Cocaine-sensitive and -insensitive dopamine uptake in prefrontal cortex, nucleus accumbens and striatum

Neurochem Int. Vol. 23, No. I, pp. 61~69, 1993 Printed m Great Britain All rights reserved

0197-0186/93 $6 00+0.00 Copyright ~ 1993Pergamon Press Ltd

COCAINE-SENSITIVE A N D -INSENSITIVE DOPAMINE UPTAKE IN PREFRONTAL CORTEX, NUCLEUS ACCUMBENS A N D STRIATUM J. D. ELSWORTH,* J. R. TAYLOR, P. BERGERt and R. H. ROTH Departments of Pharmacology and Psychiatry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, U.S.A. ( Recewed 27 July 1992 ; accepted 19 November 1992)

AMtraet--Behavioral studies have indicated that the reinforcing effects of cocaine are dependent on inhibition of dopamine uptake in nucleus accumbens and prefrontal cortex. As it has been suggested that dopamine uptake and cocaine-inhibition of dopamine uptake may differ in nucleus accumbens, prefrontal cortex and striatum, we have further characterized dopamine uptake and its susceptibility to inhibition in these three regions. Dopamine uptake was resolved into two processes, which accounts for some of the apparent reported regional differences in sensitivity of dopamine to mhibition by cocaine. One, which is probably associated with uptake into dopaminergic terminals, was sensitive to 6-hydroxydopamine lesions, cocaine, GBR 12909 or ouabain and was dependent on temperature and sodium ion concentration ; this was responsible for most of the observed uptake m tissue from striatum and nucleus accumbens, but not from prefrontal cortex. There appeared to be no regional difference in susceptibility of this mode of dopamine uptake to either cocaine or GBR 12909. The other type of dopamine uptake, which represented a significant proportion of the total in prefrontal cortex, but not in striatum or nucleus accumbens, was relatively insensitive to cocaine, GBR 12909 and ouabain and was dependent on temperature, but not sodium ion concentration. In addition, the cocaine-insensitive dopamine uptake was more sensitive to inhibition by dopamine than serotonin, but did not distinguish between dopamine and norepinephrine. The occurrence of cocaine-sensitive dopamine uptake in all examined regions and its equal sensitivity to cocaine and GBR 12909 is consistent with the involvement of nucleus accumbens and/or prefrontal cortex in the reinforcing effects of cocaine. The possibility is discussed that cocaine-insensitwe dopamine uptake may be associated with astroglia and play an important role in regulating extracellular dopamine concentrations when synaptic levels are elevated.

The powerful reinforcing properties of cocaine underlie its high potential for abuse. The mechanism thought to be involved is the ability of cocaine to inhibit the reuptake of dopamine (DA) into neurons. A substantial body of data favors the hypothesis that the crucial site of action of cocaine is the A10 D A neurons that terminate in the nucleus accumbens and prefrontal cortex, rather than the A9 D A neurons that innervate the dorsal striatum (see Wise, 1988 ; K o o b and Bloom, 1988). However, the majority of studies on D A uptake are performed with striatal tissue, with the implicit assumption that this will reliably reflect the uptake process in other regions. In fact, there have been indications that the D A uptake complex or inhibition o f D A uptake may be different in extrastriatal regions of brain that receive dopaminergic

innervation, such as nucleus accumbens (Missale et al., 1985 ; Lew et al., 1991), prefrontal cortex (Hadfield and Nugent, 1983; Izenwasser et al., 1990; Hitri et aL, 1991), olfactory bulb (Hadfield and Nugent, 1983) and median eminence and posterior pituitary (Demarest and Moore, 1979). Thus, we have characterized further the uptake of D A in the prefrontal cortex and nucleus accumbens and the susceptibility of D A uptake in these regions both to cocaine and a more specific inhibitor of D A uptake, G B R 12909 (Berger et al., 1985).

