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Focal and systemic cocaine differentially affect extracellular norepinephrine in the locus coeruleus, frontal cortex and hippocampus of the anaesthetized rat
BRAIN RESEARCH ELSEVIER
Brain Research 645 (1994) 135-142
Research Report
Focal and systemic cocaine differentially affect extracellular norepinephrine in the locus coeruleus, frontal cortex and hippocampus of the anaesthetized rat D.N. Thomas *, R.M. Post, A. Pert Biological Psychiatry Branch, National Institute of Mental Health, Bid. 10, Room 3N212, 9000 RockL,ille Pike, Bethesda, MD 20892, USA
(Accepted 25 January 1994)
Abstract
The purpose of this study was to characterize and compare the effects of cocaine on norepinephrine (NE) overflow in the forebrain and somatodendritic regions of anaesthetized rats with microdialysis. Intraperitoneal injections of cocaine (20 mg/kg) failed to increase NE overflow in the hippocampus and the frontal cortex but did elevate NE in the region of the locus coeruleus. Focal application of cocaine (1-100 /xM) via the dialysis probe into the region of the locus coeruleus also produced a concentration dependent elevation of extracellular NE. In the terminal regions the application of focal cocaine (1-100 tzM) showed a differential effect, with a concentration dependent increase in extracellular NE in the hippocampus, whilst in the frontal cortex only the highest concentration of cocaine (100 /zM) elevated extracellular NE. The regional differences seen following focal applications in this study may be related to differences in transporter function in the three brain areas or to differences in the affinity for cocaine. The inability of systemically administered cocaine to increase hippocampal and cortical NE is probably related to its predominant actions in the somatodendritic region. Key words: Norepinephrine; Microdialysis; Cocaine; Frontal cortex; Hippocarnpus; Locus coeruleus
1. Introduction
Cocaine is known to inhibit the re-uptake of dopamine [25,46], serotonin [25,46] and norepinephrine [17,25,46], such uptake blocking actions, by increasing the synaptic availability of these brain amines at both central and peripheral sites, presumably underlie the major pharmacological effects of this drug. Cocaine is relatively equipotent in blocking re-uptake of all biogenic amines [42], although the reinforcing as well as the locomotor stimulant effects generally have been attributed exclusively to its ability to enhance mesolimbic dopamine function [11,24,33,42,43,52]. While the serotonergic and noradrenergic systems may not play a prominent role in the reinforcing actions of cocaine, it is possible that they may still contribute in some way to such actions by modulating dopaminergic function [7,16,29]. In this regard it is of
* Corresponding author. Fax: (1) (301) 402 0052. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00 I50-B
interest to note that both systems project to the perikaryal regions of the nigro-striatal and mesolimbic dopamine pathways [22,51]. In addition, it is likely that alterations in serotonergic and noradrenergic function may mediate pharmacological actions of cocaine other than reinforcement. Of special interest are the effects of cocaine on the noradrenergic system originating from the locus coeruleus. The locus coeruleus has been postulated to be involved in a variety of processes including learning and memory [8,18,50], preparatory sets [53], arousal [4] and attention [44]. With regard to the latter functions, it is noteworthy that cocaine is known to enhance sensitivity to sensory stimuli in man [9] and to increase vigilance in animals [15]. Finally, the noradrenergic systems originating from the locus coeruleus also have been postulated to be involved in mediating the behavioral consequences of stress, including anxiety [41] and depression [45]. It is possible that the dysphoria and depression seen following chronic cocaine use [40] is determined at least in part through the locus coeruleus.
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D.N• Thomas et al. /Brain Research 645 (1994) 135-142
I n this r e g a r d it is of i n t e r e s t to n o t e t h a t b o t h c h r o n i c stress a n d c h r o n i c c o c a i n e [5,32,39] d o w n r e g u l a t e a z a d r e n o c e p t o r f u n c t i o n in t h e locus coeruleus. D e s p i t e t h e fact t h a t c o c a i n e is a strong i n h i b i t o r o f N E u p t a k e , its effects on t h e locus c o e r u l e u s n o r a d r e n ergic system have not b e e n fully c h a r a c t e r i z e d e x c e p t with e l e c t r o p h y s i o l o g i c a l techniques. Single cell r e c o r d ing studies have r e v e a l e d t h a t i n t r a v e n o u s injections o f c o c a i n e p r o d u c e significant d e c r e a s e s in t h e firing r a t e o f locus c o e r u l e u s n e u r o n s [10,34-38]. S i m i l a r effects have b e e n r e p o r t e d for a m p h e t a m i n e a n d n o r e p i n e p h r i n e u p t a k e i n h i b i t o r s such as d e s m e t h y l i m i p r a m i n e [12,30]. Such i n h i b i t o r y effects a p p e a r to b e m e d i a t e d by i n c r e a s e s in s o m a t o d e n d r i t i c N E which d e c r e a s e s t h e activity o f t h e locus c o e r u l e u s t h r o u g h t h e a 2 - a u t o r e c e p t o r s [35]. Since t h e t e r m i n a l a c c u m u l a t i o n o f N E in t h e presence of an u p t a k e i n h i b i t o r such as c o c a i n e is a p r o d uct o f exocytotic r e l e a s e a n d r e - u p t a k e inhibition, it is not readily apparent what consequences cocaine would have on e x t r a c e l l u l a r levels c o n s i d e r i n g its p o t e n t effects on locus c o e r u l e u s firing. T h e p u r p o s e o f this study was to c h a r a c t e r i z e t h e effects o f c o c a i n e on
e x t r a c e l l u l a r N E a c c u m u l a t i o n in b o t h the t e r m i n a l a n d p e r i k a r y a l r e g i o n s of t h e n o r a d r e n e r g i c systems o r i g i n a t i n g f r o m t h e locus c o e r u l e u s o f a n a e s t h e t i z e d rats.
2. Materials and methods 2.1. Subjects and surgery
Male Sprague-Dawley rats (Taconic Farm) weighing 300-400 g were used as subjects• The animals were individually housed and maintained for a week on a light dark cycle (lights on 07.00-19•00 h) with food and water available ad lib. Prior to surgery, animals were anaesthetized with chloral hydrate (400 mg/kg;100 mg/ml). Using standard stereotaxic procedures CMA/10 (3 mm, BAS Inc.) microdialysis probes were implanted into either the hippocampus (AP: -6.00 mm from bregma, L: - 5 mm, H: - 7 mm) or frontal cortex (AP: + 3.2 mm from bregma, L: - 1 mm, H: - 4.5 mm). In the locus coeruleus CMA12 2 mm (BAS Inc.) dialysis probes were implanted (AP: -10.2 mm from bregma, L: -1.3 mm, H: - 8 mm). All coordinates used were according to the atlas of Paxinos and Watson [32]. Anaesthesia was maintained during the course of the experiment by supplementing with chloral hydrate (90 mg/kg; 100 mg/ml) in order to abate the corneal reflex.
