Evidence for co-release of noradrenaline and dopamine from ... - Nature

Molecular Psychiatry (2001) 6, 657–664  2001 Nature Publishing Group All rights reserved 1359-4184/01 $15.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

Evidence for co-release of noradrenaline and dopamine from noradrenergic neurons in the cerebral cortex P Devoto1, G Flore1, L Pani2,3 and GL Gessa1,3 1

Department of Neuroscience ‘BB Brodie’, University of Cagliari, via Porcell 4, I-09124 Cagliari, Italy; 2CNR Center for Neuropharmacology, via Porcell 4, I-09124, Cagliari, Italy; 3Neuroscienze Scarl Via Palabanda, 9, I-09124 Cagliari, Italy The aim of this study was to determine whether extracellular dopamine (DA) in the prefrontal cortex (PFC) might originate other than from DA neurons, also from noradrenergic (NA) ones. To this aim, we compared the levels of DA and NA in the dialysates from the PFC, a cortical area innervated by NA and DA neurons, and cortices that receive NA but minor or no DA projections such as the primary motor, the occipital-retrosplenial, and the cerebellar cortex. Moreover, the effect of ␣2-ligands and D2-ligands that distinctly modify NA and DA neuronal activity on extracellular NA and DA in these areas was studied. Extracellular NA concentrations were found to be similar in the different cortices, as expected from the homogeneous NA innervation, however, unexpectedly, also DA concentrations in the PFC were not significantly different from those in the other cortices. The ␣2-adrenoceptor agonist clonidine, intraperitoneally (i.p.) injected or locally perfused into the PFC, reduced not only extracellular NA levels, as expected from its ability to inhibit NA neuron activity, but also markedly reduced extracellular DA levels. Conversely, the ␣2-adrenoceptor antagonist idazoxan, i.p. injected or locally perfused into the PFC, not only increased extracellular NA levels, in line with its ability to activate NA neuron activity, but also increased those of DA. Conversely, in contrast to its ability to inhibit DA neuronal activity, the D2 receptor agonist quinpirole only modestly and transiently reduced extracellular DA levels, while ␥-butyrolactone failed to modify DA levels in the PFC; conversely, haloperidol, at variance from its ability to activate DA neurons, failed to significantly modify extracellular DA levels in the PFC. Both haloperidol and quinpirole were totally ineffective after local perfusion into the PFC. Systemically injected or locally perfused, clonidine and idazoxan also modified both DA and NA concentrations in dialysates from primary motor, occipital-retrosplenial and cerebellar cortices as observed in the PFC. Finally, i.p. injected or locally perfused, clonidine reduced and idazoxan increased extracellular NA levels in the caudate nucleus, but neither ␣2-ligand significantly modified extracellular DA levels. Our results suggest that extracellular DA in the PFC, as well as in the other cortices, may depend on NA rather than DA innervation and activity. They suggest that dialysate DA reflects the amine released from NA neurons as well, where DA acts not only as NA precursor but also as co-transmitter. The co-release of NA and DA seems to be controlled by ␣2-receptors located on NA nerve terminals. Molecular Psychiatry (2001) 6, 657–664. Keywords: microdialysis; clonidine; idazoxan; quinpirole; haloperidol; ␥-butyrolactone; cerebellum; prefrontal cortex; primary motor cortex

Introduction The prefrontal cortex, the area situated in front of the motor and premotor cortices, receives dopaminergic (DA) and noradrenergic (NA) projections mainly from the ventral tegmental area (VTA) and locus coeruleus, respectively. While DA terminals are, in the rat, mainly confined within specific areas, such as the medial prefrontal (mPFC) and the anterior cingulate cortex, NA projections are diffuse throughout the brain cortex.1 More-

