study. In fact the outer diameter of currently available probes (0.3-0.8 ram) corresponds to about one thousand times the diameter of the synapses from which neurotransmitter release is to be monitored. On the other hand, in spite of the current use of the term 'microdialysis' for brain dialysis, dialysis probes are 10-100 times larger than other 'micro' tools (micro-pipettes, micro-electrodes) currently used by neuroscientists. These considerations point to the fact that brain dialysis with presently available probes is unable to estimate neurotransmitter release at release sites. Moreover local tissue damage, although reduced, is still expected to play a role in the results obtained.
Experimental variables Correspondence: G. Di Chiaxa, Institute of Experimental Pharmacologyand Toxicology,Universityof Cagliari, Viale A. Diaz, 182, 1-09100Cagliari, Italy.
Soon after the introduction of brain dialysis it became clear that methodological differences could result in major differences in the results obtained.
0165-0270/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
30 Thus, in freely-moving rats implanted 24 h apart with transcerebral probes, neuroleptics stimulated DA release in the caudate in a pharmacologically meaningful manner consistent with their DA-receptor blocking potencies (Imperato and Di Chiara, 1985); in contrast, in freelymoving rats implanted 2 h before with U-probes, the same neuroleptics stimulated inconsistently, in a dose-unrelated manner and, eventually, only at high doses, the output of DA in the caudate (Zetterstrom et al., 1984). In fact, a comparative analysis of the literature reveals that the results provided by brain dialysis are critically dependent upon at least 3 variables: type of probe, post-implantation interval and use of anaesthetized versus freely-moving rats. A 20 times difference between transversal and U-shaped probes has been demonstrated in the ability to recover ACh from the caudate (Damsma et al., 1988). Thus, basal ACh could be detected in dialysates obtained from transcerebral probes but not from U-shaped probes unless a cholinesterase inhibitor was added to the perfusion Ringer (Damsma et al., 1988). Direct comparison of the Ca 2÷ dependency and TTX sensitivity of DA release estimated with the two types of probes (transcerebral vs. U-shaped) in rats implanted acutely (3 h) or subchronically (24 h) reveals that with the transcerebral probe DA release is mostly Ca2+-dependent and TTX-sensitive already 3 h after the implant and totally so 24 h thereafter. Instead, with U-shaped probes DA release is Ca2÷-independent and TTX-insensitive 3 h after the implant which becomes partially Ca2÷-dependent and TTX-sensitive 24 h after (Westerink and De Vries, 1989). Therefore in acutely implanted rats some U-shaped probes seem to detect in basal conditions only an overflow of transmitter, independent from neuronal firing activity and from neurosecretion and probably arising from damaged nerve terminals. It appears therefore that post-implantation interval and type of probe can interact so that differences in probe type become critical at early post-implantation intervals. Differences in probe size and in the related tissue damage might account in part for these differences. A clue for explaining the influence of post-im-
plantation interval on brain dialysis is provided by the fact that, 2 h after the implant of a dialysis probe in the hippocampus, marked changes in glucose metabolism and blood flow are detected in the implanted area (Benveniste et al., 1987); moreover, probably as a result of massive K ÷ release from damaged elements, spreading depression and depolarization inactivation of intact neurons takes place (Benveniste et al., 1989). Recovery from the above changes appears complete 24 h after the implant of the probe (Benveniste et al., 1987). Finally, anaesthesia can drastically influence the effect of drugs which depend on an intact neuronal excitability and firing activity for their effects. Since stimulation of DA release by drugs like neuroleptics is firing-dependent (Imperato and Di Chiara, 1985), it is not unexpected that these drugs fail to consistently stimulate DA release in anaesthetized rats (Di Chiara and Waldmeier, 1984).