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D.N. Thomas et al. /Brain Research 645 (1994) 135-142 2.2. Microdialysis and sample analysis
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The dialysis probes situated in either of the three structures were perfused with a physiological medium containing 145 mM NaC1, 4 mM KCI, 1.2 mM CaC12 and 2 mM Na2HPO4, final pH of the medium being pH 7.4. The probes were dialyzed at a flow rate of 2.34 /zl/min and samples were collected every 15 min. The probes were allowed to equilibrate for 1-2 h prior to collection of baseline samples. Baseline samples were then collected until three consecutive samples did not differ by more than 10%. Cocaine (20 mg/kg) was injected intra-peritoneally and sampling was continued for a further 2 h. In a subsequent group of animals cocaine (1-100 /xM) was administered via the dialysis probe located in either the hippocampus, frontal cortex or the region of the locus coeruleus. Each concentration was applied for a period of 15 min starting with the lowest concentration of cocaine. Three sampling intervals following the application of cocaine were allowed to intervene before the next concentration was applied. The samples were analyzed for norepinephrine using HPLC with electrochemical detection. The mobile phase (75 mM NaH2PO4, 1.5 mM sodium dodecyl sulphate, 20 /xM EDTA, 100 pA/l triethylamine, 15% acetonitrile and 12% methanol, pH 5.6) was filtered and degassed before pumping at a rate of 1 ml/min through a HR-80 column (3/z BDS, 80 × 4.6 mm, ESA Inc.). Electrochemical detection was performed using a colouchem detector with Guard cell: +350 mV, Det 1 : - 4 0 mV and Det 2:+250 inV. Detection limits of the assay were 1 pg/sample.
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3. Results Fig. 1 illustrates representative probe placements in the frontal cortex, hippocampus and the region of the locus coeruleus on 20 Ixm coronal sections stained with thionin. The extent of the probe membrane is indicated with arrows. All animals included in the study had the effective region of the dialysis probe located in the appropriate structures. The effects of intraperitoneally administered cocaine (20 mg/kg) on extracellular norepinephrine in the three brain regions appear in Fig. 2. A one-way repeated measures analysis of variance which compared the pre-drug (basal) and post-drug norepinephrine levels in the hippocampus (Fig. 2A) failed to indicate a significant treatment effect (F < 1.0). The same dose of cocaine failed to alter norepinephrine levels in the frontal cortex (F < 1.0) (Fig. 2B). Norepinephrine levels in the locus coeruleus region (Fig. 2C), on the otherhand, increased (173% above baseline) following injections of cocaine, reaching a maximum 30 min later. A one-way repeated measures analysis of variance revealed a significant main effect (F = 2.411, d f = 8/43, P = 0.03). Post-hoe comparisons of norepinephrine levels following drug injections with basal values (3 samples preceding cocaine) indicated significant elevations in norepinephrine overflow at 30 and 45 min. Fig. 3 illustrates the effects of focally applied cocaine on norepinephrine overflow in the hippocampus. A two-way repeated measures analysis of variance
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Fig. 2. Effects of intraperitoneal cocaine (20 mg/kg) on extracellular norepinephrine in (A) frontal cortex (n = 4), (B) hippocampus (n = 4) and (C) region of the locus coeruleus (n = 4). All values are mean_+ S.E.M. * P < 0.05 for comparison between post-drug NE levels and basal values.
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D.N. Thomas et al. / Brain Research 645 (1994) 135-142
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which compared pre-drug norepinephrine levels with post-drug effects for all three concentrations revealed a significant effect of time ( F = 13.6, df 2/38, p < 0.001) and a significant time × concentration interaction ( F = 2.90, df 6/38, p < 0.02). The concentration effect failed to reach statistical significance ( F < 1.0). Post-hoe comparisons indicated significant peak effects following all three concentrations (1/zM, 34%; 10/zM, 86%; 100 /zM, 110% increase above baseline). In addition norepinephrine levels 15 min following application of 100 /zM cocaine was also found to be significantly elevated compared with basal levels. The focal application of cocaine to the frontal cortex had a considerably smaller effect on extracellular N E overflow than that seen in the hippocampus (Fig. 4). A two-way repeated measures analysis of variance revealed a significant time effect ( F = 6.47, df 2/32, P < 0.005) and a significant time × concentration interaction ( F = 3.16, df 6/32, P < 0.02). Post-hoc comparisons indicated that only the peak effect following application of 100 /zM cocaine to the frontal cortex 15-
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Fig. 4. Focal application of cocaine ( 1 - 1 0 0 / z m ) via the dialysis probe into the frontal cortex (n = 5). * P < 0.05 for comparison between post-drug NE levels and basal values (sampling period immediately preceding the focal application). All values are m e a n + S.E.M.
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Concentration of cocaine (uM) Fig. 6. Peak effect of cocaine ( 1 - 1 0 0 / z M ) on N E overflow at 30 min post-injection in the frontal cortex (solid columns), hippocampus (dark hatched columns) and the region of the locus coeruleus (light hatched columns). Data are expressed as peak absolute increase above baseline (pg/sample), all values are mean_+ S.E.M. (n = 5-6). A repeated measures analysis of variances revealed a significant difference among the three regions across the three concentrations ( P < 0.0004). * Locus coeruleus extracellular NE levels were transformed to compensate for the decreased dialysis m e m b r a n e surface area of the probes used in this structure (see section 3).
was significantly different from the basal values (81% increase above baseline). Fig. 5 illustrates the effects of focally applied cocaine to the region of the locus coeruleus on norepinephrine overflow. A two-way repeated analysis of variance revealed a significant time effect ( F = 61.6, df 2/32, P < 0.001), concentration effect ( F = 22.48, df 3/32, P < 0.0001) and significant time × concentration interaction ( F = 12.02, df 6/32, P < 0.001). Post hoc comparisons indicated a significant peak effect at all three concentrations (1 /zM, 113%; 10/zM, 237%; 100 ~M, 450% increase above baseline). Comparative statistical analysis of the peak effect of cocaine in the three regions (Fig. 6) by two-way A N O V A revealed a significant difference among the three regions in response to cocaine ( F = 4.69, df 2/26, P = 0.029) and among the concentrations of cocaine administered ( F = 10.58, df 2/26, P = 0.0004). Since 2 mm probes were used in the locus coeruleus and 3 mm probes were used in the other two structures, the absolute amounts of extracellular N E recovered from the locus coeruleus were adjusted proportionately to reflect the differences in probe surface area in order to make statistical comparisons among regions. This assumes that the absolute amount recovered is linear with dialysis membrane surface area.
