Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: Pharmacological and behavioral studies

Neuroscience Vol. 27, No. 3, pp. 897-904, Printed in Great Britain

0306-4522/88 $3.00 + 0.00 Pergamon Press plc 0 1988 IBRO

1988

CHARACTERIZATION OF HIPPOCAMPAL NOREPINEPHRINE RELEASE AS MEASURED BY MICRODIALYSIS PERFUSION: PHARMACOLOGICAL AND BEHAVIORAL STUDIES E. D. Departments

ABERCROMBIE,*

R. W.

KELLER

JR

and M. J.

ZIGMOND

of Behavioral

Neuroscience and Psychiatry and the Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, U.S.A.

Abstract-The release of endogenous norepinephrine in hippocampus was studied in freely moving rats with microdialysis perfusion. Using a loop-style dialysis probe, the basal amount of norepinephrine collected in 15-min fractions averaged 12 pg/25 ~1. Correcting for recovery (21%) the concentration of norepinephrine in the extracellular fluid of hippocampus under resting conditions was estimated to be approximately 14 nM. The alpha, adrenoceptor antagonist yohimbine (5.0 mg/kg, i.p.) increased norepinephrine efflux to 230% of basal levels. Clonidine (0.3 mg/kg, i.p.), an alpha, adrenoceptor agonist, decreased norepinephrine efflux to 56% of baseline. Addition of the reuptake blocker desipramine (1 .On M) to the perfusate had no significant effect on norepinephrine efflux. However, increasing the K+ concentration of the perfusate to 30mM increased norepinephrine efflux to 196% of baseline, and this effect was increased nearly two-fold by the addition of desipramine to the perfusate (364% of baseline). Restraint stress and intermittent tailshock increased norepinephrine efflux to 213% and 234% of baseline, respectively. The results suggest that microdialysis is a useful way to study norepinephrine release in hippocampus and they permit several conclusions to be drawn. First, the data obtained with systemic administration of alpha, adrenoceptor drugs emphasize the fact that a variety of regulatory mechanisms exist that may affect transmitter levels in the extracellular fluid. Second, the ratio of extracellular to intracellular norepinephrine in hippocampal tissue is considerably higher than that reported for dopamine in striatum. Coupled with the small effect of norepinephrine uptake blockade, this suggests that nerve terminal density is an important factor in determining the concentration of catecholamines in the extracellular fluid. Finally, the observed stress-induced increase in norepinephrine efflux supports the hypothesis that the locus coeruleus noradrenergic system exerts an important central influence during stressful conditions.

The noradrenergic innervation of forebrain, which originates from the pontine nucleus locus coeruleus

(LC), “.‘8,28has long been implicated in the mediation of arousal and stress.6,‘7,24.38In support of this hypothesis, it has been observed that the electrophysiological activity of these neurons is highly responsive to stressful stimulation.2 Moreover, a variety of stressors have been shown to increase norepinephrine (NE) turnover in brains~‘0*20~38~4’ and sufficiently intense stressors can even reduce the NE content of brain.7,8.45 Both electrophysiological studies and biochemical analyses of tissues, however, require that one infers changes in transmitter release and, as yet, there have been no direct demonstrations of the effects of stress on NE release in terminal areas of the LC. Recently, *To whom correspondence should be addressed at: Department of Behavioral Neuroscience, 571A Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, U.S.A. Abbreuiarions: CSF, cerebrospinal fluid; DA, dopamine; DMI, desipramine hydrochloride: DOPAC. dihvdroxvphenyla&ic acid; EDTA, ethylenediaminetetra-acetatk; 5-HIAA, 5-hydroxyindoleacetic acid; LC, locus coeruleus; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine. 897

however, several techniques have been developed to overcome this limitation by permitting the measurement of compounds in extracellular fluid of the brain in freely moving animals.25927In the present study we have utilized one such technique, perfusion of brain tissue via microdialysis tubing followed by analysis of the perfusate with high-performance liquid chromatography and electrochemical detection. Using microdialysis we have examined endogenous NE efflux in the hippocampus of unanesthetized behaving rats. First, we have examined NE concentration under basal conditions and in response to a local elevation in extracellular KC concentration. Second, we have studied the effects of pharmacological manipulations using drugs that affect noradrenergic neurotransmission. Finally, we have measured NE release in response to two stressors, restraint and intermittent tailshock. EXPERIMENTAL

