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Diffusion and uptake of dopamine in rat caudate and nucleus accumbens compared using fast cyclic voltammetry
Brain Research, 448 (1988)381-385 Elsevier
381
BRE 22899
Diffusion and uptake of dopamine in rat caudate and nucleus accumbens compared using fast cyclic voltammetry Jonathan A. Stamford 1, Zygmunt L. Kruk 1, Peter Palij 1 and Julian Millar 2 Departments of t Pharmacology and 2physiology, The London Hospital Medical College, London (U. K.)
(Accepted 9 February 1988) Key words: Dopamine uptake; Fast cyclicvoltammetry; Caudate nucleus; Nucleus accumbens; Electrical stimulation; Diffusion
Fast cyclicvoltammetry was used in the caudate and nucleus accumbens of anaesthetised rats to study the release and reuptake of dopamine following stimulation of the median forebrain bundle. Dopamine uptake was significantly slower in accumbens than caudate, indicating a lower number of functional uptake sites. This implies that dopamine may be able to diffuse further from its sites of release in nucleus accumbens than in caudate and thus may have a neuromodulator role in this region.
The sphere of effect of a neurotransmitter, in relation to its site of release, has major implications for its role within a given tissue (see ref. 12 for review). Transmitters which can act at sites distant from the synapse are unlikely to be involved in the rapid communication of neuronal information and may act in a modulatory fashion. Conversely, compounds whose action is confined to the synapse have greater potential for fast information transfer. Several factors govern the movement of neurotransmitters into and through the extracellular fluid (ECF) 21. Active uptake systems :md the volume frac6on and tortuosity of the ext'~acellular matrix all control the ability of transmitters to diffuse freely24. The present report examines the movement of dopamine (DA) in the striatal ECF. Using fast cyclic voltammetry we have, for the past few years, investigated the release and uptake of DA in the rat forebrain following electrical stimulation of the median forebrain bundle (MFB) 3°'31, In the present study we have applied the method to the simultaneous measurement of DA uptake in caudate (CPu) and nucleus accumbens (Acb). Experiments were performed in male SpragueDawley rats (250-400 g) anaesthetised with chloral
hydrate (400 mg/kg i.p.). Carbon fibre microelectrodes (exposed tip 20 x 8/zm) 2 were implanted into the Acb (AP: +3.3, L: + 1.3, V: -6.5 mm vs bregma and the corticai surface) and CPu (AP: +1.8; L: +2.8, V: -4.5 mm) according to the coordinates of Pellegrino et al. 26. Auxiliary and silver-silver chloride (Ag/AgCi) reference electrodes were placed in the neck muscle and on the skull respectively. A stimulating electrode (Rhodes SNE-100) was placed into the region of the MFB (AP: -2.2, L: + 1.2, V: -7.5 to 8.5 mm) as previously described 7. The DA release into the ECF was measured using fast cyclic voltammetry. The input voltage to the: potentiostat (+ 1 V vs Ag/AgCI, 300 V/s) was applied 20 times/s to each working electrode, giving a temp aral resolution of 50 ms. Current at the DA oxidation peak potential (+600 mV vs Ag/AgCI) was monitored using sample-and-hold circuitry 19. The output from these circuits was recorded on the disk memory of a digital storage oscilloscope (Nicolet Explorer 3). It was found to be necessary to perform data averaging to obtain adequate measurement accuracy. In each rat the averaged DA release-time profile was constructed from 5 successive MFB stimulations (50 Hz, 100-110/~A rms, 2-s train, 5 min apart).
Correspondence: J.A. Stamford. Department of Pharmacology. The London Hospital Medical College, Turner Street~ London E! 2AD. U.K.