Correspondence: P Devoto, Department of Neuroscience ‘BB Brodie’, University of Cagliari, via Porcell 4, I–09124 Cagliari, Italy. E-mail: pdevoto얀unica.it Received 13 September 2000; revised and accepted 12 February 2001

over, while NA concentrations exceed those of DA in the PFC, the rate of DA synthesis is faster than that of NA both in areas where DA and NA terminals coexist and in areas which receive NA projections but minor or no DA projections,2–4 suggesting that only a fraction of DA is converted into NA in NA neurons, the rest being either destroyed or co-released with NA from nerve terminals. The latter possibility is supported by microdialysis studies showing that different treatments or conditions that alter extracellular NA levels, including stress, different antipsychotics, antidepressants and psychostimulants, produce parallel changes in extracellular DA in the PFC.5–13 To explain the co-variations of extracellular DA and NA concentrations it has been suggested that extracellular NA concentrations would affect those of DA by competing with the same carrier at noradrenergic

Co-release of noradrenaline and dopamine in the rat cortex P Devoto et al

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terminals for which NA and DA have the same affinity.14–16 The aim of this study was to investigate the alternative possibility that extracellular DA in the PFC might originate, other than from DA neurons, also from noradrenergic ones. To this aim we compared by microdialysis DA and NA concentrations in dialysates from the PFC which receives dense DA and NA projections, with areas with the same NA innervation but sparse or no DA projections, such as primary motor, occipitalretrosplenial and cerebellar cortices.1 Moreover we investigated the effect of treatments that distinctly modify NA and DA neuronal activity, on the extracellular concentrations of DA and NA in these areas.

Materials and methods Animals Male Sprague–Dawley rats (Charles River, Calco, Italy) weighing 280–300 g, were housed in groups of five per cage prior to surgery and singly afterwards. Standard conditions of temperature and humidity were provided, with artificial light from 8 am to 8 pm; food and water were available ad libitum. The experiment was approved by the local ethical committee and performed according to the guidelines for care and use of experimental animals of the UE (EEC Council 86/609; D.L. 27/01/1992, n116). Microdialysis Animals were implanted under equithesin anesthesia with transcerebral probes with an inflow and outflow tube running in series, in the PFC, primary motor, occipital-retrosplenial and cerebellar cortices according to the co-ordinates of the atlas of Paxinos and Watson17 (A +3.0, V −2.7; A −1.0, V −2.0; A −5.0, V −1.8; A −12.8, V −3.8 from bregma, respectively), according to the technique described by Imperato et al.18 Experiments were performed 24–30 h after surgery, from 10 am to 5 pm on freely moving rats. An artificial cerebrospinal fluid (aCSF: 147 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, pH 6–6.5) was pumped through the dialysis probes at a constant rate of 1.2 ␮l min−1 via a CMA/100 microinjection pump (Carnegie Medicine, Stockholm, Sweden). Samples were collected every 20 min and directly injected into a HPLC (high performance liquid chromatography) apparatus, equipped with a 4.6 × 150 mm C18 (5 ␮m) Symmetry column (Waters, Milan, Italy) and an ESA Coulochem II detector (Chelmsford, MA, USA). The mobile phase consisted of 80 mM Na2HPO4, 0.27 mM EDTA, 0.58 mM sodium octyl sulphate, 12% methanol, pH 2.8 with H3PO4, delivered by a model 307 Gilson pump at 1 ml min−1; the Coulochem analytical cell first electrode was set at +350 mV, the second at −300 mV. In these conditions, the detection limits (signal to noise ratio 3:1) were 0.5 pg of NA and DA on column. In all experiments, dialysate was sampled for 60 min prior to any treatment to establish baseline concentrations of neurotransmitter. On termination of experiments, rats were killed with an overdose of Equithesin Molecular Psychiatry