Criteria for brain dialysis The above examples indicate the necessity of establishing specific criteria for evaluating the nature of neurotransmitter output as estimated by any given dialysis technique. In particular these criteria should be applied to the characterization of basal neurotransmitter output which is the basic reference for any experimentally induced change. Due to the 'macro' size of the probes and to their spatial relationship with nerve terminals, brain dialysis is not expected to measure neurotransmitter released at synaptic sites but rather neurotransmitter diffused into the extracellular space after its release. Nonetheless, basal output of DA, NA, ACh and 5-HT is Ca2÷-dependent and TTXsensitive to an extent which ranges from 60% (NA) (Consolo et al. 1987), to 85-100% (DA) (Imperato and Di Chiara, 1984; Westerink and De Vries, 1989), ACh (Damsma et al., 1987) and 5-HT (Carboni and Di Chiara, 1989). Thus, basal output of DA, ACh, 5-HT and NA seems to result from the sequence: action potential, terminal depolarization, activation of voltage-dependent fast Na ÷ channels, activation of voltage-dependent Ca 2+ channels, Ca 2+ influx, exocytosis, trans-
31 mitter release. In aggreement with tiffs, "t-butyrolactone, an agent known to block the firing activity of DA neurons, abolishes basal DA release (Imperato and Di Ciffara, 1984) and prevents impulse-dependent stimulation of DA release (Imperato and Di Chiara, 1985; Imperato and Di Chiara, 1986; Carbonl et al., 1989c). Given these premises, experimental conditions which are unable to reproduce these results for the above transmitters cast serious doubts on the possibility of using neurotransmitter output in dialysates as an estimate of in vivo neurotransmitter release. In the case of amino acids (glutamate, aspartate, giycine), basal output appears both TrX-insensitive and Ca2+-independent (Westerink et al., 1987), suggesting that their release is of non-neural origin or independent from physiological neural activity. One should also consider, however, the possibility of a non-terminal (e.g. dendritic) release of transmitter independent from activation of fast Na + channels and therefore TTX-insensitive. A further complication in this issue is the possibility that physiological transmitter release is carrier-mediated and independent from exocytosis or that exocytosis takes place from intracytoplasmic release of Ca 2+- rather than from Ca 2÷ influx. Therefore, although T r x sensitivity and Ca 2+ dependency should be regarded as basic criteria for brain dialysis of neurotransmitters, they should not be rigidly applied but adapte d to the physiological characteristics of the release of the neurotransmitter under study. Another currently used criterion for testing the physiological nature of neurotransmitter output in dialysates is the ability of high potassium concentrations (30-100 mM) to stimulate it (Imperato and Di Ciffara, 1984; Westerink et al., 1987; Kalrn et al., 1988). This criterion, however, simply indicates that depolarization of neural structures (not only neuronal but also glial) is capable of releasing the transmitter but tells us little about the nature of basal transmitter release. Thus, with striatal U-probes, 5-HT release is strongly stimulated by K + in spite of the fact that it is only partially TTX-sensitive (50%) (Kalrn et al.,1988). Vice-versa, basal aminoacid release is stimulated
by K + but is virtually Tl'X-insensitive and Ca 2+independent (Westerink et al., 1987). Similar considerations apply to the use of electrical or chemical stimulation of specific neural pathways as a criterion for characterizing basal neurotransmitter release in vivo (Imperato and Di Ciffara, 1984). A currently used criterion for in vivo release is the effectiveness of drugs with neurotransmitterreleasing properties [amphetamine (Zetterstrom et al., 1983; Imperato and Di Ciffara, 1984), fenfluramine (Carboni and Di Chiara, 1989), p-chloroamphetamine (Sharp et al., 1986; Kalrn et al., 1988)]. However, the releasing action of these drugs is TTX-insensitive and Ca2+-independent, in aggreement with a direct displacing action on intraneuronal amine pools (Carboni and Di Chiara, 1989; Carboni et al., 1989c). Thus, the effect of these drugs is not a criterion for physiological in vivo transmitter release. Similar considerations apply to criteria utilizing drugs which interfere with the synthesis, metabolism or compartimentalization of the transmitter (Imperato and Di Chiara, 1984). Using the above criteria for evaluating the various dialysis methods it becomes clear that much in this field has still to be done. For example, no information is available about the Ca 2÷ dependency and TTX sensitivity of neurotransmitter release estimated with concentric probes and with commercially available dialysis probes. In contrast, criteria for a physiological in vivo neurotransmitter release are satisfied by acute and chronic transcerebral probes in different laboratories and for different transmitters [DA (Imperato and Di Ciffara, 1984; Westerink and De "Cries, 1989), NA (L'Heureux et al., 1986), ACh (Consolo et al. 1987; Damsma et al., 1987), 5-HT (Carboni and Di Chiara, 1989) and by U-probes only after chronic implantation (Kalrn et al., 1988; Westerink and De Vries, 1989)].