Time (rain) Fig. 5. Focal application of into the region of the locus son betwecn post-drug NE immediately preceding the S.E.M.
cocainc (1-100/~m) via the dialysis probe coeruleus (n = 5). * P < 0.05 for comparilevels and basal valucs (sampling period focal application). All values are mean +
4. Discussion The basal levels of norepinephrine in dialysates from the hippocampus (134 fg//~l) and the frontal
D.N. Thomas et al./Brain Research 645 (1994) 135-142
cortex (120 fg//zl) approximately 2 h following the insertion of 3 mm CMA microdialysis probes are within the range reported by other groups for these structures (50-217 fg//zl for the cortex [27,49] and 104-488 f g / ~ l for the hippocampus [1,2,14,23,27,48]). Precise comparisons are not meaningful; considering differences in probe construction, perfusion rates and other methological factors. Basal levels of norepinephrine in dialysate from the region of the locus coeruleus recovered via 2 mm CMA microdialysis probes were 48 fg//~l. Following adjustment for membrane surface area and recovery rate, these levels appeared to be relatively similar to those found for the hippocampus and frontal cortex. Considering the ability of cocaine to inhibit the reuptake of norepinephrine in vitro [17,26,46] it was surprising to find that intraperitoneal administration of behaviorally effective doses were almost devoid of effects on the extracellular NE in the frontal cortex and hippocampus. Similar results have been found following administrations of desipramine, the selective NE uptake blocker (Thomas et al. unpublished data). It is likely that the lack of effect of systemically administered cocaine and desipramine on extracellular NE in these two structures is related, in part to their ability to produce elevations of NE in the somatodendritic region (locus coeruleus). There are two actions of cocaine which enter into determining its effects on the accumulation of NE in terminal regions of the locus coeruleus projection system. The first is inhibition of reuptake, the second is inhibition of noradrenergic neuronal firing. Both cocaine and amphetamine have been found to be potent inhibitors of locus coeruleus neurons following systemic injections [12,34-38]. In fact, pharmacologically relevant doses have been shown to decrease the firing rate of locus coeruleus neurons by at least 70% [10,3438]. Such effects appear to be mediated through presynaptic az-adrenoceptors [35] located in the perikaryal regions [6]. It is proposed that the accumulation of NE in the locus coeruleus region in this study by systemically administered cocaine inhibits the firing rate of these cells through az-adrenoceptors. In the presence of substantially decreased neuronal activity and exocytotic release, cocaine would be expected to have little effect on NE overflow in the projection areas. The ability of systemically administered cocaine to enhance NE overflow in the locus coeruleus region without concurrent effects in the hippocampus and frontal cortex is somewhat puzzling. However, the mechanism governing release of norepinephrine in the somatodendritic region are not fully understood. It is possible that the process is somewhat independent from that which determines release from the terminals via CaZ+-dependent exocytotic mechanisms. Such independence could account for the ability of cocaine to
139
increase NE in the locus coeruleus without having an effect in the forebrain structures. Alternatively, it is possible that such findings may be due to more efficient NE reuptake mechanisms in the projection fields relative to the locus coeruleus. The differential regional effects of cocaine on extracellular norepinephrine in this study suggest that its predominant effects are initially mediated through the somatodendritic region. Electrophysiological findings reported by Pitts and Marwah [37] also support this conclusion. These investigators found that cocaine inhibited locus coeruleus neurons but concurrently increased the activity of Purkinje cells in the cerebellum. On the surface such effects on Purkinje cells by cocaine appear contradictory since norepinephrine is known to inhibit their firing rate through/3-adrenoceptors. If the predominant effect of cocaine in these studies was to enhance accumulation of norepinephrine in the terminals of locus coeruleus neurons projecting to the cerebellum, then an inhibition of neuronal activity should be seen. These effects can be understood, however, if it is assumed that the modulatory effects of cocaine on monoaminergic synapses is greater at the level of the locus coeruleus soma than at the terminals. Inhibition of locus coeruleus neurons by cocaine might enhance the activity of the Purkinje cells by decreasing the tonic inhibitory actions of NE. In the same study, cocaine also attenuated the inhibitory effects of NE evoked by locus coeruleus stimulation and had no effect on the inhibitory effects of iontophoretically applied NE on Purkinje cells. These findings further support the proposition that the predominant actions of cocaine occur in the locus coeruleus. Focal applications of cocaine directly to the hippocampus and frontal cortex, by circumventing its effects in the locus coeruleus, should reveal the uptake inhibitory properties of this drug in these terminal regions. Focal applications of cocaine to the two regions in this study did indeed increase norepinephrine overflow in both. Surprisingly, however, the two structures showed differential responses to the focal administration. In the hippocampus, extracellular norepinephrine increased in a concentration dependent manner, whereas in the frontal cortex, only the highest concentration produced a significant elevation. There are several possible reasons for such differential actions of cocaine. First, it is possible that the noradrenergic terminals in the frontal cortex have a paucity of uptake sites relative to other regions. There is a lack of evidence, however, to support this proposition. Radioligand binding studies [47] using the highly selective noradrenergic uptake blocker nisoxetine, have not found differences in the density of binding sites in the hippocampus and cortical regions. Results from a recent study by Kuczenski and Segal [27] are consistent with the notion that uptake site density is equivalent in
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D.N. Thomas et al. / Brain Research 645 (1994) 135-142
