The Release of Noradrenaline in the Locus Coeruleus and Prefrontal Cortex Studied with Dual-Probe Microdialysis Olga L

Brain Research 906 (2001) 38±45 www.elsevier.com/locate/bres

Research report The release of noradrenaline in the locus coeruleus and prefrontal cortex studied with dual-probe microdialysis Olga L. Pudovkinaa,* , Yukie Kawahara a , Jan de Vries b , Ben H.C. Westerink a

aDepartment of Biomonitoring and Sensoring, University Center for Pharmacy, University of Groningen, Deusinglaan 1, 9712 AV Groningen, The Netherlands bDepartment of Medicinal Chemistry, University Center for Pharmacy, University of Groningen, Deusinglaan 1, 9712 AV Groningen, The Netherlands Accepted 10 April 2001

Abstract

The present study was undertaken to investigate and compare the properties of noradrenaline release in the locus coeruleus (LC) and prefrontal cortex (PFC). For that aim the dual-probe microdialysis technique was applied for simultaneous detection of noradrenaline levels in the LC and PFC in conscious rats. Calcium omission in the LC decreased noradrenaline levels in the LC, but increased its levels in the PFC. Novelty increased noradrenaline levels in both structures. Infusion of the a2-adrenoceptor agonist clonidine decreased extracellular noradrenaline in the LC as well as in the PFC. Infusion of the a2A-adrenoceptor antagonist BRL44408, or the a1-adrenoceptor agonist cirazoline into the LC or PFC caused a similar dose-dependent increase in both structures. When BRL44408 or cirazoline were infused into the LC, few effects were seen in the PFC. Infusion of the 5-HT1A -receptor agonist ¯esinoxan into the LC or the PFC decreased the release of noradrenaline in both structures. When ¯esinoxan was infused into the LC, no effects were seen in the

PFC. When the GABAA antagonist bicuculline was applied to the LC, noradrenaline increased in the LC as well as in the PFC. It is concluded that the release of noradrenaline from somatodendritic sites and nerve terminals responded in a similar manner to presynaptic receptor modulation. The possible existence of dendritic noradrenaline release is discussed.  2001 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Catecholamines

Keywords: Noradrenaline; Locus coeruleus; Prefrontal cortex; Microdialysis; Collateral; Somatodendritic release

1. Introduction renaline in the LC [15,21,29,31,35]; however, the origin and properties of the relatively high levels of extracellular The locus coeruleus (LC) is the main source of norad- noradrenaline in this somatodendritic area have not yet renergic innervation of the brain. It is involved in physio- been fully evaluated. logical processes such as the sleep±wake cycle, adaptation Studies on dopamine and serotonin neuronal systems to stress, antinociception, opioid withdrawal, etc. [3,30]. indicated that these neurotransmitters are also released in One of the methods to study LC activity is to record the somatodendritic areas such as substantia nigra and extracellular noradrenaline by voltammetry and sample nucleus raphe. This type of release has been characterized techniques such as microdialysis or push±pull perfusion. as dendritic release [11,17]. Release of noradrenaline in the Several in vivo studies have been directed to nerve LC might also derive from dendritic sites, as Ð similar to terminal areas, e.g. the prefrontal cortex (PFC) or hip- the substantia nigra reticulata Ð small granular vesicles pocampus [10,23,34,37]. Others have studied norad- were found in dendrites of the LC and dendro-dendritic contacts have been described [14,26]. Another explanation *Corresponding author. Tel.: 131-50-363-3305; fax: 131-50-363- for the high levels of extracellular noradrenaline in the LC 6908. might be found in the presence of collaterals [2,33]. E-mail address: [email protected] (O.L. Pudovkina). The present study was designed to differentiate between