Dialysis loop preparation

PROCEDURES

and calibration

Dialysis loops were constructed from a 1.5-cm piece of hollow dialysis fiber with a molecular weight cutoff of 5000 (Nephross Allegro H.F., Organon Teknika Corp.). A 2.0-cm length of Formvar-insulated nichrome wire (A-M Systems) was inserted into the fiber. Using the wire as a

E. D. ABERCROMBIEet al.

898 DIALYSIS

PROBE

~16

Gauge Stainless Steel Tube

was exposed and a small hole was drilled to allow implantation of the dialysis probe into the dentate gyrus of the dorsal hippocampus (coordinates: AP -3.8, ML k 2.0, DV -4.0 relative to Bregma). The dialysis probe was affixed to the skull with several screws and dental cement. The artificial CSF was continuously perfused through the probe at a rate of 2.0 pl/min via a fluid swivel (Spalding Medical Products) that permitted relatively unrestricted movement of the animal. The animal was allowed to recover for approximately 16 h before experiments were begun and all experiments were conducted during the first two days post-implantation. Dialysis samples were collected at 15-min intervals in plastic vials. Before initiating any experimental manipulation, NE efflux was monitored for a minimum of 1 h in order to ensure stable baseline values. Analysis qf dialysis samples

Scale

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=

t mm

Wire (0.003”dia.)

Fig. 1. Schematic showing the major

drawing of the dialysis loop probe, components utilized in the construction of the probe.

guide, each end of the dialysis tubing was threaded into a 60-cm length of polyethylene tubing (PE-IO, Clay Adams) to a depth of 5.0mm and glued into place with cyanoacrylate adhesive (Elmer’s Wonderbond Plus). After the adhesive had dried, the dialysis fiber was bent into a loop (5OG6OOnm wide). A 1.5-cm length of 16-g stainless steel tubing was placed over the free ends of the polyethylene tubing and positioned just above the dialysis loop, where it was elued into nlace with epoxy (Devcon) to provide stabilyty to the probe. The epoxy also was used to coat the dialysis fiber at its junctures with the polyethylene tubing so that the active portion of the loop was 2.0 mm in vertical distance. Figure 1 shows a schematic representation of the dialysis probe. The dialvsis nrobe was allowed to dry for at least 24 h. The recovery of NE was determined for each probe as follows. Filtered artificial cerebrospinal fluid (CSF) (189 mM NaCl, 3.9mM KC1 and 3.37 mM CaCl,; pH 6.0) was pumped through the probe at 2 nl/min with a Harvard microliter syringe pump. The probe was suspended in a beaker of the artificial CSF solution to which standards (norepinephrine, dihydroxyphenylacetic acid, S-hydroxyindoleacetic acid, and dopamine; NE, DOPAC, 5-HIAA, and DA) had been added at a concentration of 0.5 PM, and the amount of NE in the perfusate was compared to that in the buffer. The buffer was degassed thoroughly with argon during this procedure to prevent air oxidation of the catecholamines and metabolites. Diulysis loop implantation and sample collection Male SpragueeDawley rats (2755350 g) were anesthetized with chloral hydrate (400mg/kg i.p.) and placed in a stereotaxic instrument (David Kopf Instruments). The skull