the two regions. These investigators reported that amphetamine-induced increases in extracellular norepinephrine were equivalent in the frontal cortex and the hippocampus. While these findings appear incongruent with the present results, it should be noted that amphetamine is thought to produce some of its effects on catecholamine function by promoting cytoplasmic release through exchange diffusion which is carrier mediated and not Ca 2+ dependent [13,21,28]. The enhanced synaptic accumulation of catecholamines by cocaine, on the otherhand, is dependent on blockade of reuptake in the presence of Ca z+ dependent exocytotic release [18,21,26,46]. Therefore, if carrier mediated release is critical to the ability of amphetamine to enhance extracellular norepinephrine levels, the relatively similar effects of this psychomotor stimulant in the two structures would suggest that both have an equal density of uptake sites. Although the density of carriers in the two structures may not be different, it is possible that there are differences in their affinity for cocaine. It is of interest to note that concentrations of cocaine up to 1 /zM do not inhibit nisoxetine binding in the cortex [47]. In the present study, similar concentrations are achieved by diffusion of a 10 /zM solution through the dialysis probe since cocaine tissue levels within 250/xM of the probe appear to be approximately 10% of that in the dialysate (unpublished observations). At that concentration of cocaine, we failed to observe an effect on norepinephrine overflow in the frontal cortex. The differential effects of focally applied cocaine also could be related to different basal activities of locus coeruleus neurons projecting to the two regions. Cocaine-induced increases in extracellular norepinephrine are dependent almost exclusively on its ability to inhibit reuptake of this brain amine. Since cocaine induced accumulation in the presynaptic space of the hippocampus and frontal cortex is dependent on exocytotic release, the region with a higher basal output would be expected to show the largest increases in norepinephrine overflow. Focal application of cocaine to the locus coeruleus region produced significant concentration dependent increases in N E overflow in the perikaryal area indicating the prescence of prominant somatodendritic N E uptake processes. The locus coeruleus in this study appeared to be somewhat more responsive to the direct applications of cocaine than the two terminal regions. For example, when alterations in N E levels are expressed relative to the basal values, application of the 10 /zm cocaine increased extracellular N E in the frontal cortex by 16%, the hippocampus by 86% and the locus coeruleus by 237%. These findings also are consistent with the concept that the predominant actions of cocaine occur in the locus coeruleus. It is also of interest to compare the effects of
cocaine on dopamine overflow with its effects on norepinephrine overflow in this study. Systemically administered cocaine produces substantial increases in dopamine overflow in the terminal regions of the mesolimbic (n. accumbens) and nigrostriatal (striatum) dopamine pathways under similar conditions employed in this study [19,20]. Similarly, focally applied cocaine to the striatum and n. accumbens results in dramatic increases in dopamine overflow [20]. These differences in the response to cocaine can be understood in terms of the relatively high activity of mesolimbic dopamine neurons (5-8 Hz) [3,38] compared with the norepinephrine locus coeruleus neurons (1-2 Hz) [10,3436,39] and the relatively lower inhibitory actions of the drug on the basal activity of dopamine neurons [38]. Finally, it is important to emphasize that these studies were conducted in the anaesthetized rats. It is possible that quantitatively different responses may be seen in unanesthetized animals. Curtis et al. [10] for example, have recently reported that cocaine-induced inhibition of locus coeruleus neurons is quantitatively less in unanesthetized rats compared with those under halothane anaesthesia. If cocaine is less effective in depressing locus coeruleus activity in unanesthetized animals, it is possible that modest increases in hippocampal and frontal cortex norepinephrine could be realized following systemic administration. Preliminary findings from our laboratory support this prediction. Qualitatively, however, the effects of cocaine in the two preparations should be equivalent (i.e. effects in the locus coeruleus should be greater than those in the hippocampus and frontal cortex following systemic administration and the hippocampus should be more responsive to focal cocaine than the frontal cortex).
Acknowledgments This work has been partially supported by a postdoctoral training fellowship from the MacArthur Foundation Mental Health Research Network I (Psychobiology of Depression) for Dr. D.N. Thomas. We also gratefully acknowledge the expert technical assistance provided by Tim Sullivan, Judith Kransdorf and Connie Chiueh.
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intact and 6-hydroxydopamine-lesioned rats as studied by in vivo microdialysis, Brain Res., 474 (1988) 374-379. [24] Kelly, P.H. and Iversen, S.D., Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: Abolition of psychostimulant-induced locomotor activity in rats, Eur. J. Pharmacol., 40 (1976) 45-56. [25] Koe, B.K., Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain, J. Pharmacol. Exp. Then, 199 (1976) 649-661. [26] Komiskey, H.L., Miller, D.D, LaPidus, J.B. and Patil, P.N., The isomers of cocaine and tropacocaine: Effects on 3H-catecholamine uptake by rat brain synaptosomes, Life Sci., 21 (1977) 1117-1122. [27] Kuczenski, R. and Segal, D.S., Regional Norepinephrine response to amphetamine using dialysis: Comparison with caudate dopamine, Synapse, 11 (1992) 164-169. [28] Lategan, A.J., Marien, M.R. and Colpaert, F.C., Effects of locus coeruleus lesions on the release of endogenous dopamine in the rat nucleus accumbens and caudate nucleus as determined by intracerebral microdialysis, Brain Res., 523 (1990) 134-138. [29] McMiUer, B.A., German, D.C. and Share, P.A., Fuctional and pharmacological significance of brain dopamine and norepinephrine storage pools. Biochem. Pharmacol., 29 (1980) 30453050. [30] Nyback, H.V., Waiters, J.R., Aghajanian, G.K. and Roth, R.H., Tricyclic antidepressants: Effects on the firing rate of brain noradrenergic neurons, Eur. J. Pharmacol., 32 (1975) 302-312. [31] Pavcovich, L.A. and Ramirez, O.A., Time course effects of uncontrollable stress in locus coeruleus neuronal activity, Brain Res. Bull., 26 (1991) 17-21. [32] Paxinos, C. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd Edn., Academic Press, New York, 1986. [33] Pettit, H.O., Ettenburg, A., Bloom, F.E. and Koob, G.F., Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats, Psychopharmacology, 84 (1984) 167-173. [34] Pitts, D.K. and Marwah, J., Electrophysiological effects of cocaine on central monoaminergic neurons, Eur. J. Pharmacol., 131 (1986) 95-98. [35] Pitts, D.K. and Marwah, J., Effects of cocaine on the electrical activity of single noradrenergic neurons from locus coeruleus, Life Sci., 38 (1986) 1229-1234. [36] Pitts, D.K. and Marwah, J., Reciprocal pre- and postsynaptic actions of cocaine at a central noradrenergic synapse, Exp. Neurol., 98 (1987) 518-528. [37] Pitts, D.K. and Marwah, J., Electrophysiological actions of cocaine on noradreuergic neurons in rat locus coeruleus, J. Pharmacol. Exp. Then, 240 (1987) 345-351. [38] Pitts, D.K. and Marwah, J., Cocaine modulation of central monoaminergic neurotransmission, Pharmacol. Biochem. Behav., 26 ('.1987) 453-461. [39] Pitts, D.K. and Marwah, J., Chronic cocaine reduces a2-adrenoceptor elicited mydriasis and inhibition of locus coeruleus neurons, Eur. J. Pharmacol., 160 (1989) 201-209. [40] Post, R.M., Cocaine Psychoses: A continuum model, Am. J. Psych., 132 (1975) 225-231. [41] Redmond, D.E. and Huang, Y.H., Locus coeruleus and anxiety, Life Sci., 25 (1979) 2149-2169. [42] Ritz, M.C., Lamb, R.J., Goldberg, S.R. and Kuhar, M.J., Cocaine receptors on dopamine transporters are related to self administration of cocaine, Science, 237 (1987) 1219-1223. [43] Roberts, D.C.S., Koob, G.F., Klonoff, P. and Fibiger, H.C., Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens, Pharmacol Biochem. Behav., 12 (1979) 781-787. [44] Sara, S.J. and Segal, M., Plasticity of sensory responses of locus coeruleus neurons in the behaving rat: Implications for cogni-
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D.N. Thomas et al. /Brain Research 645 (1994) 135-142 tion. In C.D. Barnes and O. Pompeiano (Eds.), Progress in Brain Research, Elsevier, Amsterdam, 1991, pp. 571-585. Simson, P.E. and Weiss, J.M., Altered activity of the locus coeruleus in an animal model of depression, Neuropsychopharmacology, 1 (1988) 287-295. Taylor, D. and Ho, B.T., Comparison of inhibition of monoamine uptake by cocaine, methyphenidate and amphetamine, Res. Commun. Chem. Path. Physiol., 21 (1978) 67-75. Tejani-Butt, S.M., [3H]-Nisoxetine: A radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding, J. Pharmacol. Exp. Ther., 260 (1992) 427436. Thomas, D.N. and Holman, R.B., A microdialysis study of the regulation of endogenous noradrenaline release in the rat hippocampus, J. Neurochem., 56 (1991) 1741-1746. Velhuizen, M.J.A., Feenstra, M.G.P., Boer, G.J. and Westerink, B.H.C., Microdialysis studies on cortical noradrenaline release:
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basic characteristics, significance of extracellular calcium and massive post-mortem increase, Neurosci. Let., 119 (1990) 233236. Velley, L., Cardo, B., Kempf, E., Mormede, P., NassifCaudarella, S. and Velly, J., Facilitation of learning consecutive to electrical stimulation of the locus coeruleus: Cognitive alteration or stress-reduction? In C.D. Barnes and O. Pompeiano (Eds.), Progress in Brain Research, Elsevier, Amsterdam, 1991, pp. 555-569. Vertes, R.P., A PHA-L analysis of ascending projections of the DR in the rat, J. Comp. Neurol., 313 (1991) 643-668. Wise, R.A. and Bozarth, M.A., A psychomotor stimulant theory of addiction, Psychol. Rev, 94 (1987) 469-492. Yamamoto, K.-I. and Ozawa, N., Increased firing of locus coeruleus neurons associated with preparatory set in rats, Neurosci. Lett., 106 (1989) 112-118.
Brain Research 645 (1994) 135-142
Research Report
Focal and systemic cocaine differentially affect extracellular norepinephrine in the locus coeruleus, frontal cortex and hippocampus of the anaesthetized rat D.N. Thomas *, R.M. Post, A. Pert Biological Psychiatry Branch, National Institute of Mental Health, Bid. 10, Room 3N212, 9000 RockL,ille Pike, Bethesda, MD 20892, USA
(Accepted 25 January 1994)
Abstract
The purpose of this study was to characterize and compare the effects of cocaine on norepinephrine (NE) overflow in the forebrain and somatodendritic regions of anaesthetized rats with microdialysis. Intraperitoneal injections of cocaine (20 mg/kg) failed to increase NE overflow in the hippocampus and the frontal cortex but did elevate NE in the region of the locus coeruleus. Focal application of cocaine (1-100 /xM) via the dialysis probe into the region of the locus coeruleus also produced a concentration dependent elevation of extracellular NE. In the terminal regions the application of focal cocaine (1-100 tzM) showed a differential effect, with a concentration dependent increase in extracellular NE in the hippocampus, whilst in the frontal cortex only the highest concentration of cocaine (100 /zM) elevated extracellular NE. The regional differences seen following focal applications in this study may be related to differences in transporter function in the three brain areas or to differences in the affinity for cocaine. The inability of systemically administered cocaine to increase hippocampal and cortical NE is probably related to its predominant actions in the somatodendritic region. Key words: Norepinephrine; Microdialysis; Cocaine; Frontal cortex; Hippocarnpus; Locus coeruleus
1. Introduction
Cocaine is known to inhibit the re-uptake of dopamine [25,46], serotonin [25,46] and norepinephrine [17,25,46], such uptake blocking actions, by increasing the synaptic availability of these brain amines at both central and peripheral sites, presumably underlie the major pharmacological effects of this drug. Cocaine is relatively equipotent in blocking re-uptake of all biogenic amines [42], although the reinforcing as well as the locomotor stimulant effects generally have been attributed exclusively to its ability to enhance mesolimbic dopamine function [11,24,33,42,43,52]. While the serotonergic and noradrenergic systems may not play a prominent role in the reinforcing actions of cocaine, it is possible that they may still contribute in some way to such actions by modulating dopaminergic function [7,16,29]. In this regard it is of
* Corresponding author. Fax: (1) (301) 402 0052. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00 I50-B
interest to note that both systems project to the perikaryal regions of the nigro-striatal and mesolimbic dopamine pathways [22,51]. In addition, it is likely that alterations in serotonergic and noradrenergic function may mediate pharmacological actions of cocaine other than reinforcement. Of special interest are the effects of cocaine on the noradrenergic system originating from the locus coeruleus. The locus coeruleus has been postulated to be involved in a variety of processes including learning and memory [8,18,50], preparatory sets [53], arousal [4] and attention [44]. With regard to the latter functions, it is noteworthy that cocaine is known to enhance sensitivity to sensory stimuli in man [9] and to increase vigilance in animals [15]. Finally, the noradrenergic systems originating from the locus coeruleus also have been postulated to be involved in mediating the behavioral consequences of stress, including anxiety [41] and depression [45]. It is possible that the dysphoria and depression seen following chronic cocaine use [40] is determined at least in part through the locus coeruleus.