Some applications of brain dialysis Brain dialysis in freely-moving rats has given major contributions to the knowledge of the mechanism of action of centrally acting drugs by
32 providing direct information on the effect of the drugs on the release of specific neurotransmitters.
Receptor agonists and antagonists Brain dialysis in freely-moving, sub-chronically implanted rats has provided the long sought but never provided evidence for a stimulating effect of systemic neuroleptics on DA release (Imperato and Di Chiara, 1985). It should pointed out that the failure to obtain consistent effects by neuroleptics on DA release in anaesthetized, acutely implanted rats (Di Chiara and Waldmeier, 1984) was instrumental for developing transcerebral dialysis in sub-chronically implanted freely-moving rats (Imperato and Di Chiara, 1985). Both D-2 (Imperato and Di Chiara, 1985) and D-1 receptor antagonists (Imperato et al., 1987) stimulate DA release to a similar extent while DA metabolism is known to be more effectively stimulated by D-2 antagonists. Rapid tolerance takes place to the stimulatory effect of neuroleptics on DA release in the caudate since a second dose of neuroleptic given 3-6 h after the first fails to stimulate DA release (Di Chiara and Imperato, 1985). This process develops during the time of action of a single dose of neuroleptic and explains the observation that stimulation of DA release by classic neuroleptics is short-lasting in comparison with stimulation of DA metabolite output and with motor inhibition. Depolarization blockade of a subpopulation of DA neurons has been suggested as the mechanism of this tolerance (Di Chiara and Imperato, 1985). In contrast to neuroleptics, dopamine receptor agonists, active on D-2 receptors, reduce DA release (Imperato et al., 1988) in aggreement with their ability to inhibit dopaminergic firing activity and to stimulate DA autoreceptors. The doses at which DA-receptor agonists and antagonists (neuroleptics) affect DA release are quite low; thus, agonists like LY 171555, pergolide, apomorphine and BHT 920 act at threshold doses around 25 /~g/kg s.c. (Imperato et al., 1988) while antagonists like haloperidol and SCH 23390 act at threshold doses of 12.5-25 # g / k g s.c. (Imperato and Di Chiara, 1985; Imperato et al., 1987). These doses are in the range of those at which these drugs produce effects in the most sensitive in vivo tests
for central activity. This means that the study of DA release with brain dialysis is among the most sensitive in vivo methods for detecting drug actions at central dopaminergic receptors. By correlating drug effects on DA release to behaviour it is possible to characterize the profile of drugs acting on dopaminergic receptors and to distinguish at least 4 classes of DA agonists (Imperato et al., 1988) differing for their intrinsic activity at D-2 receptors.