EXPERIMENTALPROCEDURES Matertals Cocaine hydrochloride and desipramine hydrochloride (DMI) were purchased from Sigma Chemical Co. (St Louis, MO). GBR 12909 and nialamide were purchased from Research Biochemicals Inc. (Natick, MA). [3H]DA ([7-3H] ; 30-40 Ci/mmol) was purchased from Dupont NEN (Boston,

* To whom correspondence should be addressed. tPresent address, V. A. Medical Center, Department of Psychiatry, San Francisco, CA 94121. 61

62

J. 1) ELSW()RIH{'/ C,'[

MA). Ouabam octahydratc was purchased from Aldrich ChemmaI Co (Milwaukee, Wl) I~,plaLe as'su v Male Sprague Dawley rats (275 350 g ; Camm) were used Brain regions for assay were removed bilaterally, as preciously described (Deutch et al., 1985) As the amount of tissue from the nucleus accumbens (3 mg wet wt) and prefrontal corte~ (20 mg wet wt) of one rat was insufficient for the anal~sis, pooled tissue from 6 rats was used for these regions in each assay. Dissected tissue was placed in ice-cold sucrose (0.32 M) and homogenized, using a glass homogemzing tube and a teflon pestle. Following centrifugation at 1000 g l\~r 10 mln at 4 C, the pellet was resuspended m (t.32 M sucrose and centrifuged at 23,000 g for 20 min at 4 C The resulting P2 pellet was resuspended m ice-cold sucrose (0.32 M) and used in the assay. A 15 min period was allowed to elapse before the P2 preparation was added to assay tubes, as we and others (Cooper and Carlson, 1983) have found that an enhanced, but unstable, rate of uptake occurs immediately after resuspension of the P2 pellet The assay was performed in a Krebs- phosphate buffer (pH 7.4, adjusted with HC1 when necessary) thai contained the following (final concentrations given) 126 mM NaC1, 16 mM Na2HPO4, 4 8 mM KCI, 1.2 mM MgSO4, 0 75 mM CaCI> 1 1 mM ascorbic acid, 10 mM glucose and 12 5 #M nialamide. In some instances, when the effect of sodium ton concentration was tested, sodium chloride was replaced by choline chloride. To prevent uptake into norepinephrme terminals, DMI was added to the assay. As DMI inhibits the uptake of norepmephrine 1000 times more potently than DA (Horn et al, 1971), the concentration used to ensure blockade of the norepmephrme transporter m the assay (100 riM) was that which inhibited DA uptake 5 10% in striatal tissue; the IC~,~ alue for DMI inhibition of DA uptake was 5 ILM. The initial assay' mixture comprised duplicates of the following' 730/,1 buffer, 50 td DM1, 50 1~1 cocaine (or water) or GBR 12909 (or its ~ehlcle, I mM HCII and 50 /~1 second inhibitor (or vehicle) These components were mixed m each tube on ice prior to the addition of 100 /tl tissue preparation, which contained 50 100 #g protein for striatum and nucleus accumbens and 150 200/~g protein for prefrontal cortex (Lowry et al, 1951 ). After incubation at 37 C for 5 mm, 20 Id [~H]DA (40 nM) was added. Two minutes later the uptake was terminated by rapid filtration through glass fiber filters (Whatman GF/C) In each assay the temperature-independent uptake or accumulation of [3H]DA was assessed by incubation of the assay components on ice Cocaine-sensitive [~H]DA uptake was detined as accumulation that was sensitl~e to 100 #M cocaine Uptake was found not to be hnear after 3 mm mcubatmn at 3 7 C The retained portion was washed 3 times with 3 ml ice-cold buffer. The filtration and washing took less than 15 s. The radioactivity trapped on the filter was counted after addition of 5 ml scintillation fluid Solutmns of GBR 12909 (in 1 mM HCII were used on the day of preparation, as they were found to be unstable on storage. HPLC analysis (Elsworth el al., 1989) was used to determine tissue DA and NE concentration and to check periodically the stability of [~H]DA Ci'yoprescq'l'UtlOH Since cryopreserved brain tissue has been shown to retain the ability to take up and release varmus neurotransmitters (Hardy et al., 1983: Haberland and Hetey. 1987: Miiller-