0006-8993/88/$03.511 (~) 1988Elsevier Science Publishers B.V. (Biomedical Divisioa)
382 In each nucleus, 3 parameters were measured: (1) peak DA release (maximum ECF concentration of DA determined by post experiment calibrations), (2) peak time (the time, after cessation of stimulation, at which peak ECF DA levels are attained), and (3) DA uptake (the rate, in :~M/s, of DA uptake during the decline half-time). The values represented solely the observed r a t e s - no-assumptions were made about the number of different uptake types in either nucleus. All statistical comparisons were made by paired t-test. Fig. 1 shows representative DA release-time profiles recorded in CPu and Acb during stimulation of the MFB. In CPu the ECF DA concentration fell almost immediately upon cessation of stimulation. By contrast, DA release in Acb did not reach maximum until shortly after the end of the stimulation period. The rate of DA uptake was also slower. Table 1 shows the group data. DA release showed much variation between animals. The variation was greater in CPu than Acb, presumably because of the greater anatomical heterogeneity of this nucleus 25. Mean DA release was not significantly different in the two nuclei. The peak time, however, was more than 3 times as long in Acb
DA 1pM Pu
STIMULATION PERIOD i,
Time : seconds
Fig. 1. Dopamine (DA) release in caudate (CPu) and nucleus
accumbens (Acb) following stimulation of the median forebrain bundle (50 Hz, 100/~Arms, 2-s train) during the period of the horizontal bar. The data are in the form of current at the DA oxidation peak potential plotted against time. Representative examples from a singleexperiment are shown.
TABLE I
Release and uptake of DA All values are mean _+ S.E.M. (n = 36). P values calculated by paired t-test. Peak DA release: maximum ECF concentration of DA determined by post-experiment calibrations in vitio. Peak time: the ume, after cessation of stimulation, at which peak ECF DA levels are attained. DA uptake rate: the rate of DA uptake during the DA concentration decline half-time.
CPu Acb P
Peak DA release (/~M)
Peak time (ms)
DA uptake O~M/s)
3.79 + 0.43 2.91 + 0.25 n.s.
42 + 10 151 + 21 0.01
3.22 + 0.23 1.61 + 0.15 0.01
than CPu.(P < 0.01) and the observed rate of DA uptake was slower in Acb than CPu (P < 0.01). The peak DA release attained by a given set of stimulus parameters is, in part at least, a measure of the dopaminergic innervation density of the nuclei. Despite discrepancies between individual studies, DA levels in Acb and CPu are similar 15"33'36although striatal levels show marked regional variation 5"37. Morphologically too, there appear to be few differences between the nuclei. In both cases approximately 50% of all nerve terminals detected at the electron microscope level stain positively for tyrosine hydroxylase I. The present finding of similar DA release in both nuclei also closely matches the results of a previous study 16 and are as expected in view of the similarities in the levels of DA in the two nuclei, The principal differences between the two nuclei lie in the removal of DA from the ECF. It is unlikely that these apparent differences are the result of electrochemical artefacts since the carbon fibre working electrodes used in each nucleus had the same size and geometry. Calibrations after removal from the brain showed no differences in sensitivity or response time between electrodes from CPu or Acb. Electrical crosstalk between working electrodes was undetectable. The electrical properties of nigrostriatal and mesolimbic DA ~erves show little difference. Basal firing rates, optimal stimulation frequency, conduction velocity, and action potential duration are similar in both pathways H).11.16.34. In both cases action potentials invade the nerve terminals in CPu and Acb within about 10 ms of their initiation in the MFB, too fast to account for the observed differences.
383 TABLE II Diffusion of DA
Mean diffusion distances were calculated usingthe equation I = (Dt) ~ where ! = distance, D = "apparent" diffusion coefficient for DA, and t = peak time as previously described~7"32. Study
D
Calculated diffusion distances Oim) CPu
Rice et al.2~ 6.8 × 10 -7 cm2s -I 1.7 Dayton et al.3 2 x l0 -ocm2s-I 2.9 Gerhardt and Adams~ 6 × l0-6cm2s-I 5.0
Acb
3.2 5.5 9.5