and perfused intracardially with 10% formalin in 0.2 M phosphate buffer. The brain was removed; coronal sections (40 ␮m thick) were made with a Vibratome, oriented according to the atlas of Paxinos and Watson (1997),17 stained with fast cresyl violet stain and the location of probes was verified according to the above atlas. If any portion of the active membrane was found to be outside the target location, data from that animal were excluded. Figure 1 shows the localization of the dialysing segment in the cortices. Tissue catecholamine levels Rats were killed by decapitation and the brains rapidly removed and placed in an ice-cold brain slicer. From each brain, 2-mm slices were cut in correspondence to PFC, primary motor, occipital-retrosplenial and cerebellar cortices, according to the co-ordinates of the atlas of Paxinos and Watson17 (antero-posteriority: +3.0, −1.0, −5.0, and −12.8 from bregma, respectively), corresponding to the cortical areas from which extracellular fluid was collected. The cortices were dissected out and frozen on dry ice. For NA and DA evaluation, tissue was homogenised by sonication in 0.1 N HClO4 (1:20, weight/volume), centrifuged at 10 000 × g, the surnatant filtered on microspin centrifuge tubes (0.22-␮m filter) and directly injected in the HPLC, with analytical conditions as described for microdialysis experiments. Drugs and treatments Clonidine HCl was purchased from Tocris Cookson (Bristol, UK); haloperidol, quinpirole and idazoxan HCl from Sigma-Aldrich (Milan, Italy), ␥-butyrolactone (GBL) from Laboratorio Farmaceutico CT (Sanremo, IM, Italy). Haloperidol was dissolved in 10 ␮l of glacial acetic acid, diluted in saline (for i.p. administration) or in aCSF (for inverse dialysis) and buffered to pH 6.0– 6.5 with NaOH. The other drugs were directly dissolved in saline for i.p. administration and in aCSF for inverse dialysis. Pilot experiments showed that neither i.p. vehicle administration nor the intracortical perfusion with buffered aCSF modified basal extracellular NA and DA levels. Expression of the results and statistics For tissue concentration assays, catecholamine levels are expressed as pg mg−1 of tissue (fresh weight). For microdialysis experiments, the average concentration of three stable samples (less than 10% variation) before treatment was considered as the control and was defined as 100%. Thus, values are expressed as percentages of controls ± SEM. Statistical significance was evaluated by analysis of variance (ANOVA) for repeated measures, followed by Dunnett multiple comparison test or Student–Newman–Keuls test as posthoc.

Results The cortical areas from which the extracellular fluid was collected, and the relative tissue concentrations of


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Figure 1 Schematic representations (redrawn from Paxinos and Watson’s Atlas17) of the areas implanted with transcerebral dialysis probes, and where DA and NA tissue levels were evaluated.

NA and DA are presented in Figure 1 and Table 1, respectively. It appears that NA concentrations were similar in the different cortices, being about 10–30 times higher than that of DA in all cortical regions tested. On the other hand, considerable differences in tissue DA concentration among the different cortices were present, DA being highest in the PFC and lowest in the cerebellar cortex. Mean basal extracellular NA and DA levels, as recovered by transversal microdialysis probes located in the different cortices, are reported in Table 2. As expected from the widespread homogeneous distribution of NA projections, extracellular NA concentrations were similar in different areas, being about three times higher than those of DA in all cortices. On the other hand, unexpectedly, extracellular DA concentrations in the PFC were not significantly different from those in dialysates from primary motor, occipitalretrosplenial and cerebellar cortices. Extracellular DA in the dialysate from the latter

Table 2 Basal dopamine and noradrenaline concentrations in dialysates from different cortical areas Cerebral area

NA

DA

Prefrontal cortex (23) Primary motor cortex (8) Occipital retrosplenial cortex (8) Cerebellar cortex (24)

0.81 ± 0.07 0.64 ± 0.12 0.80 ± 0.12

1.96 ± 0.12 1.57 ± 0.20 1.77 ± 0.22

0.78 ± 0.08

1.58 ± 0.12

Values are expressed as fmol ␮l−1 min−1, and are the means ± SEM of data obtained on the number of animals indicated in parentheses. ANOVA evidenced no significant differences among cortical NA or DA values (NA: F(3,59) = 1.849, P ⬎ 0.05; DA: F(3,59) = 0.4577, P ⬎ 0.05).

Table 1 Tissue NA and DA concentration in different cerebral cortices Cerebral area Prefrontal cortex Primary motor cortex Occipital retrosplenial cortex Cerebellar cortex

NA

DA

338.46 ± 9.42 438.73 ± 11.44**

31.62 ± 3.37 23.17 ± 2.06*

377.71 ± 8.66*

14.64 ± 2.10**

222.99 ± 12.41**

8.00 ± 0.60**

Values are expressed as pg mg−1 of tissue and are the mean ± SEM of 10 rats. *P ⬍ 0.05 with respect to PFC (Student–Newman–Keuls multiple comparison test). **P ⬍ 0.001 with respect to PFC (Student–Newman–Keuls multiple comparison test).