SchwemltTel, 1988, Schwltzkowsky and Hetty, 1989). cl5o preserved tissue wa,, u,,ed m some of the uptake assay~ after prdlmmary m\.estlgatlons indicated thai there was nt, dflTerence from non-cryopreserved tissue in terms of Ihc rate of uptake of DA (mean uptake pmol,,mg proteul 2 m m + SE at 4(1 nM DA, cryopreserved strlatum 42.8 ± 5.9./~ 7. fresh striatum 38.1_+3.1, n = 11) and m the susccpllhhty of DA uptake to lnhlbltors ([:lg. It Subsequent cstunate~ o[ the Km and 1",,~, for DA uptake m fresh and cryopreserved strlatum (from 3 assays whlch incorporated a range of concentratlons DA) showed no statistically significant changes m l ",,,~, or K,,,, although a tendency toward a decrease m I ",.... was noted The use of cryopreserved brain samples greatly expands the number of tissues that can be studied postmortem, by allowing more than one uptake assa\ to be performed on tissues fronl each animal. Without cryopreservation, uptake studies ha~e to be performed munediately on freshly collected tissue and so are limited to the number of samples thai can be processed m a single assa}. The cryopreservatmn procedure involved transfer of the tissue to a liquid nitrogen proof polypropylene tube containing [ ml ice-cold 0 32 M sucrose containing 5% dimeth.~lsulfoxlde, as a cryoprotectant (Schwilzkowsky and [fetey, 1989) The amount of tissue in each tube was between 25 and 75 mg wet wt After 10 20 min, excess solution a b m c the tissue was removed and the tube placed m an adjustable freezing tray (Taylor-Wharton, Theodore, ALL The freezing tray' was inserted in a compatible liquid nitrogen tank (35 VHC, Taylor Wharton, Theodore, ALL The tray wa~ lowered into the tank to achlew the desired cooling rate At the end of the coohng period the tube was removed from the tray and stored m contact with liquid nitrogen To tha~ a sample prior to use, the lube was removed from the liquid nitrogen container and the frozen tissue transferred Io 1 ml 0.32 M sucrose at 37 C m a shaking water bath Aftra I mm, the sucrose solution was removed and replaced with ice-cold sucrose m preparation for homogenization, as a b m e The temperature and coohng rate during cryopreservation were standardized b y ( l ) loading the tray. with a hxed nulnber of samples, (2) having a lixed w>lume of hqmd nitrogen m the container and (3) lowering the tray into the tank using a predetermined schedule The cooling (0.5 10 ( ' r a m to - 3 0 C) was chosen based on indications m the literature (Farrant, 1980) and our prehmmary studies. The freezing cycle revolved lowering the tray 3 trams, leawng 15 rain between each stage, so that the procedure took 45 mm PreJ/'onla[ COl lea [{2~,1011 The following procedure was used to create a lesion of the DA mnervation to the prefrontal cortex which spared the norepinephrmc lnnervatlon Rats (300 400 g) were anesthetized with chloral hydrate (400 mg/kg, 3 ml/kg J.p ). Thirty minutes before infusion of 6-hydroxydopamlne hydrobromide (6-OHDA, Sigma Chemical Co., St Louis, MO), DMI (15 mg/kg, 0.1 ml/kg l.p., Sigma Chemical Co., St Louts, MO) was administered. Bilateral infusions of 6O H D A were made at two sites in the prefrontal cortex (site I AP+3.1 mm and M L + 0 75 mm from bregma, D V - 4 7 mm relative to dura: site 2 A P + 3 . 5 mm and ML + 0 75 mm from bregma, DV - 4 4 mm relative to dural Two injection cannulae (28 gauge) were cemented together I 5 mm apart, to enable the two sites with the same AP coordinates to bc refused simultaneously. Due to the susceptibility ot 6-OHDA to oxidation, a fresh solution of 6-

Cocaine inhibition of dopamine uptake 100' 80'

60

40-~

20

;

cryo~scrv~

o

-9

-8

-7

-6 -5 -4 -3 Cocaine(logM) Fig. 1. No difference m susceptibihty of [3H]DA uptake to inhibition by cocaine m fresh or cryopreserved striatum (mean + SE of 3 separate determinations). OHDA (8/~g//ll in 0.05% ascorbic acid, double the infused concentration) was prepared immediately before each infusion. To protect NE terminals from neurotoxic effects, 6-OHDA was infused with DMI (final concentration, 4 pg base of each drug in 1/~1of saline containing 0.025% ascorbic acid). The 6-OHDA/DMI infusion was preceded, in the same syringe, by 4 #g DMI in 0.5/tl saline. The two solutions were separated by a small air bubble ( < 0.1 #1), and delivered at 0.2 #l/min. In some cases, saline was substituted for DMI. Two weeks later animals were sacrificed. The prefrontal cortex dissected and processed for assay of either DA and NE content by HPLC (Elsworth et al., 1989) or [3H]DA uptake. Slattsttcs