The dialysate was injected directly (25 ~1) into a highperformance liquid chromatography system consisting of a Waters M45 Solvent Delivery System, a Waters U6K injector and a Velosep RP-18 column (100 x 3.2mm, 3pm; Brownlee Labs). The mobile phase consisted of 0.1 M sodium phosphate buffer pH 3.6, 100 p M EDTA, 1.O mM sodium octyl-sulfate and 9% (v/v) methanol. The flow rate through the system was 70pl/min. Electrochemical detection was accomplished with an ESA Coulochem model 5100A detector operated in differential mode whereby the catecholamines and metabolites underwent sequential oxidation and reduction. The potential of the first (oxidizing) detector cell was set at f0.37 V, and the potential of the second (reducing) detector cell at -0.26 V. Figure 2 shows chromatograms obtained from an injection of standards and from a hippocampal dialysate sample utilizing these assay parameters. Pharmucological treatments Drugs used in these studies were obtained from Sigma Chemical Co. and included yohimbine hydrochloride, an alpha> adrenoceptor antagonist,” clonidine hydrochloride, an alpha, agonisti and desipramine hydrochloride (DMI), an inhibitor of high affinity NE uptake.” Yohimbine and clonidine were administered systemically in 0.5 ml propylene glycol and l.Oml/kg 0.9% NaCl, respectively; DMI was added directly to the artificial CSF solution used to perfuse the tissue. Stressors Intermittent tailshock. This was delivered via a tail cuff containing two stainless steel contact electrodes on opposite sides of the rat’s tail. Through this cuff, animals received constant current pulses of l.O-mA intensity passed for 1 s every 10 s for a duration of 1 min. This process was repeated every 5 min during the 30-min stress period. Restraint. Restraint was produced by placing a metal loop over the neck of the animal and inserting it into predrilled holes in a wooden platform. The limbs were secured with adhesive tape to four metal clips. The animal was left otherwise undisturbed for 30 min. Hislological analysis Upon completion of some experiments, rats were perfused intracardially with saline containing 8% formalin and the dialysis probe implantation site was verified histologically in coronal sections after Cresyl Violet staining. Figure 3 shows an example of the dialysis probe placement. Dora analysis The NE in each sample was expressed as a percentage of the average NE concentration for four baseline samples. Data are mean k S.E.M. of the results obtained from groups of n animals, The effects of the experimental manipulations on hippocampal NE efflux were analysed by comparing the mean baseline NE concentration and that of

Hippocampal NE release

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Fig. 2. Chromatogram of 50-pg standards (left) and 25-~1 hippocampal dialysate (right). The hippocampal dialysate sample shown contained 12.0 pg NE. 2 x and 0.5 x indicate multiples of the amplification at which the standard chromatogram was obtained (1 x ).

Fig. 3. Coronal section through dorsal hippocampus, histologically verifying the implantation site of the dialysis probe. The section was obtained from an experimental animal approximately 36 h after probe implantation. The probe was removed after in situ formalin fixation of the brain.

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experimental samples using one-way repeated measures analysis of variance coupled with Dunnett’s test.

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0.3MG/KG

CLON

RESULTS

Basal monoamine content of extracellular fluid from hippocampus

The amount of NE obtained in 25~1 hippocampal dialysate samples collected from resting animals was 12.2 + 0.6 pg. When corrected for recovery (21.2 f 0.9%) the extracellular concentration of NE in hippocampus was estimated to be 14 + 1 nM. Although epinephrine, dopamine and serotonin were not detected in these samples, we were able to detect DOPAC and 5-HIAA, and estimated their concentration to be 14 f 2 nM (recovery 24.2 + 0.6%) and 613 f 39 nM (recovery 20.9 + 1.1%) respectively. Effects of systemic yohimbine and clonidine

Administration of yohimbine (5.0 mg/kg, i.p.) caused an increase in hippocampal NE efflux which reached a maximum of 230% of basal levels and then returned to baseline over the course of 8 h [F(l7,51) = 8.31, P < O.OOOl](Fig. 4). At a lower dose of yohimbine (2.0 mg/kg, i.p.), we observed no significant change in NE [F(8,24) = 2.10, P = 0.221 (Fig. 4). The systemic injection of clonidine (0.3 mg/kg, i.p.) resulted in a significant decrease in hippocampal NE efflux to 56% of baseline [F(l9,38) = 10.07, P < O.OOOl] that gradually returned to baseline over the course of 4 h (Fig. 5). Lower doses of clonidine (0.025 mg/kg, n = 1; 0.1 mg/kg, n = 3) failed to produce an observable effect on hippocampal NE efflux (data not shown).