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D.N• Thomas et al. /Brain Research 645 (1994) 135-142
I n this r e g a r d it is of i n t e r e s t to n o t e t h a t b o t h c h r o n i c stress a n d c h r o n i c c o c a i n e [5,32,39] d o w n r e g u l a t e a z a d r e n o c e p t o r f u n c t i o n in t h e locus coeruleus. D e s p i t e t h e fact t h a t c o c a i n e is a strong i n h i b i t o r o f N E u p t a k e , its effects on t h e locus c o e r u l e u s n o r a d r e n ergic system have not b e e n fully c h a r a c t e r i z e d e x c e p t with e l e c t r o p h y s i o l o g i c a l techniques. Single cell r e c o r d ing studies have r e v e a l e d t h a t i n t r a v e n o u s injections o f c o c a i n e p r o d u c e significant d e c r e a s e s in t h e firing r a t e o f locus c o e r u l e u s n e u r o n s [10,34-38]. S i m i l a r effects have b e e n r e p o r t e d for a m p h e t a m i n e a n d n o r e p i n e p h r i n e u p t a k e i n h i b i t o r s such as d e s m e t h y l i m i p r a m i n e [12,30]. Such i n h i b i t o r y effects a p p e a r to b e m e d i a t e d by i n c r e a s e s in s o m a t o d e n d r i t i c N E which d e c r e a s e s t h e activity o f t h e locus c o e r u l e u s t h r o u g h t h e a 2 - a u t o r e c e p t o r s [35]. Since t h e t e r m i n a l a c c u m u l a t i o n o f N E in t h e presence of an u p t a k e i n h i b i t o r such as c o c a i n e is a p r o d uct o f exocytotic r e l e a s e a n d r e - u p t a k e inhibition, it is not readily apparent what consequences cocaine would have on e x t r a c e l l u l a r levels c o n s i d e r i n g its p o t e n t effects on locus c o e r u l e u s firing. T h e p u r p o s e o f this study was to c h a r a c t e r i z e t h e effects o f c o c a i n e on
e x t r a c e l l u l a r N E a c c u m u l a t i o n in b o t h the t e r m i n a l a n d p e r i k a r y a l r e g i o n s of t h e n o r a d r e n e r g i c systems o r i g i n a t i n g f r o m t h e locus c o e r u l e u s o f a n a e s t h e t i z e d rats.
2. Materials and methods 2.1. Subjects and surgery
Male Sprague-Dawley rats (Taconic Farm) weighing 300-400 g were used as subjects• The animals were individually housed and maintained for a week on a light dark cycle (lights on 07.00-19•00 h) with food and water available ad lib. Prior to surgery, animals were anaesthetized with chloral hydrate (400 mg/kg;100 mg/ml). Using standard stereotaxic procedures CMA/10 (3 mm, BAS Inc.) microdialysis probes were implanted into either the hippocampus (AP: -6.00 mm from bregma, L: - 5 mm, H: - 7 mm) or frontal cortex (AP: + 3.2 mm from bregma, L: - 1 mm, H: - 4.5 mm). In the locus coeruleus CMA12 2 mm (BAS Inc.) dialysis probes were implanted (AP: -10.2 mm from bregma, L: -1.3 mm, H: - 8 mm). All coordinates used were according to the atlas of Paxinos and Watson [32]. Anaesthesia was maintained during the course of the experiment by supplementing with chloral hydrate (90 mg/kg; 100 mg/ml) in order to abate the corneal reflex.
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7 Fig. 1. Representative probe placement in (a) frontal cortex, (b) hippocampus and (c) region of the locus coeruleus on 20 ~m coronal sections stained with thionin. Arrows indicate the extent of the dialysis probe membrane.
D.N. Thomas et al. /Brain Research 645 (1994) 135-142 2.2. Microdialysis and sample analysis
137
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The dialysis probes situated in either of the three structures were perfused with a physiological medium containing 145 mM NaC1, 4 mM KCI, 1.2 mM CaC12 and 2 mM Na2HPO4, final pH of the medium being pH 7.4. The probes were dialyzed at a flow rate of 2.34 /zl/min and samples were collected every 15 min. The probes were allowed to equilibrate for 1-2 h prior to collection of baseline samples. Baseline samples were then collected until three consecutive samples did not differ by more than 10%. Cocaine (20 mg/kg) was injected intra-peritoneally and sampling was continued for a further 2 h. In a subsequent group of animals cocaine (1-100 /xM) was administered via the dialysis probe located in either the hippocampus, frontal cortex or the region of the locus coeruleus. Each concentration was applied for a period of 15 min starting with the lowest concentration of cocaine. Three sampling intervals following the application of cocaine were allowed to intervene before the next concentration was applied. The samples were analyzed for norepinephrine using HPLC with electrochemical detection. The mobile phase (75 mM NaH2PO4, 1.5 mM sodium dodecyl sulphate, 20 /xM EDTA, 100 pA/l triethylamine, 15% acetonitrile and 12% methanol, pH 5.6) was filtered and degassed before pumping at a rate of 1 ml/min through a HR-80 column (3/z BDS, 80 × 4.6 mm, ESA Inc.). Electrochemical detection was performed using a colouchem detector with Guard cell: +350 mV, Det 1 : - 4 0 mV and Det 2:+250 inV. Detection limits of the assay were 1 pg/sample.
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3. Results Fig. 1 illustrates representative probe placements in the frontal cortex, hippocampus and the region of the locus coeruleus on 20 Ixm coronal sections stained with thionin. The extent of the probe membrane is indicated with arrows. All animals included in the study had the effective region of the dialysis probe located in the appropriate structures. The effects of intraperitoneally administered cocaine (20 mg/kg) on extracellular norepinephrine in the three brain regions appear in Fig. 2. A one-way repeated measures analysis of variance which compared the pre-drug (basal) and post-drug norepinephrine levels in the hippocampus (Fig. 2A) failed to indicate a significant treatment effect (F < 1.0). The same dose of cocaine failed to alter norepinephrine levels in the frontal cortex (F < 1.0) (Fig. 2B). Norepinephrine levels in the locus coeruleus region (Fig. 2C), on the otherhand, increased (173% above baseline) following injections of cocaine, reaching a maximum 30 min later. A one-way repeated measures analysis of variance revealed a significant main effect (F = 2.411, d f = 8/43, P = 0.03). Post-hoe comparisons of norepinephrine levels following drug injections with basal values (3 samples preceding cocaine) indicated significant elevations in norepinephrine overflow at 30 and 45 min. Fig. 3 illustrates the effects of focally applied cocaine on norepinephrine overflow in the hippocampus. A two-way repeated measures analysis of variance
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Fig. 2. Effects of intraperitoneal cocaine (20 mg/kg) on extracellular norepinephrine in (A) frontal cortex (n = 4), (B) hippocampus (n = 4) and (C) region of the locus coeruleus (n = 4). All values are mean_+ S.E.M. * P < 0.05 for comparison between post-drug NE levels and basal values.