0006-8993/01/$ ± see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993(01)02553-7 O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45 39 dendritic and collateral release mechanisms for nor- mm and V/D 8.3 mm from lambda and surface of the adrenaline in the LC. For that aim one microdialysis probe skull, respectively. Another probe (exposed length 4 mm) was implanted in the LC and a second in the ipsilateral was placed in the ipsilateral PFC. Co-ordinates of im- PFC. In most of the experiments noradrenaline was plantation were as follows: A/P 3.3 mm, L/M 1.2 mm and detected in both probes. Various behavioral (novelty) or V/D 5.0 mm from bregma and dura. The probes were pharmacological conditions (infusion of receptor speci®c implanted under chloral hydrate anesthesia (400 mg/kg, compounds) were applied and the release of noradrenaline i.p.). from the LC was compared with that from the PFC as an Microdialysis experiments were carried out 24±48 h indicator of electrical activity of LC neurons [5]. For a after implantation of the probes. An on-line approach was better interpretation of the dual-probe experiments used in which the probes were perfused with a Ringer's BRL44408, cirazoline and ¯esinoxan were also infused solution at a ¯ow rate of 2.0 ml/min (BeeHave infusion into the PFC. The release of noradrenaline recorded in the pump, BAS, West Lafayette, IN, USA) and 15-min frac- PFC was considered as an example of terminal release. tions were collected. The composition of the Ringer's

The following pharmacological conditions were studied: solution was (in mM): NaCl 140.0; KCl 3.0; CaCl2 1.2; calcium omission, infusion of tetrodotoxin (TTX), the MgCl2 1.0. The composition of the Ca-free Ringer's a12-adrenoceptor agonist cirazoline, the a -adrenoceptor solution was (in mM): NaCl 141.8; KCl 3.0; MgCl2 1.0. agonist clonidine, the a2A-adrenoceptor antagonist Fractions of 15 min were on-line collected in the sample BRL44408, the GABAA antagonist bicuculline and the loop of a high-performance liquid chromatography 5-HT1A serotonin receptor agonist ¯esinoxan. (HPLC) system. Connections to the infusion pump and HPLC were made with ¯exible tubing (Peek, i.d. 0.12 mm). Before the experiments were ®nished, implantation 2. Materials and methods of the LC probe was functionally evaluated by infusion of 100 mM clonidine (45 min) into the LC probe [36]. A 2.1. Animals, drug treatment and doses decrease in extracellular noradrenaline in the PFC to at least 30% of controls was considered as an appropriate Male albino rats of a Wistar-derived strain (285±340 g; implantation. When the experiment was terminated, the rat Harlan, Zeist, The Netherlands) were used for the experi- was given an overdose of chloral hydrate and the brain was ments. The rats were housed in plastic cages (20330370 ®xed with 4% paraformaldehyde via intracardiac perfu- cm), with lights on from 07:00 to 19:00 h and had free sion. Coronal sections (40 mm thick) were made, and access to food and water. After probe implantation and dialysis probe placement was localized according to the during the experiments the rats were individually housed in atlas of Paxinos [24]. a plastic cage (25325330 cm). Experiments were carried out in the light cycle. 2.3. Chemical assays The experiments were approved by the Animal Care Committee of the Faculty of Mathematics and Natural Noradrenaline was quanti®ed by HPLC with electro- Science of the University of Groningen. chemical detection. A Shimadzu LC-10AD pump (Kyoto, The following drugs were used: clonidine HCl, (2)- Japan) was used in conjunction with an electrochemical bicuculline methylchloride, TTX (Research Biochemicals detector (ESA, Bedford, MA). The oxidizing potential of International, Natick, MA, USA), cirazoline hydrochloride, the cell was 1175 mV; the reduction potential of the cell BRL 44408 maleate (Tocris Cookson, Bristol, UK) and was 2200 mV. A reverse-phase column (15034.6 mm, ¯esinoxan (Solvay Pharmaceuticals, Weesp, The Nether- Supelco LC18: Belleofonte, PA, USA) was used. The lands). All drugs were dissolved in Ringer's solution and mobile phase consisted of a mixture of 4.1 g sodium were applied to the LC via retrograde microdialysis. acetate, 140 mg 1-octanesulfonic acid, 50 mg EDTA and

Concentrations of clonidine, bicuculline, TTX and 7% of methanol in 1000 ml H2 O (pH 4.4). The ¯ow rate ¯esinoxan were based on earlier microdialysis experiments was 1.2 ml/min. The detection limit of the assay was 3 [7,36,38]. fmol per sample (on-column).