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Fig. 4. The effect of an alpha, adrenoceptor antagonist on hippocampal NE release. Yohimbine (YOH, 2.0 or 5.0 mg/kg) was administered intraperitoneally at the time indicated by the arrow. The 2.0mg/kg dose (filled diamonds) produced a brief increase in NE efflux to levels 125% of baseline (n = 4). The 5.0 mg/kg dose (filled circles) produced a larger increase (maximum increase to 230% of baseline) in extracellular NE levels that returned to baseline over the course of 8 h (n = 4). In these experiments, samples were collected at 30-min intervals. Results are mean k S.E.M.; *P < 0.05 versus basal values.

2.5 t

Fig. 5. The effect of an alpha, adrenoceptor agonist hippocampal NE release. Clonidine (CLON, 0.3 mg/kg) administered intraperitoneally at the time indicated by arrow (n = 3). NE efflux was decreased to a maximum 56% of baseline by this manipulation and gradually turned to baseline over the course of 4 h. Results mean + S.E.M.; *P < 0.05 versus basal values.

on was the of reare

Effects of desipramine hydrochloride and increased K+ concentration in the perfusate

To determine the role of high-affinity NE uptake in establishing the extracellular concentration of NE, DMI was added to the perfusate to a concentration of 1.0 PM. In the presence of DMI alone, no significant change in NE efflux was observed [F( 15,45) = 1.43, P = 0.181 (Fig. 6a). Elevating the K+ concentration of the artificial CSF perfusate to 30 mM significantly increased NE efflux to 196% of baseline [F(7,21) = 6.53, P = 0.0004]. This K+-induced elevation of extracellular NE was increased to 364% of baseline by the addition of DMI [F(7,21) = 8.20, P = O.OOOl](Fig. 6b, c). Eflects of acute stress

A significant increase in hippocampal NE efflux was observed in response to restraint [F(9,36) = 11.68, P < O.OOOI].NE levels were maximal in the first and second 15-min fractions, reaching a peak of 213% of baseline. At the end of the second fraction the restraint was terminated and NE levels returned to baseline within 15-30 min (Fig. 7). Tailshock stress was also associated with a significant increase in hippocampal NE efflux [F(9,36) = 14.41, P < O.OOOl].In this case NE levels in extracellular fluid continued to rise throughout 30min of shock, reaching a maximum of 234% of baseline during the 15-min fraction collected after the shock session. Control levels of NE were achieved within the next 15min (Fig. 8). DISCUSSION

The present studies demonstrate that in uiuo microdialysis is a suitable method with which to study NE overflow into the extracellular space of the hippocampus. The loop-style dialysis probes that were employed provided a relative recovery for NE of

901

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Fig. 6. Effect of reuptake blockade on hippocampal NE release under basal conditions and in the presence of elevated K+. The addition of the reuptake blocker desipramine (DMI) to the perfusate in a concentration of 1.OPM (shaded bars, panel A) was not associated with a significant increase in NE efflux (125% of baseline, n = 4). When the potassium (K+) concentration in the perfusate was increased to 30 mM (shaded bars, panel B), NE efflux was increased to a maximum of 196% of baseline (n = 4). The combination of 1.0 PM DMI and 30 mM K+ in the perfusate (shaded bars, panel C) resulted in a large increase in NE efflux. In this condition, the maximum increase in extracellular NE observed was 364% of baseline (n = 4). Results are mean k S.E.M.; *P < 0.05.

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Fig. 7. Effect of restraint stress on hippocampal NE release. Rats were restrained in a prone position for 30 min (shaded bars). This resulted in a maximum increase in NE efflux to 213% of baseline (n = 5). Results are mean * S.E.M.; ‘P < 0.05.

TIME (h)

Fig. 8. Effect of tailshock stress on hippocampal NE release. Through a tail cuff, animals received intermittent shocks for a total of 30 min (shaded bars). This manipulation produced a maximum increase in NE efflux to 234% of baseline (n = 5). Results are mean k S.E.M.; *P < 0.05.