Figure 2 Potassium-induced DA and NA release in the primary motor cortex. Twenty-four hours after probe implantation, potassium (60 mM) was infused directly into the cortex via the microdialysis probe for 20 min (black bar). Values are expressed as the mean ± SEM of three animals. Closed symbols indicate P ⬍ 0.01 with respect to basal values (Dunnett test). Similar results were observed in occipitalretrosplenial and cerebellar cortex (data not shown). Molecular Psychiatry


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Figure 3 Effect of the D2 receptor agonist quinpirole, ␥-butyrolactone (GBL) and haloperidol on extracellular DA and NA concentrations in the PFC. Values are expressed as the mean ± SEM of at least four rats. DA values differed from baseline at the point indicated by a closed symbol (P ⬍ 0.05, Dunnett test). At the 0 time, quinpirole, GBL and haloperidol were injected i.p. at doses of 0.5, 200 and 1.0 mg kg−1, respectively.

cortices was of neuronal origin, as indicated by the fact that 60 mM potassium infusion produced a three-fold increase in both extracellular NA and DA (Figure 2). The effect of treatments that distinctly modify DA and NA neuronal activity on the extracellular DA and NA concentrations in the PFC was analyzed. As shown in Figure 3, in contrast to their ability to suppress the activity of DA neurons,19,20 the D2 receptor agonist quinpirole (0.5 mg kg−1, i.p.) produced only a modest (22%) and transient (about 20 min) reduction of extracellular DA, while GBL (200 mg kg−1, i.p.) was totally ineffective. Neither treatment modified NA levels. Conversely, the D2 receptor antagonist haloperidol (1.0 mg kg−1, i.p.) failed to significantly increase DA levels, in contrast to its ability to stimulate meso-cortical DA neurons;21 moreover, it did not modify extracellular NA levels. On the other hand, the i.p. administration of the ␣2adrenoceptor agonist clonidine (0.15 mg kg−1) not only reduced extracellular NA levels by about 80%, as expected from its ability to suppress the electrical activity of NA neurons,22 but also reduced extracellular DA concentrations near or below detection limits (Figure 4, left panel).

Conversely, the administration of the ␣2-adrenoceptor antagonist idazoxan (1.5 mg kg−1, i.p.) not only increased extracellular NA concentrations up to 170% of basal values, which is consistent with its ability to activate NA neurons,23 but also increased extracellular DA to 165% of basal values (Figure 4, right panel). To determine if ␣2-adrenoceptors controlling extracellular DA and NA concentrations were localised in the cortex, clonidine and idazoxan were locally perfused via reverse dialysis. As shown in Figure 5, locally perfused into the PFC, clonidine (10−8 to 10−6 M, left panel) reduced to almost zero not only NA but also DA levels; while idazoxan (10−6 to 10−4 M, right panel) increased both extracellular NA and DA levels up to 280% of basal levels. On the other hand, locally perfused into the PFC, neither quinpirole nor haloperidol (up to a concentration of 10−4 and 10−5 M, respectively) modified extracellular DA or NA levels (Figure 6). The effect of ␣2-ligands on extracellular NA and DA concentrations in different cortices besides PFC, and, for comparison, in the caudate nucleus, was investigated. As shown in Figure 7, i.p. injected clonidine reduced, while idazoxan increased, both NA and DA

Figure 4 Effects of clonidine (0.15 mg kg−1 i.p., left panel) and idazoxan (1.5 mg kg−1 i.p., right panel) on extracellular NA and DA concentrations in the PFC. Data are means ± SEM of five and four rats, respectively. Closed symbols indicate P ⬍ 0.01 with respect to basal values (Dunnett test). Molecular Psychiatry


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Figure 5 Effect of the perfusion of clonidine (10−8 to 10−6 M) and idazoxan (10−6 to 10−4 M) on the extracellular concentrations of NA and DA in the PFC. Data are expressed as means ± SEM of six and eight animals, respectively. Drugs were dissolved in aCSF and perfused via reverse dialysis into the PFC, at the time indicated by the black bars. Closed symbols indicate P ⬍ 0.05 with respect to basal values (Dunnett test).