Data were analyzed by one-way or two-way ANOVA, followed by Student Newman-Keuls test where appropriate (SuperANOVA program for Macintosh computer, Abacus Concepts Inc., Berkeley, CA). RESULTS When administered in combination with D M I (peripherally and locally), 6 - O H D A resulted in a significant loss of DA, but not NE, in the prefrontal cortex (Table 1). [3H]DA uptake in prefrontal cortex was significantly reduced ( 3 4 + 8 % of control) in Table 1. DA and NE concentrationsin prefrontalcortex after lesions with 6-OHDAand DMI Group Control Sahne+6-OHDA DMI+6-OHDA

NE level (% of control) 100+4.8 (12) 6.3_+5.3 (5)* 86.1 _+6.3(7)

DA level (% of control) 100± 10.6(12) 31.0+ 16.8 (5)* 47 8_+62 (7)*

* Indicatessignificantlydifferentfrom control group. (ANOVA, Student-Newman Keuls, P < 0.05). Control value for NE, 4.16_+0.20 ng/mg protein, DA, 1.13+0.12 ng/mg protein Number in parentheses refer to number of samples used to derive mean

63

another group of rats treated similarly with 6 - O H D A and DMI. The results below derive from studies using normal rat tissue. All uptake experiments with prefrontal cortex tissue described below were performed in the presence of D M I (100 nM). When D M I was omitted from the incubation, [3H]DA uptake was increased by a factor of 1.9 + 0.2 (mean + SE of 6 assays in duplicate). A high concentration of cocaine (100 #M) or G B R 12909 (10 #M) inhibited temperature-dependent D A uptake by approx 90% in striatum and nucleus accumbens; however, in prefrontal cortex this concentration of cocaine or G B R 12909 resulted in only 50% inhibition of temperature-dependent D A uptake (Fig. 2). A two-way A N O V A (on arcsin transformed data, to normalize the distribution of percentages) showed that for cocaine inhibition of temperaturedependent DA uptake there was a significant effect of drug concentration (F = 92, df 6,90, P < 0.0001) and region ( F = 28.4, df 2,90, P < 0.0001), and a significant interaction between drug concentration and region ( F = 2.6, df 11,90, P < 0.01) (see Fig. 2 for direction of significant differences). A two-way A N O V A for G B R 12909 inhibition of temperaturedependent D A uptake showed that there was a significant effect of drug concentration ( F = 37.4, df 5,70~ P < 0 . 0 0 0 1 ) and region ( F = 67.2, df 2,70, P < 0.0001), but no interaction between drug concentration and region (F = 1.3, df 9,70, P = 0.27) (see Fig. 2 for direction of significant differences). In the presence of ouabain (250/~M) greater than 90% of temperature-dependent DA uptake in striatum and nucleus accumbens was prevented, yet when prefrontal cortex was exposed to ouabain, approx 50% of control temperature-dependent D A uptake still occurred (Fig. 3). Temperature-dependent D A uptake was markedly reduced in striatum and nucleus accumbens when the assay was conducted in a buffer containing a low concentration of sodium. In prefrontal cortex only 50% inhibition of temperature-dependent DA uptake was observed in the low sodium buffer (Fig. 3). The reduction in uptake observed in the low sodium buffer was not altered in any region when the incubation mixture contained 10/~M G B R 12909 in addition to low sodium concentration (Fig. 3). The rate of temperature-dependent DA uptake and DA concentration was highest in the striatum, intermediate in the nucleus accumbens (53% of striatum) and lowest in prefrontal cortex (2.6% of striatum) (Table 2). When analyzed in terms of cocaine-sensitive uptake this pattern of regional uptake was not altered,

,1 D EI.S~,~()e,TH et a/

64

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STR

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Fig. 3. Comparatwe effect of 10 #M GBR 12909 (n = 8- 10), 250 #M ouabain (n = 3-4), low sodium concentration (32 mM, n = 3 5) or a GBR 12909 plus low sodium concentratlon (n = 3 5) on temperature-dependent [3HIDA uptake as a percentage of control (n = 8-10) in stnatum (STR), nucleus accumbens (NAC) and prefrontal cortex (PFC). *Indicates significantly different from prefrontal cortex for that condition (ANOVA, followed by Student-Newman Keul's test, P < 0.05) In addition, all manipulations (GBR 12909, ouabam, low sodium and low sodium plus GBR) significantly reduced [3H]DA uptake compared to the respective control for that region.