2.2. Surgery and brain dialysis 2.4. Expression of results and statistics

Microdialysis was performed with two I-shaped probes. All values given are expressed as percentages of The dialysis tube was prepared from polyacrylonitrile/ control6S.E.M. The average of concentration of four sodium methalyl sulfonate copolymer (i.d.: 0.22 mm; o.d.: stable baseline samples before drug administration was 0.31 mm; AN 69, Hospal, Bologna, Italy). One probe considered as a control and was de®ned as 100%. A (exposed length 1.5 mm) was implanted in the vicinity of statistical program (Sigmastat 1.0) was used to calculate the right LC at the angle of 158. Co-ordinates of the the statistics. Data were analyzed by a non-parametric implantation were as follows: A/P 23.3 mm, L/M 1.3 repeated measurement one-way analysis of variance on 40 O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45 ranks (Dunnett's test). The level of signi®cance was set at P,0.05.

3. Results

3.1. Success rate of implantation and basal values

Noradrenaline was only detectable (.90% of the im- plantations) when the probe was localized in the vicinity of the LC. Basal values of noradrenaline in the LC (6S.E.M.) were 9.7260.7 fmol per sample (n545). The basal level in the LC in control rats often ¯uctuates during the ex- perimental period (6±8 h). In general lower levels were recorded when the rats were in the sleeping position. The basal values of noradrenaline in the PFC (6S.E.M.) were Fig. 2. Effect of Ca-depletion (black bar) in the LC during 45 min on 12.560.5 fmol per sample (n538). extracellular noradrenaline in the LC (d) and PFC (s). Data are given as % of basal levels6S.E.M. (n54). *P,0.05 compared to basal levels. 3.2. Effect of TTX infused into the LC on the extracellular noradrenaline in the LC 45 min caused a decrease of the extracellular noradrenaline Infusion of TTX in the LC (1 mM, 60 min) caused a levels in the LC to 20% of the basal levels (Fig. 2). This decrease of extracellular noradrenaline in the LC to 20% of decrease was statistically signi®cant between 15 and 60 basal levels (Fig. 1). The decrease was statistically signi®- min after the start of the infusion. During Ca depletion the cant between 15 and 120 min after the start of the infusion. extracellular noradrenaline in the PFC was increased to TTX infusions caused little inhibitory effect on the loco- about 150% of the basal levels. This increase was statisti- motion function. The decrease of noradrenaline was very cally signi®cant between 30 and 90 min after the start of similar to results of a dual-probe experiment (infusion of the infusion and at this time strong behavioral activation TTX into the LC and recording in the PFC) that have been was always observed. Animals exhibited constant turning, carried out earlier in our laboratory [36]. rearing and snif®ng. Noradrenaline output in the LC returned quicker to basal values as compared to the PFC 3.3. Effect of calcium-free Ringer's solution applied to when Ca21 -containing Ringer's solution was reintroduced. the LC on extracellular noradrenaline in the LC and PFC 3.4. Effect of novelty on extracellular noradrenaline in the LC and PFC Infusion of Ca-free Ringer's solution in the LC during To evaluate responsiveness of the LC to mild arousal, rats were exposed to a new cage during 15 min. The new cage was similar to the home cage. Exposure an animal to a new cage is considered to be mild stressful stimulus (novelty), which induces investigating behavior during the ®rst 5±10 min. This procedure enhanced the extracellular levels of noradrenaline in the LC to about 175% of the basal levels (Fig. 3). The increase of the extracellular noradrenaline was statistically signi®cant between 15 and 45 min after the start of novelty. In the PFC the level of noradrenaline was elevated to about 155% of the controls. This increase was statistically signi®cant between 15 and 45 min after the start of novelty.

3.5. Effect of clonidine, infused into the LC, on extracellular noradrenaline in the LC and PFC

Infusion of the a -adrenoceptor agonist clonidine (100 Fig. 1. Effect of tetrodotoxin (TTX) infusion (1 mM, black bar) into the 2 LC during 60 min on extracellular noradrenaline in the LC. Data are mM, 60 min) into the LC, caused a similar decrease of given as % of basal levels6S.E.M. (n57). *P,0.05 compared to basal noradrenaline in the LC and PFC to about 20% of the basal levels. levels (Fig. 4). The decrease was statistically signi®cant O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45 41

Fig. 3. Effect of novelty for 15 min on extracellular noradrenaline in the LC (d) and PFC (s). Data are given as % of basal levels6S.E.M. (n55). *P,0.05 compared to basal levels. between 30 and 120 min after the start of the infusion. No behavioral changes were seen.