One major difference in the control of the two DA pathways lies in the neurotml feedback loop. While the substantia nigra receives a reciprocal topographic innervation from the CPu 6 the striatofugal efferents from Acb project mainly to A9 rather than A10 cell groups 22"29. Despite these differences, both pathways show marked depression of basal firing rates immediately after cessation of a train of stimulation t8"35. Thus, continued firing of the cells can be excluded as a cause of the different time courses. The simplest explanation of the data is that there are differences in the uptake and diffusion of DA in the ECF of the two nuclei. Kuhr et al. 16, also using voltammetric detection of stimulated DA release, stated that the decline in ECF DA concentration was slower in Acb than CPu. A recent comparative study of in vitro DA uptake in CPu and Acb slices 2° reported similar findings. Despite similar Km values for DA (0.635/~M: CPu, 0.514/~M: Acb) there was a much lower V°m,~xvalue in Acb (36 pmol/mg protein/5 min) than CPu (183 pmol/mg protein/5 min). Since the Vmaxvalue is a direct measure of the number of uptake sites ~3the data indicate that there are fewer in Acb than CPu. Such a finding provides a good explanation for the data of the present study. The existence of fewer uptake sites in relation to the innervation d~nsity would be expected to influence the mass transport of DA through the ECF of Acb. This is reflected in the longer time to peak observed in Acb. If one assumes that neuronal activity essentially stops upon cessation of stimulation ~8'35the lag period reflects the average time that DA takes to reach the detector electrode from its release sites. A longer time thus represents a greater distance. With a knowledge of the apparent diffusion coefficient (D)
for DA in vivo the distances can be estimated. Table II shows values for the distance based on the values for D in the literature 3"9"2s. Despite wide variation in the reported value of D, it is clear that DA is being measured following release from sites within a few microns of the electrode surface and that the distance is greater in Acb than CPu. It should be stressed that these are apparent and not real values of D. The real diffusion coefficient of a compound is an intrinsic physical property which can be altered by pH, ionic strength and charge. However the apparent diffusion coefficient in vivo is also influenced by the volume fraction, tortuosity and uptake processes of the medium in which diffusion occurs. In all probability the greater apparent diffusion distance of DA in Acb than CPu is the result of the lower density of uptake sites. In other words, if there are fewer uptake sites removing DA it should exist longer in the ECF, as observed. A longer residence time would thus allow DA to travel further while propelled by the same concentration gradient. However, one cannot dismiss the possibility that there are differences in the ECF volume or in the tortuosity of the diffusion path in the two nuclei. The ECF volume fraction is typically about 20% 23.27.38. Although minor ECF volume changes occur with neuronal dep~larisation 4a4 it is generally held that volume fraction and tortuosity do not vary much across species and preparations 8"24. Thus, although this cannot be excluded as a cause of the greater apparent diffusion distance in Acb, the most likely explanation remains the lower density of uptake sites. In conclusion, the present study demonstrates a lower density of functional DA uptake sites in Acb than CPu. Consequently the mass transport of DA is greater in the ECF of Acb. DA may therefore act over a greater distance than in CPu, possibly behaving more as a neuromodulator. It may be possible to exploit this difference by means of selective pharmacological intervention. The results also demonstrate the validity of fast cyclic voltammetry as a method to study DA function with high spatial and temporal resolution. This research was funded by the Weilcome Trust. We thank Dr. Margaret Rice for data on extracellular fluid volume. Thanks also go to Mary Miller for typing tht~ manuscript.
384 1 Arluison, M., Dietl, M. and Thibault, J., Ultrastructural morphology of dopaminergic nerve terminals and synapses in the striatum of the rat using tyrosine hydroxylase immunocytochemistry: a topographical study, Brain Res. Bull., 13 (1984) 269-285. 2 Armstrong James, M. and Millar, J., Carbon fibre microelectrodes, J. Neurosci. Methods, 1 (1975)279-287. 3 Dayton, M.A., Ewing, A.G. and Wightman, R.M., Diffusion processes measured at microvoltammetric electrodes in brain tissue, J. Electroanal. Chem., 146 (1983) 189-200. 4 Dietzei, I. and Heinemann, U., Dynamic variations of the brain cell microenvironment in relation to neuronal hyperactivity. Ann. N. Y. Acad. Sci., 481 (1987)72-84. 5 Di Paolo, T., Daigle, M. and Du Pont, A., Distribution of dopamine in 35 subregions of the rat caudate-putamen: a high performance liquid chromatography with electrochemical detection analysis, J. Can. Sci. Neurol., 9 (1982) 421-427. 