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D.N. Thomas et al. / Brain Research 645 (1994) 135-142
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which compared pre-drug norepinephrine levels with post-drug effects for all three concentrations revealed a significant effect of time ( F = 13.6, df 2/38, p < 0.001) and a significant time × concentration interaction ( F = 2.90, df 6/38, p < 0.02). The concentration effect failed to reach statistical significance ( F < 1.0). Post-hoe comparisons indicated significant peak effects following all three concentrations (1/zM, 34%; 10/zM, 86%; 100 /zM, 110% increase above baseline). In addition norepinephrine levels 15 min following application of 100 /zM cocaine was also found to be significantly elevated compared with basal levels. The focal application of cocaine to the frontal cortex had a considerably smaller effect on extracellular N E overflow than that seen in the hippocampus (Fig. 4). A two-way repeated measures analysis of variance revealed a significant time effect ( F = 6.47, df 2/32, P < 0.005) and a significant time × concentration interaction ( F = 3.16, df 6/32, P < 0.02). Post-hoc comparisons indicated that only the peak effect following application of 100 /zM cocaine to the frontal cortex 15-
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Concentration of cocaine (uM) Fig. 6. Peak effect of cocaine ( 1 - 1 0 0 / z M ) on N E overflow at 30 min post-injection in the frontal cortex (solid columns), hippocampus (dark hatched columns) and the region of the locus coeruleus (light hatched columns). Data are expressed as peak absolute increase above baseline (pg/sample), all values are mean_+ S.E.M. (n = 5-6). A repeated measures analysis of variances revealed a significant difference among the three regions across the three concentrations ( P < 0.0004). * Locus coeruleus extracellular NE levels were transformed to compensate for the decreased dialysis m e m b r a n e surface area of the probes used in this structure (see section 3).
was significantly different from the basal values (81% increase above baseline). Fig. 5 illustrates the effects of focally applied cocaine to the region of the locus coeruleus on norepinephrine overflow. A two-way repeated analysis of variance revealed a significant time effect ( F = 61.6, df 2/32, P < 0.001), concentration effect ( F = 22.48, df 3/32, P < 0.0001) and significant time × concentration interaction ( F = 12.02, df 6/32, P < 0.001). Post hoc comparisons indicated a significant peak effect at all three concentrations (1 /zM, 113%; 10/zM, 237%; 100 ~M, 450% increase above baseline). Comparative statistical analysis of the peak effect of cocaine in the three regions (Fig. 6) by two-way A N O V A revealed a significant difference among the three regions in response to cocaine ( F = 4.69, df 2/26, P = 0.029) and among the concentrations of cocaine administered ( F = 10.58, df 2/26, P = 0.0004). Since 2 mm probes were used in the locus coeruleus and 3 mm probes were used in the other two structures, the absolute amounts of extracellular N E recovered from the locus coeruleus were adjusted proportionately to reflect the differences in probe surface area in order to make statistical comparisons among regions. This assumes that the absolute amount recovered is linear with dialysis membrane surface area.
Time (rain) Fig. 5. Focal application of into the region of the locus son betwecn post-drug NE immediately preceding the S.E.M.
cocainc (1-100/~m) via the dialysis probe coeruleus (n = 5). * P < 0.05 for comparilevels and basal valucs (sampling period focal application). All values are mean +
4. Discussion The basal levels of norepinephrine in dialysates from the hippocampus (134 fg//~l) and the frontal
D.N. Thomas et al./Brain Research 645 (1994) 135-142
cortex (120 fg//zl) approximately 2 h following the insertion of 3 mm CMA microdialysis probes are within the range reported by other groups for these structures (50-217 fg//zl for the cortex [27,49] and 104-488 f g / ~ l for the hippocampus [1,2,14,23,27,48]). Precise comparisons are not meaningful; considering differences in probe construction, perfusion rates and other methological factors. Basal levels of norepinephrine in dialysate from the region of the locus coeruleus recovered via 2 mm CMA microdialysis probes were 48 fg//~l. Following adjustment for membrane surface area and recovery rate, these levels appeared to be relatively similar to those found for the hippocampus and frontal cortex. Considering the ability of cocaine to inhibit the reuptake of norepinephrine in vitro [17,26,46] it was surprising to find that intraperitoneal administration of behaviorally effective doses were almost devoid of effects on the extracellular NE in the frontal cortex and hippocampus. Similar results have been found following administrations of desipramine, the selective NE uptake blocker (Thomas et al. unpublished data). It is likely that the lack of effect of systemically administered cocaine and desipramine on extracellular NE in these two structures is related, in part to their ability to produce elevations of NE in the somatodendritic region (locus coeruleus). There are two actions of cocaine which enter into determining its effects on the accumulation of NE in terminal regions of the locus coeruleus projection system. The first is inhibition of reuptake, the second is inhibition of noradrenergic neuronal firing. Both cocaine and amphetamine have been found to be potent inhibitors of locus coeruleus neurons following systemic injections [12,34-38]. In fact, pharmacologically relevant doses have been shown to decrease the firing rate of locus coeruleus neurons by at least 70% [10,3438]. Such effects appear to be mediated through presynaptic az-adrenoceptors [35] located in the perikaryal regions [6]. It is proposed that the accumulation of NE in the locus coeruleus region in this study by systemically administered cocaine inhibits the firing rate of these cells through az-adrenoceptors. In the presence of substantially decreased neuronal activity and exocytotic release, cocaine would be expected to have little effect on NE overflow in the projection areas. The ability of systemically administered cocaine to enhance NE overflow in the locus coeruleus region without concurrent effects in the hippocampus and frontal cortex is somewhat puzzling. However, the mechanism governing release of norepinephrine in the somatodendritic region are not fully understood. It is possible that the process is somewhat independent from that which determines release from the terminals via CaZ+-dependent exocytotic mechanisms. Such independence could account for the ability of cocaine to
139