902

E. D. ABERCROMBIE et al

2 1%. This high recovery value, combined with the sensitivity of the assay, allowed us to detect basal levels of NE in dialysis samples collected every 15 min. NE levels in the perfusate remained stable for approximately 2 days after implantation of the dialysis probe. The basal value of 12.2 pg NE per 25~1 fraction that was obtained in the present study is quite comparable to that recently reported for cortical NE efflux using microdialysis coupled with radioenzymatic assay of NE.** We believe that the NE obtained in the dialysis samples was derived primarily from local NE nerve terminals in hippocampus rather than from the sympathoadrenal system. First, although NE was readily detectable, a peak corresponding to epinephrine was never observed in the chromatographic assay. Since epinephrine is a significant component of plasma, particularly during stress, this suggests that peripheral catecholamine made little if any contribution to our measurements. Furthermore, the increment in NE that we observed in response to stress was considerably smaller than that which has been observed in plasma. 2’,29Moreover, a marked increase in NE efflux was observed in response to increased K+ concentration in the perfusate, also supporting a neuronal origin for the observed NE. Finally, in preliminary studies we have observed that extensive destruction (>95%) of the NE afferents to hippocampus, produced by local injection of the neurotoxin 6-hydroxydopamine into the dorsal noradrenergic bundle, reduces the NE concentration in our samples to undetectable levels.3 Furthermore, the stress- and K+-induced increases in NE release are no longer observable in these animals. These and other experiments (see Ref. 42) suggest that the dialysis probe is functionally within the blood-brain barrier. Using the basal concentration of NE that we obtained in our dialysis samples and correcting for relative recovery of the probe, the extracellular concentration of NE in hippocampus was estimated to be approximately 14 nM. Previous microdialysis studies have estimated the extracellular concentration of dopamine (DA) in striatum to be 2&50 nM.“.‘5,19.33 Considering that tissue levels of DA in striatum are approximately 25-fold greater than are tissue levels of NE in hippocampus,3.‘6 a difference of only two- to five-fold in extracellular transmitter levels in the two structures was unexpected. A possible explanation for this phenomenon relates to the density of DA nerve terminals in the striatum, which is much greater than that of NE nerve terminals in hippocampus. A relative absence of high-affinity uptake sites in hippocampus would allow released NE to diffuse more freely in extracellular space, since uptake by neighboring nerve terminals would be less likely. Findings concerning the relative effects of uptake inhibitors on extracellular NE and DA concentrations are consistent with this explanation (see below). Drugs that stimulate central alpha, adrenoceptors decrease the firing rate of NE neurons in LC, while

drugs that block these receptors increase the firing rate of these neurons.‘.2a,30.40We wished to determine whether our measure of extracellular NE was correlated with these known electrophysiological changes. In the present experiment, systemic administration of the alpha, adrenoceptor antagonist yohimbine elicited a dose-dependent increase in hippocampal NE efflux. In addition, clonidine, an alpha, adrenoceptor agonist, produced a decrease in extracellular NE levels. These results are in agreement with recent microdialysis studies.22,30 It is interesting to note, however, that the doses of yohimbine and clonidine required to elicit significant changes in hippocampal NE efflux were in excess of the doses necessary to effect changes in the neuronal activity of LC neurons.26,30,“” A similar situation appears to exist in the case of the nigrostriatal DA system. 9.34.43,44 This may be due in part to the fact that microdialysis measures the amount of neurotransmitter that overflows into the extracellular space, not the actual concentration of neurotransmitter in the synapse, thus requiring relatively large changes to occur before they can be detected. It also is likely that firing rate and transmitter release do not always co-vary and that the alterations in hippocampal NE release observed after systemic administration of yohimbine and clonidine are determined at least partly by the blockade and stimulation, respectively, of alpha, adrenoceptors located on NE terminals. There is evidence suggesting that these pre-synaptic alpha, adrenoceptors play a prominent role in the regulation of NE release in response to alpha, adrenergic drugs.‘*.” Thus, the overall effect of systemic administration of alpha, adrenergic antagonists and agonists on NE release is probably a consequence of combined effects on both cell body and nerve terminal alpha, adrenoceptors. In addition to autoreceptor influences, NE overflow into the extracellular space should be affected by transmitter reuptake processes. It is thought that a major route of inactivation for NE in the brain is the reuptake of transmitter into the nerve terminal where it is subsequently recycled or degraded.3s.36 In the present study, we observed that under basal conditions the level of extracellular NE was not significantly altered by the addition of the NE reuptake blocker DMI to the perfusate, although DMI did increase K+ stimulated NE release. Like the relatively high basal level of NE, the relatively weak effect of DMI may also be accounted for by the low density of NE terminals in the hippocampus. This explanation is supported by observations that local reuptake blockade in the striatal DA system, where terminal density is great, results in lo- to 20-fold increases in extracellular DA concentrations.‘9,39 The fact that the K+-stimulated efflux of NE was potentiated by DMI suggests that as the amount of NE in the extracellular fluid becomes greater, reuptake plays a larger role in regulating NE concentration and the influence of reuptake processes is revealed.