Fig. 2. Inhibition of temperature-dependent [3H]DA uptake by cocaine or GBR 12909 in stnatum (;l = 6 or 7), nucleus accumbens (n = 7) and prefrontal cortex (n = 4 6).

except that the rate of D A uptake in the prefrontal cortex was only 1.0% of that occurring in the striatum (Table 2). However, there was no significant difference in the cocaine-insensitive c o m p o n e n t of D A uptake a m o n g the regions (Table 2). The cocaine-insensitive D A uptake in prefrontal

cortex was inhibited by a large c o n c e n t r a t t o n (100 l~M) of D A or norepinephrine, but not by serotonm. In fact, serotonin did not significantly affect total temp e r a t u r e - d e p e n d e n t [3H]DA uptake (Fig. 4). A two-way A N O V A o n arcsin t r a n s f o r m e d d a t a to test the effect of cocaine c o n c e n t r a t i o n a n d brain region on cocaine-sensitive D A uptake revealed a main effect o f drug c o n c e n t r a t i o n ( F = 244, d f 5,96,

Table 2 [3H]DA Uptake and DA concentration m examined brain regions Temperature-dependent [3H]DA uptake (pmol/mg protein/2 mm) Region Stnatum Nucleus accumbens Prefrontal cortex

Total

Cocaine-sensitive

Cocaine-insensitive

52 9 + 3 9 (10) 27 9 + 4 4 (8) I 38+0 13 (10)

51 8 + 3 9 26 7+4.4 0.53+009

0 96_+0 22 1 24_+0 29 085_+0 12

All regions slgmficantly dtfferent from each other for "Total" and "Cocalne-sensalve" uptake * Converted from mg protein for nucleus accumbens (Lentner, 1981 I Numbers in parentheses refer to number of assays used to derive the mean

DA concentration (,ug/g*tmue) 13.8_+0.5 (6) I 1.5_+0 4 (6) 0 14-+001 (6)

Cocaine inhibition of dopamine uptake

65

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NE

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Cocaine

Fig. 4. Comparative effect of DA, norepinephrine (NE), serotonin (5HT), GBR 12909 (GBR) and cocaine on temperature-dependent [3H]DA uptake (n = 3) in prefrontal cortex. Bars with a different letter are significantly different from each other (ANOVA, followed by Student-Newman-Keul's test, P < 0.05). P < 0.0001), and a main effect of brain region ( F = 3.3, df 2,96, P = 0.04), but no interaction between drug concentration and brain region ( F = 1.3, df 10,96, P = 0.27). However, the subsequent post-hoc test ( S t u d e n t - N e w m a n - K e u l s ) failed to identify a significant difference (P < 0.05) between regions. There was no significant difference (one-way ANOVA, F = 0.29, df2,19, P = NS) among the regions in the IC50 values of cocaine as an inhibitor of cocaine-sensitive D A uptake (Fig. 5): the IC50 values (nM + SE) were as follows ; striatum 294 + 46 (n = 7), nucleus accumbens 2 9 4 + 5 5 (n = 7), prefrontal cortex 245 + 48 (n = 6). A two-way A N O V A to test the effect of G B R 12909 concentration and brain region on cocaine-sensitive D A uptake revealed a main effect of drug concentration ( F = 132, df 5,78, P < 0.0001), and a main effect of brain region (F = 3.1, df2,78, P = 0.05), but no interaction between drug concentration and brain region (F = 0.91, df 10,78, P = 0.53). However, the subsequent post-hoc test ( S t u d e n t - N e w m a n - K e u l s ) failed to identify a significant difference (P < 0.05) between regions. There was no significant difference (one-way ANOVA, F = 0 . 9 0 , df 2,14, P = N S ) between the three regions for the ICs0 values of G B R 12909 as an inhibitor of cocaine-sensitive D A uptake (Fig. 5) : the IC50 values ( n M + SE) were as follows ; triatum 1.2 + 0.1 (n = 6), nucleus accumbens 1.8 + 0.5 (n = 7), prefrontal cortex 1.9_+ 0.3 (n = 4). DISCUSSION