6 Dray, A.. The striatum and substantia nigra: a commentary on their relationships, Neuroscience, 4 (1979) 14•7-1439. 7 Ewing. A.G., Bigelow, .I.C. and Wightman, R.M., Direct in vivo monitoring of d~3pamine released ftvm two striatal compartments, Science, 221 (1983) 169-171. 8 Fenstermacher, J.D. and Padlak, C.S., The exchange of materials between cerebrospinal fluid and brain. In H.F. Cserr, J.D. Fenstermacher and V. Fend (Eds.), The Fluid Environment of the Brain, Academic, New York, 1975, pp. 201-214. 9 Gerhardt, G. and Adams, R.N., Determination of diffusion coefficients bv flow injection analysis, Anal. Chem., 54 (1982) 2618-2620. 10 Grace, A.A. and Bunney, B.S., lntraceliular and extraceilular electrophysiology of nigral dopaminergic neurones. I. Identification and charaeterisation, Neuroscience, 10 (1983) 301-315. 11 Guyenet, P.G. and Aghajanian, G.K., Antidromic identification of dopaminergic and other output neurones of the rat substantia nigra, Brain Research, 150 (1978) 69-84. 12 Herkenham, M., Mismatches between neurotransmitter and receptor Iocalisations in brain: observations and implications, Neuroscience, 23 (1987) 1-38. 13 Horn, A.S., Characteristics of dopamine uptake. In A.S. Horn, J. Korf and B.H.C. Westermk (Eds.), The Neurobiology of Dopamine, Academic, London, 1979, pp. 217-235. 14 Kent, T.A., Nagy, G., Oke, A.F., Preskorn, S.H. and Adams, R.N., Effect of CO, on a brain extracellular space marker and evidence of its neuronal modulation, Brain Research, 342 (1985) 141-144. 15 Kilts, C.D., Breese, G.R. and Mailman, R.B., Simultaneous quantification of dopamine, 5-hydroxytryptamine, and four metabolically related compounds by means of reversed-phase high performance liquid chromatography with electrochemical detection, J. Chromatogr., 225 (1981) 347-357. 16 Kuhr, W.G., Bigelow, J.C. and Wightman, R.M., In vivo comparison of the regulation of releasable dopam[ne in the caudate nucleus and nucleus accumbens, J. Neurosci., 6 (1986) 974-982. 17 Kuhr, W.G. and Wightman, R.M., Real-time measurement of dopamine release in rat brain, Brain Research, 381 (1986) 168-171. 18 Kuhr, W.G., Wightman, R.M. and Rebec, G.V., Dopaminergic neurones: simultaneous measurements of dopa-
mine release and single-unit activity during stimulation of the median forebrain bundle, Brain Research, 418 (1986) 122-128. 19 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological, and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the median forebrain bundle, Eur. J. Ph,lrmacol., 109 (1985) 341-348. 20 Missale, C., Castelletti, L., Govoni, S., Spano, P.F., Trabucchi, M. and Hanbauer, I., Dopamine uptake is differentially regulated in rat striatum and nucleus accumbens, J. Neurochem., 45 (1985) 51-56. 21 Moghaddam, B. and Adams, R.N., Recent developments of in vivo voltammetry: applications to studies of chemical dynamics in the neuronal microenvironment, Ann. N.Y. Acad. Sci., 481 (1987) 106-114. 22 Nauta, W.J.H. and Domesick, V.B., Afferent and efferent relationships of the basal ganglia. In Function of the Basal Ganglia, London Ciba Foundation Symposium 107, i:itman, London, 1984, pp. 3--29. 23 Nevis, A.H. and Collins, G.H., Electrical impedance and volume changes in brain during preparation for electron microscopy, Brain Research, 5 (1967) 57-85. 24 Nicholson, C. and Rice, M.E., The migration of substances in the neuronal microenvironment, Ann. H.Y. Acad. Sci., 481 (1987) 55-68. 25 Olson, L., Seiger, A. and Fuxe, K., Heterogeneity of striata! and limbic dopam;ne innervation: highly fluorescent islands in developing and adult rats, Brain Research, 44 (1972) 283-288. 26 Pellegrino, L.J., Peilegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Appleton-CenturyCrofts, New York, 1979. 27 Rees, S., Cragg, B.G. and Everitt, A.V., Comparison of extracellular space in the mature and ageing brain using a new technique, J. Neurol. Sci., 53 (1982) 347-357. 28 Rice, M.E., Gerhardt, G.A., H~erl, P.M., Nagy, G. and Adams, R.N., Diffusion coefficients of neurotransrlitters and their metabolites in brain extracellular fluid space, Neuroscience, 15 (1985) 891-902. 29 Scheel-Kruger, J., Dopamine-GABA interactions: evidence that GABA transmits, modulates and mediates dopaminergic functions in the basal ganglia and limbic system, Acta Neurol. Scand., 73 Suppl. 107 (1986). 30 Stamford, J.A., Kruk, Z.L., Millar, J. and Wightman, R.M., Striatal dopamine uptake in the rat: in vivo analysis by fast cyclic voltammetry, Neurosci. Lett., 51 (1984) 133-138. 31 Stamford, J.A., Kruk. Z.L. and Millar, J., In vivo voltammetric characterisation of low affinity striatal dopamine uptake: drug inhibition profile and relation to dopaminergic innervation density, Brain Research, 373 (1986) 85-91. 32 Stamford, J.A., Kruk, Z.L. and Millar, J., Sub-second striatal dopamine release measured by in vivo voltammetry, Brain Research, 381 (1086) 351-355. 33 Versteeg, D.H.G., Van Der Gu~ten, J., De Jong, W. and Palkovits, M., Regional concentrations of noradrenaline and dopamine in rat brain, Brain Research, 113 (1976) 563-574. 34 Wang, R.Y., Dopaminergic neurones in the rat ventral tegmental area. I. Identification and characterisation, Brain Res. Rev., 3 (1981) 123-140. 35 Wang, R.Y., Dopaminergic neurones in the rat ventral teg-