3.6. Effect of BRL 44408, infused into the LC, on extracellular noradrenaline in the LC and PFC

Infusion of the speci®c a2A-adrenoceptor antagonist BRL 44408, in a concentration of 1, 10 and 100 mM(60 min each dose) into the LC, caused an increase of noradrenaline in the LC to about 130%, 180% and 680% Fig. 5. (a) Effect of BRL 44408 infusion (10, 100, 500 mM, black bars) into the LC during 60 min each dose, on extracellular noradrenaline in the of basal levels, respectively (Fig. 5a). The increases were LC (d) and PFC (s). Data are given as % of basal levels6S.E.M. statistically signi®cant at least between 75 and 225 min (n55). *P,0.05 compared to basal levels. (b) Effect of BRL 44408 after the start of the infusions. During infusion of 1 and 10 infusion (10, 100, 500 mM, black bars) into the PFC during 60 min each mM BRL 44408 extracellular noradrenaline in the PFC dose, on extracellular noradrenaline in the PFC (s). Data are given as % was unchanged. When 100 mM BRL 44408 was infused, of basal levels6S.E.M. (n55). *P,0.05 compared to basal levels. noradrenaline levels in the PFC rose to about 160% of basal levels. The increase was statistically signi®cant at 180 min after the start of the infusions. No behavioral changes were seen.

3.7. Effect of BRL 44408 infused into the PFC on extracellular noradrenaline in the PFC

Infusion of BRL 44408 into the PFC, in a concentration of 1, 10 and 100 mM (60 min each dose), caused an increase of noradrenaline in the PFC to about 125, 230, and 480% of basal levels, respectively (Fig. 5b). The increases were statistically signi®cant between 60 and 235 min after the start of the infusion. The release of noradrenaline in the LC was not affected by infusion into the PFC (data not shown). No behavioral changes were seen during BRL 44408 administration.

3.8. Effect of bicuculline, infused into the LC, on Fig. 4. Effect of clonidine infusion (100 mM, black bar) into the LC during 60 min on extracellular noradrenaline in the LC (d) and PFC (s). extracellular noradrenaline in the LC and PFC Data are given as % of basal levels6S.E.M. (n57). *P,0.05 compared to basal levels. Infusion of GABAA receptor antagonist bicuculline into 42 O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45 the LC, in a concentration of 50 mM (during 75 min), caused an increase of noradrenaline in the LC and PFC to about 185% and 155% of basal levels, respectively (Fig. 6). The increases were statistically signi®cant between 15 and 90 min after the start of the infusion. No behavioral changes were seen.

3.9. Effect of cirazoline, infused into the LC, on extracellular noradrenaline in the LC and PFC

Infusion of a1 adrenoceptor agonist cirazoline into the LC, in a concentration of 10 and 100 mM (60 min each dose), caused an increase of noradrenaline in the LC to 220% and 440% of the basal levels, respectively (Fig. 7a). The increases were statistically signi®cant between 15 and 150 min after the start of the infusion. Infusion of cirazoline into the LC did not alter the level of noradrenaline in the PFC. No behavioral changes were seen.

3.10. Effect of cirazoline, infused into the PFC, on extracellular noradrenaline in the PFC

Infusion of cirazoline into the PFC, in a concentration of 100 mM during 60 min, increased the extracellular noradrenaline levels in the PFC to 450% of the basal levels (Fig. 7b). The increase was statistically signi®cant between 15 and 90 min after the start of the infusion. The release of noradrenaline in the LC was not affected by infusion of cirazoline into the PFC (data not shown). No behavioral changes were observed. Fig. 7. (a) Effect of cirazoline infusion (10 and 100 mM, black bars) into 3.11. Effect of ¯esinoxan, infused into the LC, on the LC during 60 min each dose on extracellular noradrenaline in the LC (d) and PFC (s). Data are given as % of basal levels6S.E.M. (n55). extracellular noradrenaline in the LC and PFC *P,0.05 compared to basal levels. (b) Effect of cirazoline infusion (10 and 100 mM, black bars) into the PFC during 60 min each dose on s Infusion of the speci®c 5-HT1A -receptor agonist extracellular noradrenaline in the PFC ( ). Data are given as % of basal ¯esinoxan into the LC, in a concentration of 1 mM during levels6S.E.M. (n55). *P,0.05 compared to basal levels.