Unlike in striatum or nucleus accumbens, temperature-dependent DA uptake in prefrontal cortex

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Fig. 5. Inhibition of cocaine-sensitive [3H]DA uptake by cocaine or GBR 12909 in striatum (n = 6 or 7), nucleus accumbens (n = 7) and prefrontal cortex (n = 4 6). IC50 values given in Results.

was only partially inhibited (about 50%) by large concentrations of cocaine or G B R 12909. This has been observed previously (Hadfield and Nugent, 1983; Izenwasser et al., 1990) but no further investigation of the cocaine-resistant uptake has been carfled out. Hadfield and Nugent (1983), who originally observed the difference in cocaine inhibition of D A uptake in prefrontal cortex, considered that in prefrontal cortex either (1) D A uptake occurred into neurotransmitter terminals that were not affected by cocaine, or that (2) non-specific uptake of D A occurred into structures that were more a b u n d a n t in prefrontal cortex than striatum, or that (3) the D A uptake site in prefrontal cortex was different than in striatum. Izenwasser et al. (1990), based on studies that did not differentiate uptake into D A or norepinephrine terminals, concluded that D A uptake into

()6

J D t~ISW()R[tl t'/ d/

other monoamme terminals was the most hkel> reason wh> only a portion of DA uptake in prefrontal cortex was sensitive to GBR 12909. The present data suggests that it is unlikely that tissue from norepinephrine neurons was responsible for the cocaine-resistant accumulation of [~H]DA reported here, since data are derived from incubatmns conducted m the presence of a selective concentration of DMI. When DMI was omitted [~H]DA uptake in prefrontal cortex was doubled, whereas no difference was detected in strlatum or nucleus accumbens, as would be predicted from the relative noradrenerglc and dopaminergic innervatlons to these areas (Bj6rklund and Lindvall, 1984). The persistence of 50% of total temperature-dependent [~H]DA uptake in buffer containing low sodium concentratmn in the prefrontal cortex, but not in striatum (90°0 inhibited) or nucleus accumbens (85% inhibited) together with the occurrence of the same pomon (50°'o) ol" total temperature-dependent [~H]DA uptake in the presence of ouabam in the prefi'ontal cortex, but not m strlatum or nucleus accumbcns, showed that th~s represents [~H]DA uptake by a mechanism not requiring sodium-potassmm ATPase (Tissari and Bogdansk~, 1971). Thus, Jt IS unlikely that this uptake represents actwe transport into other monoamine neurons. The results of the DMI-6OHDA lesion experiments, which showed a significant decrease in cocaine-sensitive [~H]DA uptake in the prefrontal cortex in the presence ofa stgnificant DA-selecnvc lesion, confirms that DA is indeed a substrate for the transporter on 6-OHDA-sensmvc DA terminals in the prefrontal cortex The cocaine-insensitive part of [~H]DA uptake comprises a significant proportion of total [)H]DA uptake in prefrontal cortex, compared w~th the other regions examined. However, when analyzed in terms of actual rate (rather than proportion), the sodiumindependent, ouabain resistant, cocaine- and GBRinsenmive [~H]DA uptake, was not different between the three regions. As the assays were done with a fixed concentration of [3H]DA, the relative densities of the two processes cannot be determined from the present data. However, it does appear that the cocaine-insenstove [~H]DA uptake is present to the same degree in all examined regmns. At 40 nM [~H]DA, cocaineinsensitive [~H]DA uptake represcnts a greater proportion of the total temperaturc-dependent [3H]DA uptake in prefrontal cortex, because the total temperature-dependent [~H]DA uptake is much lower in the prefrontal cortex than in striatum or nucleus accumbens. Besides ves~culated synaptlc nerve terminals, synaptosome preparations probably include a mixture of