385 mental area. I!. Evidence for autoregulation, Brain Res. Rev., 3 fi981) 141-151. 36 Westerink, B.H.C. and Mulder, T.B.A., Determination of picomole amounts of dopamine, noradrenaline, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenyi acetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid in nervous tissue after one-step purification of sephadex G-10, using high performance. Iiqui~ chromatography with a novel
type of electrochemical detection, J. Neurochem., 36 (1981) 1449-1462. 37 Widmann, R. and Sperk, (3., Topographical distribution of amines and major amine metaboiites in the rat stfiatum, Brain Research, 367 (1986) 244-249. 38 Woodward, D.L., Reed, D.J. and Woodbury. D.M., Extracellular space of rat cerebral cortex, Am. J. Physiol., 212 (1967) 367-370.
,t
381
BRE 22899
Diffusion and uptake of dopamine in rat caudate and nucleus accumbens compared using fast cyclic voltammetry Jonathan A. Stamford 1, Zygmunt L. Kruk 1, Peter Palij 1 and Julian Millar 2 Departments of t Pharmacology and 2physiology, The London Hospital Medical College, London (U. K.)
(Accepted 9 February 1988) Key words: Dopamine uptake; Fast cyclicvoltammetry; Caudate nucleus; Nucleus accumbens; Electrical stimulation; Diffusion
Fast cyclicvoltammetry was used in the caudate and nucleus accumbens of anaesthetised rats to study the release and reuptake of dopamine following stimulation of the median forebrain bundle. Dopamine uptake was significantly slower in accumbens than caudate, indicating a lower number of functional uptake sites. This implies that dopamine may be able to diffuse further from its sites of release in nucleus accumbens than in caudate and thus may have a neuromodulator role in this region.
The sphere of effect of a neurotransmitter, in relation to its site of release, has major implications for its role within a given tissue (see ref. 12 for review). Transmitters which can act at sites distant from the synapse are unlikely to be involved in the rapid communication of neuronal information and may act in a modulatory fashion. Conversely, compounds whose action is confined to the synapse have greater potential for fast information transfer. Several factors govern the movement of neurotransmitters into and through the extracellular fluid (ECF) 21. Active uptake systems :md the volume frac6on and tortuosity of the ext'~acellular matrix all control the ability of transmitters to diffuse freely24. The present report examines the movement of dopamine (DA) in the striatal ECF. Using fast cyclic voltammetry we have, for the past few years, investigated the release and uptake of DA in the rat forebrain following electrical stimulation of the median forebrain bundle (MFB) 3°'31, In the present study we have applied the method to the simultaneous measurement of DA uptake in caudate (CPu) and nucleus accumbens (Acb). Experiments were performed in male SpragueDawley rats (250-400 g) anaesthetised with chloral
hydrate (400 mg/kg i.p.). Carbon fibre microelectrodes (exposed tip 20 x 8/zm) 2 were implanted into the Acb (AP: +3.3, L: + 1.3, V: -6.5 mm vs bregma and the corticai surface) and CPu (AP: +1.8; L: +2.8, V: -4.5 mm) according to the coordinates of Pellegrino et al. 26. Auxiliary and silver-silver chloride (Ag/AgCi) reference electrodes were placed in the neck muscle and on the skull respectively. A stimulating electrode (Rhodes SNE-100) was placed into the region of the MFB (AP: -2.2, L: + 1.2, V: -7.5 to 8.5 mm) as previously described 7. The DA release into the ECF was measured using fast cyclic voltammetry. The input voltage to the: potentiostat (+ 1 V vs Ag/AgCI, 300 V/s) was applied 20 times/s to each working electrode, giving a temp aral resolution of 50 ms. Current at the DA oxidation peak potential (+600 mV vs Ag/AgCI) was monitored using sample-and-hold circuitry 19. The output from these circuits was recorded on the disk memory of a digital storage oscilloscope (Nicolet Explorer 3). It was found to be necessary to perform data averaging to obtain adequate measurement accuracy. In each rat the averaged DA release-time profile was constructed from 5 successive MFB stimulations (50 Hz, 100-110/~A rms, 2-s train, 5 min apart).