75 min, suppressed extracellular noradrenaline in the LC to about 20% of basal levels (Fig. 8a). The decrease was statistically signi®cant between 45 and 120 min after the start of the infusion. No changes in the extracellular levels of noradrenaline in the PFC were observed during ¯esinoxan infusion. No behavioral changes were seen.

3.12. Effect of ¯esinoxan infused into the PFC on extracellular noradrenaline in the PFC

Infusion of ¯esinoxan into the PFC, in a concentration of 1 mM during 75 min, suppressed extracellular levels of noradrenaline in the PFC to about 30% of basal levels (Fig. 8b). The decrease was statistically signi®cant between 45 and 120 min after the start of the infusion. The Fig. 6. Effect of bicuculline infusion (50 mM, black bar) into the LC during 75 min on extracellular noradrenaline in the LC (d) and PFC (s). release of noradrenaline in the LC was not affected by Data are given as % of basal levels6S.E.M. (n55). *P,0.05: compared infusion of ¯esinoxan into the PFC (data not shown). No to basal levels. behavioral changes were seen. O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45 43

observed during the calcium-free perfusion. This activation might have increased extracellular noradrenaline in the PFC by indirect mechanisms. It is generally accepted that LC noradrenaline neurons respond strongly to certain arousal behaviors, novelty or stress [1,3,34]. Novelty caused an increase in extracellular levels of noradrenaline in the LC as well as in the PFC. The present results show that changes in release of noradrenaline in these brain areas were parallel. Recently, Berridge and Abercrombie have demonstrated that a linear relationship exists between the LC activity and noradrenaline dialysate concentrations in the PFC [5]. Thus, changes in noradrenaline after novelty probably re¯ected alteration in impulse ¯ow activity of noradrenergic neurons. A similar conclusion can been draw from the results

of experiments with GABAA -receptor antagonist bicuculline. Infusion of bicuculline into the LC caused a similar increase in extracellular levels of noradrenaline in the LC and the PFC. This ®nding is in line with a recent study performed by push±pull superfusion of the LC with bicuculline [34] and supports the evidence that the GABAergic afferents to the LC exert tonic inhibitory in¯uence on the activity of LC neurons [12,15]. Infusion of bicuculline into the PFC was not performed due to strong behavioral activation of the animals.

It is well known that a2-adrenoceptors play an important inhibitory role during various stimulatory conditions [16,28]. Earlier studies have shown that infusions of clonidine in the cortex or hippocampus decreased and infusions of idazoxan in the cortex increased extracellular Fig. 8. (a) Effect of ¯esinoxan infusion (1 mM, black bar) into the LC levels of noradrenaline in those projecting areas of the LC during 75 min on the extracellular noradrenaline in the LC (d) and PFC via a2-adrenoceptors presynaptically located on norad- (s). Data are given as % of basal levels6S.E.M. (n57). *P,0.05: renergic axon terminals [8,10,37]. In the present study compared to basal levels. (b) Effect of ¯esinoxan infusion (1 mM, black infusion of clonidine in the LC strongly decreased the bar) into the PFC during 75 min on the extracellular noradrenaline in the extracellular levels of noradrenaline in the LC as well as in PFC (s). Data are given as % of basal levels6S.E.M. (n57). *P,0.05 compared to basal levels. the PFC (Fig. 4). The observed decrease of noradrenaline in the PFC during clonidine infusion into the LC probably re¯ects inhibition of impulse ¯ow [32], whereas the 4. Discussion decrease of noradrenaline in the LC might be related to both decreased impulse ¯ow and inhibition of terminal The ®nding that both calcium omission from the perfu- release. sion ¯uid and TTX application decreased extracellular To further investigate the regulatory mechanism of noradrenaline in the LC, suggests that extracellular nor- autoreceptors on the LC cell bodies a2-adrenoceptor adrenaline sampled in the cell body region is indeed blockade was applied. Immunohistochemical studies have released by classical release mechanism involving ex- revealed that both a2A- and a 2B/C-adrenoceptor subtypes ocytosis. A small part of noradrenaline is probably derived are present in the LC [19,20]. Evidence was provided that from non-neuronal pool, as it did not respond to infusion noradrenaline release in terminal areas (cingulate cortex) is of TTX or Ca-free solution. tonically modulated by somatodendritic a2A-adrenoceptor In contrast to the LC, extracellular noradrenaline in the subtype [22]. Local administration of the speci®c a2A- ipsilateral PFC increased during calcium free infusion. adrenoceptor antagonist BRL 44408 into the LC caused a This somewhat paradoxical ®nding suggest that calcium concentration-dependent increase in the release of nor- depletion stimulated LC neurons. This result is in agree- adrenaline in the LC (Fig. 5a). This increase is explained ment with the observation that calcium depletion potently by the inhibition of a2A-adrenoceptors localized pre- increases the discharge rate of LC neurons [27]. Another synaptically on dendrites and/or collaterals of LC neurons explanation for the present observations in the PFC might [19]. A slight but statistically signi®cant increase of be found in the strong behavioral activation that was noradrenaline was observed in the PFC after infusion of 44 O.L. Pudovkina et al. / Brain Research 906 (2001) 38 ±45