axonal and dendrmc proccsscs, ghal proccsscs and other vesiculated plasma membrane fractions (Kunelberg, 1986). Thus, tile cocaine-insensitive component of DA accumulanon may reflect uptake or bmding to these o. other elements. Although the present data do not allow' identification of this other mechamsm or s~te, It is interesting to consider further some of lhe alternatives. A compartmental analysis of synaptosomal DA uptake (Schoemaker and Nickolson, 1983) found that DA uptake was best described by a 3-compartment in-series configuration, which may be related to the pool outside the synaptosomal membrane, the pool in synaptosomal cytoplasm and the pool in synapnc vesicles. The properties of uptake into synapnc vesicles are consistent with some of the characteristics of cocaine-insensitive uptake described here (eg. sodium-insensltwity), although vesicular uptake seems unlikely to account for the cocaineinsensitive uptake observed, as access to the vesicles ~s secondary to cocaine-sensitive uptake across the synaptosomal membrane, and isolated synaptic yesroles would not be present in the membrane preparatlon used (De Robems et a l , 1962), unless lysis somehow occurred after forming the P2 pellet It is interesting that the properties of the cocaine-insensitive uptake bear a striking resemblance to the "Tacihtared-diffusion'" mechanism of uptake described for DA and norepmephrme m astroglia (Paterson and Hertz, 1989; Kimelberg, 1986: Pelton el al., lq81l. This astroghal uptake is temperature-dependent, sod> um-independent and resistant to mhlbitmn by desipramine or ouabain. In addition, astroglial uptake of norepinephrine and DA is saturable, being inhibited by large concentrations of DA or norepinephrme, but not by 5-hydroxytryptamme. Similarly, the cocaineinsensitive uptake described here is specific since ~t is inhibited by DA or norepinephrine, but not 5-hydroxytryptamine. Thus, it is concmvable that uptake into glia explams the cocaine-insensitive uptake. In fact, even though this uptake represents a small fraction of total uptake in some regions, it may be thai other assay' conditions or preparations would emphasize this component of uptake more (Hansson et al., 1984), In tentatively suggesting that glial uptake of DA may be responsible for the presently observed cocaine-resistant uptake, it should be noted that there is good evidence that glial elements ("gliasomes") are indeed present following subcellular fractmnatlon to obtain a crude synaptosomal preparatmn (Kimelberg, 1996 Henn et al., 1976). The perisynaptic location of astroc>tes is appropriate for a role m regulating thc concentration and dispersion of extracellular catccholamines by uptake and metabolism. Astroglia

Cocaine inhibition of dopamine uptake uptake may function as an extraneuronal system that operates either in conjunction with the neuronal uptake system or functioning as a supplement to neuronal uptake that becomes most significant when synaptic concentrations are elevated. In fact, it has been suggested that presynaptic neuronal uptake may be unable to cope immediately with the relatively large amount of DA released following depolarization (Stamford et al., 1984; Woodward et al., 1986). It is possible that the cocaine-insensitive DA uptake characterized here shares identity with the extraneuronal uptake described for normetanephrine in rat brain (Hendley et al., 1970), which was temperature-dependent, sodium-independent, insensitive to DMI and cocaine, and was most noticeable in cortex. The comparative rates of [3H]DA uptake in the three regions examined differed markedly. The rate of all temperature-dependent DA uptake in prefrontal cortex was 38 times less than in striatum. However, because of the large proportion of cocaine-insensitive DA uptake in the prefrontal cortex, the rate of cocaine-sensitive DA uptake was 98 times less in prefrontal cortex than m striatum. The rate of all temperaturedependent uptake or just cocaine-sensitive uptake of DA in nucleus accumbens was half that observed in striatum. Previous studies of the regional distribution of the DA uptake carrier using radioactive labelled ligands have demonstrated a greater number of uptake sites in striatum compared to nucleus accumbens (Dawson et al., 1986; Leroux-Nicollet and Costentin, 1988; De Keyser et al., 1989; Marshall et al., 1990). These data may be a reflection that DA uptake per terminal is lower in nucleus accumbens than striatum. In fact, voltammetric studies have shown that DA survives in the extracellular space of the nucleus accumbens longer than in striatum (Stamford et al., 1988). If similar regional differences in the density of uptake sites exist in the primate, which some data suggest may be the case (Elsworth et al., 1989; Graybiel and Moratalla, 1989), a less avid uptake of substrates in DA neurons innervating the accumbens may explain the relative resistance of this region to the effects of l-methyl-4-phenyl-l,2,3,6tetrahydropyridine, a neurotoxin that relies on the DA uptake system for entry of its toxic metabolite into neurons. The present observation that cocaine was equally effective at inhibiting [3H]DA uptake in striatum and nucleus accumbens agrees with other recent studies' by us and others (Boja and Kuhar, 1989; Berger et al., 1990 ; Izenwasser et al., 1990), but contrast with an earlier study, using different conditions, which found that cocaine was relatively weak at inhibiting DA