Correspondence: J.A. Stamford. Department of Pharmacology. The London Hospital Medical College, Turner Street~ London E! 2AD. U.K.
0006-8993/88/$03.511 (~) 1988Elsevier Science Publishers B.V. (Biomedical Divisioa)
382 In each nucleus, 3 parameters were measured: (1) peak DA release (maximum ECF concentration of DA determined by post experiment calibrations), (2) peak time (the time, after cessation of stimulation, at which peak ECF DA levels are attained), and (3) DA uptake (the rate, in :~M/s, of DA uptake during the decline half-time). The values represented solely the observed r a t e s - no-assumptions were made about the number of different uptake types in either nucleus. All statistical comparisons were made by paired t-test. Fig. 1 shows representative DA release-time profiles recorded in CPu and Acb during stimulation of the MFB. In CPu the ECF DA concentration fell almost immediately upon cessation of stimulation. By contrast, DA release in Acb did not reach maximum until shortly after the end of the stimulation period. The rate of DA uptake was also slower. Table 1 shows the group data. DA release showed much variation between animals. The variation was greater in CPu than Acb, presumably because of the greater anatomical heterogeneity of this nucleus 25. Mean DA release was not significantly different in the two nuclei. The peak time, however, was more than 3 times as long in Acb
DA 1pM Pu
STIMULATION PERIOD i,
Time : seconds
Fig. 1. Dopamine (DA) release in caudate (CPu) and nucleus
accumbens (Acb) following stimulation of the median forebrain bundle (50 Hz, 100/~Arms, 2-s train) during the period of the horizontal bar. The data are in the form of current at the DA oxidation peak potential plotted against time. Representative examples from a singleexperiment are shown.
TABLE I
Release and uptake of DA All values are mean _+ S.E.M. (n = 36). P values calculated by paired t-test. Peak DA release: maximum ECF concentration of DA determined by post-experiment calibrations in vitio. Peak time: the ume, after cessation of stimulation, at which peak ECF DA levels are attained. DA uptake rate: the rate of DA uptake during the DA concentration decline half-time.
CPu Acb P
Peak DA release (/~M)
Peak time (ms)
DA uptake O~M/s)
3.79 + 0.43 2.91 + 0.25 n.s.
42 + 10 151 + 21 0.01
3.22 + 0.23 1.61 + 0.15 0.01
than CPu.(P < 0.01) and the observed rate of DA uptake was slower in Acb than CPu (P < 0.01). The peak DA release attained by a given set of stimulus parameters is, in part at least, a measure of the dopaminergic innervation density of the nuclei. Despite discrepancies between individual studies, DA levels in Acb and CPu are similar 15"33'36although striatal levels show marked regional variation 5"37. Morphologically too, there appear to be few differences between the nuclei. In both cases approximately 50% of all nerve terminals detected at the electron microscope level stain positively for tyrosine hydroxylase I. The present finding of similar DA release in both nuclei also closely matches the results of a previous study 16 and are as expected in view of the similarities in the levels of DA in the two nuclei, The principal differences between the two nuclei lie in the removal of DA from the ECF. It is unlikely that these apparent differences are the result of electrochemical artefacts since the carbon fibre working electrodes used in each nucleus had the same size and geometry. Calibrations after removal from the brain showed no differences in sensitivity or response time between electrodes from CPu or Acb. Electrical crosstalk between working electrodes was undetectable. The electrical properties of nigrostriatal and mesolimbic DA ~erves show little difference. Basal firing rates, optimal stimulation frequency, conduction velocity, and action potential duration are similar in both pathways H).11.16.34. In both cases action potentials invade the nerve terminals in CPu and Acb within about 10 ms of their initiation in the MFB, too fast to account for the observed differences.