Hippocampal NE release

Stress increased extracellular hippocampal NE levels by approximately two-fold. This provides further support for the hypothesis that the NE system originating in the LC is importantly involved in the response to stressful conditions. By modulating the activity of target neurons and thereby facilitating the CNS response to stress, the LC may function in a manner analogous to the peripheral sympathetic NE neurons.4.46 The stress-induced increase in NE efflux observed in the present study is in good agreement, both in direction and magnitude, with recent electrophysiological data.2 In that study, a two- to three-fold increase in the neuronal activity of LC neurons was reported in response to acute stressor application in freely moving cats. CONCLUSION

The present study demonstrates that the effects of both pharmacological and behavioral variables on NE efflux in hippocampus can be studied using microdialysis. The results obtained with pharma-

903

cological agents emphasize the need for careful interpretation of results. The exact relation between changes in transmitter efflux that occur in the extracellular fluid as measured by microdialysis and changes in transmitter release in the synapse may be obscured by the involvement of various regulatory mechanisms such as autoreceptors and reuptake carriers. Given this precaution, it appears that microdialysis will provide important new data regarding dynamic changes in neurotransmitter output in response to specific pharmacological and behavioral events.

thank Edward M. Stricker for helpful discussions. These data were reported in November 1987, at the 17th Annual Meeting of the Society for Neuroscience, New Orleans, LA, and in January 1988, at the 21st Annual Meeting of the Winter Conference on Brain Research, Steamboat Springs, Co. This research was funded in part by USPHS grants NS19608, MH30915. E. D. A. was supported by an NIMH Postdoctoral Traineeship (MH18273) and an MHCRC Grant (MH30915), and M. J. 2. by a Research Scientist Award (MHO0058). Acknowledgements-We

REFERENCES 1.

Abercrombie E. D. and Jacobs B. L. (1987) Microinjected clonidine inhibits noradrenergic neurons of the locus coeruleus in freely moving cats. Neurosci. Left. 76, 203-208. 2. Abercrombie E. D. and Jacobs B. L. (1987) Single-unit response of noradrenergic neurons in the locus coeruleus of freely moving cats. I. Acutely presented stressful and non-stressful stimuli. J. Neurosci. 7, 2837-2843. 3. Abercrombie E. D. and Zigmond M. J. (1988) Hippocampal norepinephrine and dihydroxyphenylacetic acid release following damage to the dorsal noradrenergic bundle: in uiuo microdialysis studies. Sot. Neurosci. Abstr. 14, (in press). 4. Amaral D. G. and Sinnamon H. M. (1977) The locus coeruleus: neurobiology of a central noradrenergic nucleus. Prog. Newrobiol. 9, 147-196. 5. Anisman H., Kokkinidis L. and Sklar L. S. (1984) Neurochemical consequences of stress: contributions of adaptive processes. In Psychological and Physiological Interaction in Response to Stress (ed. Burchfield S.), pp. 67-98.

Hemisphere, New York. 6. Aston-Jones G. (1985) Behavioral functions of locus coeruleus derived from cellular attributes. Physiol. Psychol. 13, 118-126. 7. Barchas J. D. and Freedman D. X. (1963) Brain amines: response to physiological stress. Biochem. Pharmacol. 12, 1232-1235. 8. Bliss E. I. and Zwanziger J. (1966) Brain amines and emotional stress. J. Psychiar. Res. 4, 189-198. 9. Bunney B. S., Walters J. R., Roth R. H. and Aghajanian G. K. (1973) Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. J. Pharmacol. exp. Ther. 185, 56&571.