increase NE in the locus coeruleus without having an effect in the forebrain structures. Alternatively, it is possible that such findings may be due to more efficient NE reuptake mechanisms in the projection fields relative to the locus coeruleus. The differential regional effects of cocaine on extracellular norepinephrine in this study suggest that its predominant effects are initially mediated through the somatodendritic region. Electrophysiological findings reported by Pitts and Marwah [37] also support this conclusion. These investigators found that cocaine inhibited locus coeruleus neurons but concurrently increased the activity of Purkinje cells in the cerebellum. On the surface such effects on Purkinje cells by cocaine appear contradictory since norepinephrine is known to inhibit their firing rate through/3-adrenoceptors. If the predominant effect of cocaine in these studies was to enhance accumulation of norepinephrine in the terminals of locus coeruleus neurons projecting to the cerebellum, then an inhibition of neuronal activity should be seen. These effects can be understood, however, if it is assumed that the modulatory effects of cocaine on monoaminergic synapses is greater at the level of the locus coeruleus soma than at the terminals. Inhibition of locus coeruleus neurons by cocaine might enhance the activity of the Purkinje cells by decreasing the tonic inhibitory actions of NE. In the same study, cocaine also attenuated the inhibitory effects of NE evoked by locus coeruleus stimulation and had no effect on the inhibitory effects of iontophoretically applied NE on Purkinje cells. These findings further support the proposition that the predominant actions of cocaine occur in the locus coeruleus. Focal applications of cocaine directly to the hippocampus and frontal cortex, by circumventing its effects in the locus coeruleus, should reveal the uptake inhibitory properties of this drug in these terminal regions. Focal applications of cocaine to the two regions in this study did indeed increase norepinephrine overflow in both. Surprisingly, however, the two structures showed differential responses to the focal administration. In the hippocampus, extracellular norepinephrine increased in a concentration dependent manner, whereas in the frontal cortex, only the highest concentration produced a significant elevation. There are several possible reasons for such differential actions of cocaine. First, it is possible that the noradrenergic terminals in the frontal cortex have a paucity of uptake sites relative to other regions. There is a lack of evidence, however, to support this proposition. Radioligand binding studies [47] using the highly selective noradrenergic uptake blocker nisoxetine, have not found differences in the density of binding sites in the hippocampus and cortical regions. Results from a recent study by Kuczenski and Segal [27] are consistent with the notion that uptake site density is equivalent in
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the two regions. These investigators reported that amphetamine-induced increases in extracellular norepinephrine were equivalent in the frontal cortex and the hippocampus. While these findings appear incongruent with the present results, it should be noted that amphetamine is thought to produce some of its effects on catecholamine function by promoting cytoplasmic release through exchange diffusion which is carrier mediated and not Ca 2+ dependent [13,21,28]. The enhanced synaptic accumulation of catecholamines by cocaine, on the otherhand, is dependent on blockade of reuptake in the presence of Ca z+ dependent exocytotic release [18,21,26,46]. Therefore, if carrier mediated release is critical to the ability of amphetamine to enhance extracellular norepinephrine levels, the relatively similar effects of this psychomotor stimulant in the two structures would suggest that both have an equal density of uptake sites. Although the density of carriers in the two structures may not be different, it is possible that there are differences in their affinity for cocaine. It is of interest to note that concentrations of cocaine up to 1 /zM do not inhibit nisoxetine binding in the cortex [47]. In the present study, similar concentrations are achieved by diffusion of a 10 /zM solution through the dialysis probe since cocaine tissue levels within 250/xM of the probe appear to be approximately 10% of that in the dialysate (unpublished observations). At that concentration of cocaine, we failed to observe an effect on norepinephrine overflow in the frontal cortex. The differential effects of focally applied cocaine also could be related to different basal activities of locus coeruleus neurons projecting to the two regions. Cocaine-induced increases in extracellular norepinephrine are dependent almost exclusively on its ability to inhibit reuptake of this brain amine. Since cocaine induced accumulation in the presynaptic space of the hippocampus and frontal cortex is dependent on exocytotic release, the region with a higher basal output would be expected to show the largest increases in norepinephrine overflow. Focal application of cocaine to the locus coeruleus region produced significant concentration dependent increases in N E overflow in the perikaryal area indicating the prescence of prominant somatodendritic N E uptake processes. The locus coeruleus in this study appeared to be somewhat more responsive to the direct applications of cocaine than the two terminal regions. For example, when alterations in N E levels are expressed relative to the basal values, application of the 10 /zm cocaine increased extracellular N E in the frontal cortex by 16%, the hippocampus by 86% and the locus coeruleus by 237%. These findings also are consistent with the concept that the predominant actions of cocaine occur in the locus coeruleus. It is also of interest to compare the effects of
cocaine on dopamine overflow with its effects on norepinephrine overflow in this study. Systemically administered cocaine produces substantial increases in dopamine overflow in the terminal regions of the mesolimbic (n. accumbens) and nigrostriatal (striatum) dopamine pathways under similar conditions employed in this study [19,20]. Similarly, focally applied cocaine to the striatum and n. accumbens results in dramatic increases in dopamine overflow [20]. These differences in the response to cocaine can be understood in terms of the relatively high activity of mesolimbic dopamine neurons (5-8 Hz) [3,38] compared with the norepinephrine locus coeruleus neurons (1-2 Hz) [10,3436,39] and the relatively lower inhibitory actions of the drug on the basal activity of dopamine neurons [38]. Finally, it is important to emphasize that these studies were conducted in the anaesthetized rats. It is possible that quantitatively different responses may be seen in unanesthetized animals. Curtis et al. [10] for example, have recently reported that cocaine-induced inhibition of locus coeruleus neurons is quantitatively less in unanesthetized rats compared with those under halothane anaesthesia. If cocaine is less effective in depressing locus coeruleus activity in unanesthetized animals, it is possible that modest increases in hippocampal and frontal cortex norepinephrine could be realized following systemic administration. Preliminary findings from our laboratory support this prediction. Qualitatively, however, the effects of cocaine in the two preparations should be equivalent (i.e. effects in the locus coeruleus should be greater than those in the hippocampus and frontal cortex following systemic administration and the hippocampus should be more responsive to focal cocaine than the frontal cortex).
Acknowledgments This work has been partially supported by a postdoctoral training fellowship from the MacArthur Foundation Mental Health Research Network I (Psychobiology of Depression) for Dr. D.N. Thomas. We also gratefully acknowledge the expert technical assistance provided by Tim Sullivan, Judith Kransdorf and Connie Chiueh.
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