BRL 44408 in the LC in a concentration of 100 mM. This evidence for an exocytotic-like release mechanism for result is an evidence for a tonic inhibitory in¯uence of noradrenaline in the LC. Furthermore, the present study a2-adrenoceptors on the activity of LC neurons. Interest- has identi®ed several pharmacological conditions (infusion ingly, the present ®ndings differ from the study of Mateo of BRL 44408, cirazoline or ¯esinoxan into the LC), and co-workers [22]. They found that BRL 44408 infused during which the release of noradrenaline in the LC was into the LC induced an increase of noradrenaline in the affected without a concomitant effect on the release of the PFC to 250% of controls upon a concentration of 1 mM. transmitter in the PFC. In these experiments changes in the When BRL 44408 was infused into the PFC (Fig. 5b), a release of noradrenaline were apparently not related to the dose±response curve for noradrenaline was obtained that impulse ¯ow activity of noradrenaline LC neurons [5]. The was similar to that in the LC. This ®nding indicates that similar changes in the release of noradreneline were the somatodendritic area of noradrenaline neurons re- observed after application of BRL 44408, cirazoline and sponds in a similar way as the terminal area to a2A- ¯esinoxan into the PFC. In those experiments the altera- adrenoceptor blockade. tions in the noradrenaline release are explained by a

a1-Adrenoceptors are considered to play a minor role presynaptic regulatory mechanism without involvement of within the somatodendritic region of the LC [25]. How- electrical activity of the LC. This assumption would be in ever, our experiments proved otherwise. The present study accordance with the earlier study that provided evidence demonstrates for the ®rst time that the infusion of the that the noradrenaline release in hippocampus might be a1-adrenoceptor agonist cirazoline into the LC induced the uncoupled to impulse activity of LC neurons [9]. large increase of the release of noradrenaline that was The question remains whether dendrites contributed to comparable with the increase observed after local applica- the release of noradrenaline in the LC. One of the tion of cirazoline into the PFC. Apparently a1-adreno- properties of dendritic release is its impulse ¯ow driven ceptors located presynaptically on the noradrenergic nerve nature [18]. If we de®ne dendritic release by impulse terminals in the PFC and noradrenergic nerve terminals/ driven, it is evident that in the above-mentioned experi- dendrites in the LC strongly regulate the release of ments the drug-induced modulation of noradrenaline re- noradrenaline. lease in the LC does not ful®ll this criterion. The changes The fact that during infusion of cirazoline into the LC in release of noradrenaline in the LC are therefore most no changes in noradrenaline were observed in the PFC, easily interpreted by release derived from collaterals. questions the presence of a1-adrenoceptors that regulate However the ®nding from Lee and co-workers that colla- the impulse activity of LC neurons. However, the large terals in the LC do not possess a2-adrenoreceptors [19], increase of noradrenaline in the LC might have decreased supports a dendritic origin of noradrenaline. It is empha- noradrenaline levels in the PFC, via stimulation of a2- sized that if dendritic release also implies impulse ¯ow autoreceptors. In other words, the effects of a1-adreno- independent release (modi®ed by auto- and heteroreceptors ceptors could have been abolished by the stimulation of like on nerve terminals), then this type of release could not a2-adrenoceptors. Further studies are necessary to investi- be discriminated from nerve terminal release by the present gate this matter. approach.

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