10. Cassens G., Roffman M., Kuruc A., Orsulak P. J. and Schildkraut J. J. (1980) Alterations in brain noreoinenhrine metabolism induced by environmental stimuli previously paired with inescapable shock. Science 209, 1138-l 1’40. 11. Church W. H., Justice J. B. and Neil1 D. B. (1987) Detecting behaviorally relevant changes in extracellular dopamine with microdialysis. Brain Res. 412, 397-399. 12. Curet O., Dennis T. and Scatton B. (1987) Evidence for the involvement of presynaptic alpha-2 adrenoceptors in the regulation of norepinephrine metabolism in the rat brain. J. Pharmacol. exp. Ther. 240, 327-336. 13. Dennis T., L’Heureux R., Carter C. and Scatton B. (1987) Presynaptic alpha-2 adrenoceptors play a major role in the effects of idazoxan on cortical noradrenaline release (as measured by in viuo dialysis) in the rat. J. Pharmacol. exp. Ther. 241, 642-619. 14. Fuxe K., Hambuerger B. and Hiikfelt T. (1968) Distribution of noradrenaline nerve terminals in cortical areas of the rat. Brain Res. 8, 125-131. 15. Hernandez L., Stanley B. G. and Hoebel B. G. (1986) A small removable microdialysis probe. Life Sci. 39,2629-2637. 16. Jacobowitz D. M. and Richardson J. S. (1978) Method for the rapid determination of norepinephrine, dopamine and serotonin in the same brain region. Pharmacol. Biochem. Eehav. 8, 515-519. 17. Jones B. E. (1972) The respective involvement of noradrenaline and its deaminated metabolites in waking and paradoxical sleep: a neuropharmacological model. Brain Res. 39, 121-136. 18. Jones B. E. and Moore R. Y. (1977) Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study. Brain Res. 127, 23-53.

19. Keller R. W., Hucke C. L., Zigmond M. J. and Stricker E. M. (1985) Dialysis perfusion studies of the effects of amphetamine on the striatum. Sot. Neurosci. Absfr. 11, 671. 20. Korf J., Aghajanian G. K. and Roth R. H. (1973) Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus. Neuropharmacol. 12, 933-938. 21. Kvetnansky R., Sun C. L., Lake C. R., Thoa N., Torda T. and Kopin I. J. (1978) Effect of handling and forced immobilization on rat plasma levels of epinephrine, norepinephrine, and dopamine#-hydroxylase. Endocrinol. 103, 1868-1874.

904

E. D.

ABERCR~MBIE

ef

al.

22. L’Heureux R., Dennis T., Curet 0. and Scatton B. (1986) Measurement of endogenous noradrenaline

release in the rat cerebral cortex in uivo by transcortical dialysis: effects of drugs affecting noradrenergic transmission. J. Neurochem.