383 TABLE II Diffusion of DA
Mean diffusion distances were calculated usingthe equation I = (Dt) ~ where ! = distance, D = "apparent" diffusion coefficient for DA, and t = peak time as previously described~7"32. Study
D
Calculated diffusion distances Oim) CPu
Rice et al.2~ 6.8 × 10 -7 cm2s -I 1.7 Dayton et al.3 2 x l0 -ocm2s-I 2.9 Gerhardt and Adams~ 6 × l0-6cm2s-I 5.0
Acb
3.2 5.5 9.5
One major difference in the control of the two DA pathways lies in the neurotml feedback loop. While the substantia nigra receives a reciprocal topographic innervation from the CPu 6 the striatofugal efferents from Acb project mainly to A9 rather than A10 cell groups 22"29. Despite these differences, both pathways show marked depression of basal firing rates immediately after cessation of a train of stimulation t8"35. Thus, continued firing of the cells can be excluded as a cause of the different time courses. The simplest explanation of the data is that there are differences in the uptake and diffusion of DA in the ECF of the two nuclei. Kuhr et al. 16, also using voltammetric detection of stimulated DA release, stated that the decline in ECF DA concentration was slower in Acb than CPu. A recent comparative study of in vitro DA uptake in CPu and Acb slices 2° reported similar findings. Despite similar Km values for DA (0.635/~M: CPu, 0.514/~M: Acb) there was a much lower V°m,~xvalue in Acb (36 pmol/mg protein/5 min) than CPu (183 pmol/mg protein/5 min). Since the Vmaxvalue is a direct measure of the number of uptake sites ~3the data indicate that there are fewer in Acb than CPu. Such a finding provides a good explanation for the data of the present study. The existence of fewer uptake sites in relation to the innervation d~nsity would be expected to influence the mass transport of DA through the ECF of Acb. This is reflected in the longer time to peak observed in Acb. If one assumes that neuronal activity essentially stops upon cessation of stimulation ~8'35the lag period reflects the average time that DA takes to reach the detector electrode from its release sites. A longer time thus represents a greater distance. With a knowledge of the apparent diffusion coefficient (D)
for DA in vivo the distances can be estimated. Table II shows values for the distance based on the values for D in the literature 3"9"2s. Despite wide variation in the reported value of D, it is clear that DA is being measured following release from sites within a few microns of the electrode surface and that the distance is greater in Acb than CPu. It should be stressed that these are apparent and not real values of D. The real diffusion coefficient of a compound is an intrinsic physical property which can be altered by pH, ionic strength and charge. However the apparent diffusion coefficient in vivo is also influenced by the volume fraction, tortuosity and uptake processes of the medium in which diffusion occurs. In all probability the greater apparent diffusion distance of DA in Acb than CPu is the result of the lower density of uptake sites. In other words, if there are fewer uptake sites removing DA it should exist longer in the ECF, as observed. A longer residence time would thus allow DA to travel further while propelled by the same concentration gradient. However, one cannot dismiss the possibility that there are differences in the ECF volume or in the tortuosity of the diffusion path in the two nuclei. The ECF volume fraction is typically about 20% 23.27.38. Although minor ECF volume changes occur with neuronal dep~larisation 4a4 it is generally held that volume fraction and tortuosity do not vary much across species and preparations 8"24. Thus, although this cannot be excluded as a cause of the greater apparent diffusion distance in Acb, the most likely explanation remains the lower density of uptake sites. In conclusion, the present study demonstrates a lower density of functional DA uptake sites in Acb than CPu. Consequently the mass transport of DA is greater in the ECF of Acb. DA may therefore act over a greater distance than in CPu, possibly behaving more as a neuromodulator. It may be possible to exploit this difference by means of selective pharmacological intervention. The results also demonstrate the validity of fast cyclic voltammetry as a method to study DA function with high spatial and temporal resolution. This research was funded by the Weilcome Trust. We thank Dr. Margaret Rice for data on extracellular fluid volume. Thanks also go to Mary Miller for typing tht~ manuscript.
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