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uptake in nucleus accumbens (Missale et al., 1985). In addition, the more selective DA uptake inhibitor, GBR 12909, blocked [3H]DA uptake with the same potency in striatum and nucleus accumbens (Fig. 5). These findings, together with the reports that extracellular DA concentrations are raised in nucleus accumbens and striatum following systemic cocaine administration (Church et al., 1987 ; Di Chiara and Imperato, 1988) are consistent with the hypothesis that inhibition of DA uptake in the nucleus accumbens is an important biochemical substrate of the behavioral effects of cocaine. In addition, the present data are consistent with the observation that GBR 12909 has a behavioral profile in rats similar to other psychostimulants (Kelley and Lang, 1989). A recent study suggested that the molecular weight of the DA transporter in nucleus accumbens and striatum may be different (Lew et al., 1991), however, the present data do not support the existence of regional isoforms of the dopamine transporter that have different sensitivities to pharmacological inhibition. The observation that rats will self-administer cocaine injections into the prefrontal cortex has been the foundation of the hypothesis that this region may have an important role in the reinforcing effects of cocaine (Goeders and Smith, 1983; 1986). However, Moghaddam and Bunney (1989) found that 2 mg/kg i.v. cocaine produced a relatively small increase in extracellular DA levels in prefrontal cortex (3-fold higher than baseline) compared to the response observed in nucleus accumbens (7-fold higher than baseline). Maisonneuve et al. (1990) reported cocaine (20 mg/kg by i.p. route) produced 10-fold increase in extracellular DA levels in prefrontal cortex, but did not compare the response with other regions. In the present studies, GBR 12909 and cocaine were found to inhibit cocaine-sensitive DA uptake equally well in prefrontal cortex as in nucleus accumbens and striatum, indicating that there is no regional difference in the affinity of the DA uptake site on DA terminals for cocaine and GBR 12909. However, a possible explanation for a relatively low elevation of extracellular DA levels in prefrontal cortex following cocaine injection is that a relatively large proportion of cocaine-insensitive uptake in this region limits the cocaine-induced increase in DA levels compared to nucleus accumbens or striatum. In addition, the lower affinity of cocaine for the norepinephrine, compared to the DA, transporter (Ritz et al., 1990), suggests that at certain doses of cocaine, the uptake of DA into norepinephrine neurons (Snyder and Coyle, 1969; Carboni et al., 1990) may conceivably occur and attenuate the extracellular levels of DA in regions receiving

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J. I) ELSWORTHct a/

substantml noradrenergm and dopaminergic innervations. In summary, two modes o f D A uptake were identified in tissue prepared from rat brain. One, which was probably associated with uptake into dopaminergic nerve terminals, was very sensitive to inhibition by cocaine, G B R 12909 or ouabain, and was dependent on temperature and sodium mn concentration. This cocaine-sensitive DA uptake appeared to account for the majority o f the transport o f d o p a m i n e in striatum and nucleus accumbens, but not in prefrontal cortex. There was no regional difference in the susceptibility o f cocame-sensitive uptake to mhlbitmn by cocame or G B R 12909. Cocaine-sensitive DA uptake in prefrontal cortex was susceptible to a dopamme-selecUve 6 - O H D A lesion o f the prefrontal cortex. The other process, which concmvably may be extraneuronal, was relatwely insensitive to cocaine, G B R 12909 or ouabmn and persisted m a buffer o f low sodium ~on concentration, but was temperature dependent and showed preference for d o p a m i n e and norepinephrine, compared to serotonin. U n d e r the assay conditions employed here, cocaine-insensitive DA uptake accounted for only a small fraction o f the total observed uptake in striatum and nucleus accumbens, but represented approx 50% o f the total m prefrontal cortex by DA 05119 We thank Katahn Plros and Janice Abele for excellent techmcal assistance Acknowh, dgement--Supported

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