46, 1794-1801. 23. Langer S. Z., Raisman

R. and Briley M. (1981) High-affinity [3H]DMI binding is associated with neuronal noradrenaline uptake in the periphery and the central nervous system. Eur. J. Pharmacol. 72, 423-424. 24. Lidbrink P. and Fuxe K. (1973) Effects of intra~erebral injections of 6-hydroxydopamine on sleep and waking in the rat. J. Pharm. Pharmacol. 25, 84-87. 25. Marsden C. A. (1984) Measurement qf Neuroiransmitter Release in vivo. Wiley, New York. 26. Marwaha J. and Aghajanian G. K. (1982) Relative potencies of alpha-l and alpha-2 antagonists in the locus coeruleus, dorsal raphe and dorsal lateral geniculate nuclei: an electrophysiological study. J. Pharmacol. exp. Ther. 222, 287-293. 27. Myers R. D. and Knott P. J. (1986) Neurochemical Analysis of the Conscious Brain. Ann. N. Y. Acad. Sci. Vol. 473. 28. Pickel V. M., Segal M. and Bloom F. E. (1974) A radioautographic study of the efferent pathways of the nucleus locus coeruleus. J. camp. Neuroi. 155, 15-42. 29. Popper C. W., Chiueh C. C. and Kopin I. J. (1977) Plasma ~t~holamine ~on~ntrations in unan~thetiz~ rats during sleep, wakefulness, immobilization and after decapitation. J. Pharmac. exp. Ther. 202, 144-148. 30. Rasmussen K. and Jacobs B. L. (1986) Single unit activity of locus coeruleus neurons in the freely moving cat. II. Conditioning and pharmacologic studies. Brain Res. 371, 335-345. 31. Routledge C. and Marsden C. A. (1987) Comparison of the effects of selected drugs on the release of hypothalamic adrenaline and noradrenaline measured in viuo. Brain Res. 426, 103-I I I. hydrochloride (ST 32. Schmitt H. and Schmitt H. (1970) Interactions between 2-(2,6-dichlorophenylamine)2-imidazoline 155, Catapressan) and alpha-ad~nergic blocking drugs. Eur. J. P~armaco~. 9, 7-13. 33. Sharp T., Zetterstrom T. and Ungerstedt U. (1986) An in t&o study of dopamine release and metabolism in rat brain regions using intracerebral dialysis. f. Neurochem. 47, 113-122. 34. Skirboll L. R., Grace A. A. and Bunney B. S. (1979) Dopamine auto- and postsynaptic receptors: electrophysiological evidence for differential sensitivity to dopamine agonists. Science 206, 80-82. 35. Snyder S. H. and Coyle J. T. (1969) Regional differences in 3H-norepinephrine and ‘H-dopamine uptake into rat brain homogenates. J. Pharmacoi. exp. Ther. 165, 78-86. 36. Snyder S. H., Green A. 1. and Hendley E. D. (1968) Kinetics of ‘H-norepinephrine accumulation into slices from different regions of the rat brain. J. ~~arrnac~~. exp. Ther. 164, 9itIO2. 37. Starke K., Borowski E. and Endo T. (1975) Preferential blockade of presynaptic alpha receptors by yohimbine. Eur. J. Pharmacol. 34, 385-388. 38. Stone E. A. (1975) Stress and catecholamines. In Catechoiamines and Behavior, Vol. 2 (ed. FriedhotTA. J.), pp. 31-72.

Plenum, New York. 39. Strecker R. E., Sharp T., Brundin P., Zetterstrom

T., Ungerstedt U. and Bjorklund A. (1987) Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 22,

1699178. 40. Svensson T. H., Bunney B. S. and Aghajanian G. K. (1975) Inhibition of both noradrener~c and serotonergic neurons in the brain by the alpha-adrenergic agonist clonidine. Brain Res. 92, 291-308. 41. Tsuda A., Tanaka M., Kohno Y., Nishikawa T., Iimori K., Nakagawa R., Hoaki Y., Ida Y. and Nagasaki N. (1982) Marked enhancement of noradrenaline turnover in extensive brain regions after activity-stress in rats. Physiol. Behav. 29, 337-341. 42. Ungerstedt U. (1984) Measurement of neurotransmitter release by intracranial dialysis. In Measurement of Neurotransmitter Refease in vivo (ed. Marsden C. A.), pp. 81-106. Wiley, New York. 43. Zetterstrom T. and Ungerstedt U. (1984) Effects of apomorphine on the in vivo release of dopamine and its metabolites, studied by brain dialysis. Eur. J. Phar~laco~. 97, 29-36. 44. Zetterstrom T., Sharp T. and Ungerstedt U. (1984) Effect of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat studied by intracerebral dialysis. Eur. J. Pharmacol. 106, 27-37. 45. Zigmond M. J. and Harvey J. A. (1970) Resistance to central norepinephrine depletion and decreased mortality in rats chronically exposed to electric foot shock. J. Nemo-vise. Relations 31, 3733381. 46. Zigmond M. J. and Stricker E. M. (1974) Ingestive behavior following damage to central dopamine neurons: implications for homeostasis and recovery of function. In Neuropsychopharmaco~agy c# Mono~ine.~ and Their Regu~atary Enzymes (ed. Usdin E.), pp. 385402. Raven Press, New York. (Accepted 25 May 1988)