Monoamine reuptake blockers and releasers Blockade of amine reuptake increases its extracellular concentrations as estimated by brain dialysis; thus, cocaine, nomifensine, methylphenidate and phencyclidine increase DA output (Church et al., 1987; Carboni et al., 1989c), desipramine increases NA output (L'Heureux et al., 1986) and chlorimipramine and indalpine increase 5-HT output in dialysates (Kal~n et al., 1988; Carboni and Di Chiara, 1989). The time-course of cocaine concentrations in the extracellular fluid sampled by dialysis appear to match quite nicely that of the increase in DA output (Hurd et al., 1988; Nicolaysen et al., 1988). Brain dialysis has provided clear-cut in vivo evidence for differentiating monoamine reuptake blockers from releasers. Thus, the effect of cocaine, phencyclidine, nomifensine on DA output (Carboni et al., 1989) and of chlorimipramine (Carboni and Di Chiara, 1989) on 5-HT output is blocked by T I X and by lack of Ca 2÷. In contrast, the effect of amphetamine (Carboni et al., 1989c), which releases DA, and of fenfluramine (Carboni and Di Chiara, 1989), which releases 5-HT, is TTX-insensitive and Ca2+-independent. The reason for this difference is readily apparent if one considers that reuptake blockers do not interfere with the release mechanism but simply prevent removal from the extracellular space of the transmitter released by nerve impulses; in contrast, amphetamine and fenfluramine, by displacing amines from vesicular pools into the cytoplasm, make them available for active transport out of the terminal by the reuptake carrier. Transmitter-precursors Administration of the precursor aminoacids (tryptophan for serotonin and tyrosine for dopa-
33 mine) is known to result in a stimulation of the synthesis of the respective transmitters because of the lack of saturation of the rate-limiting enzymes (tryptophan-hydroxylase and tyrosine-hydroxylase) by the in vivo concentration of the precursor aminoacids. Brain dialysis studies have revealed that administration of tryptophan increases extraceUular concentrations of serotonin (Carboni et al., 1989). This effect does not seem the result of an overflow of transmitter as, at least in the case of tryptophan (Carboni et al., 1989), retains the physiological characteristics of basal output, namely, calcium dependency and TITK sensitivity. These results indicate that administration of amino-acid precursors might provide an efficient means for stimulating serotonergic or dopaminergic neurotransmission in experimental or clinical conditions.
Serotonin via excitatory 5-HT3 receptors located in the VTA, might exert a permissive role for the ability of drug stimuli to activate the firing activity of DA neurons. Thus, 5-HT3 antagonists like M D L 72222 and ICS 205-930 prevent the stimulation of DA release by drugs, like morphine and nicotine, which stimulate the firing activity of mesolimbic DA neurons, but not of amphetamine, which stimulates DA release independently from neuronal firing (Carboni et al., 1989a). Correlated with this is the property of 5-HT3 antagonists to block the rewarding properties of morphine and nicotine but not of amphetamine (Carboni et al., 1989b). These results have opened the possibility of utilizing 5-HT3 antagonists in clinical conditions characterized by disorders of motivation and affection such as anxiety, drug dependence, schizo-affective syndromes, etc. etc.
Motivational properties of drugs
References
Various centrally acting drugs are provided with motivational properties which can be positive (rewarding) or negative (aversive). Drugs abused by humans show positive reinforcing properties while drugs like naloxone, picrotoxin, K-opioid agonist are aversive. Brain dialysis has provided seminal evidence of the neurochemical basis of these properties. Drugs abused by humans such a s narcotic analgetics (Di Chiara a n d Imperato,'1988); central stimulants [amphetamine, cocaine, phencyclidine (Carboni et al.,1989c)], ethanol (impetato argd Di Chiara, 1986) and nicotine (Imperato et~ ak, 1986) have in common the property of increasing extracellular DA concentrations preferentially in the limbic n. accumbens as compared to the dorsal caudate (Di Chiara and Imperato, 1988). Therefore, a property of drugs of abuse might be the ability to stimulate DA release preferentially in the mesolimbic DA system. This property is probably related to the psychomotor stimulant and, eventually, to the rewarding properties of these drugs: in fact, blockade of DA receptors (particularly of D-1 receptors) (Leone and Di Chiara, 1987) prevents the motor-stimulant and rewarding effects of drugs of abuse (Wise and Bozarth, 1987).
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