Figure 6 Effect of the perfusion of quinpirole (10−6 to 10−4 M) and haloperidol (10−7 to 10−5 M) on the extracellular concentrations of NA and DA in the PFC. The data are means ± SEM of six animals. Quinpirole and haloperidol were dissolved in aCSF and perfused via inverse dialysis, at the time indicated by the black bars.

Figure 7 Effects of clonidine (0.15 mg kg−1, i.p., left panel) and idazoxan (1.5 mg kg−1, i.p., right panel) on extracellular NA and DA concentrations in the occipital retrosplenial cortex. Data are means ± SEM of five and four rats, respectively. Closed symbols indicate P ⬍ 0.01 with respect to basal values (Dunnett test).

in the dialysate from occipital retrosplenial cortex, to a similar extent as in the PFC. Similar results were observed in the primary motor and cerebellar cortex (results not shown).

In the caudate nucleus the effect of ␣2-ligands on extracellular NA was the same as that observed in the cortex, but their effect on DA was quite different. Namely, i.p. injected clonidine produced a modest Molecular Psychiatry


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Figure 8 Effects of clonidine (0.15 mg kg−1, i.p.) on extracellular NA and DA concentrations in the caudate nucleus. Data are means ± SEM of four rats. Closed symbols indicate P ⬍ 0.01 with respect to basal values (Dunnett test).

reduction in extracellular DA (Figure 8), while locally perfused was totally ineffective (Figure 9). Idazoxan, locally perfused, increased extracellular DA to a much smaller extent than in the PFC (Figure 9), as previously reported by Hertel et al.24

Discussion As expected from the evenly homogeneous distribution of NA projections in the brain cortex,1 tissue NA concentrations were similar in different cerebral cortices, while cerebellar cortex had lower NA content, consistent with its lower NA innervation.25 On the other hand, considerable differences in tissue DA concentrations among the different cortices were present, DA being highest in the PFC and lowest in the cerebellar cortex. Tissue DA concentrations should reflect both DA present in nerve terminals and that present in NA terminals, where it functions, at least in part, as NA precursor.

As for tissue NA levels, extracellular NA concentrations in different cortices were similar, including the cerebellar cortex, in spite of the lower NA content, likely because extracellular NA levels depend on innervation density, as well as on nerve activity, reuptake etc. The most interesting outcome of our study is that: (a) DA concentration in the dialysate from the PFC was not significantly different from that obtained from cortices that receive NA but minor or no DA projections; and (b) extracellular DA concentration in the PFC, as well as in the other cortices, was influenced by treatments that modify NA but not DA neuronal activity. Thus, in contrast to their ability to suppress the firing of DA neurons,19,20 the D2 receptor agonist quinpirole and GBL produced a very modest or no reduction in DA levels in the dialysates from the PFC, whereas haloperidol, in contrast to its ability to stimulate DA mesocortical neurons,21 failed to significantly modify extracellular DA in the PFC. Instead, the ␣2-adrenoceptor agonist clonidine not only profoundly reduced NA concentrations, consistent with its ability to suppress NA neuronal activity,22 but also reduced to almost zero DA concentrations in the dialysates from PFC. Conversely, the ␣2-adrenoceptor-antagonist idazoxan not only increased NA concentrations, in line with its ability to activate NA neurons,23 but also markedly increased DA levels in dialysates from PFC. The results suggest that: (a) DA concentration in the dialysate from different cortices does not correlate with DA innervation and DA activity but with the presence and activity of NA neurons, implying that DA might be co-released with NA from noradrenergic terminals; (b) the majority of DA measured in the dialysate from the PFC could represent the amine released from NA rather than DA terminals. To explain this finding, it may be suggested that the re-uptake of DA supposedly co-released with NA is hampered by the latter amine

Figure 9 Effect of the perfusion of clonidine (10−6 M) and idazoxan (10−4 M) on the extracellular concentrations of NA and DA in the caudate nucleus. Data are expressed as means ± SEM of four and five animals, respectively. Clonidine treatment basal values were 0.48 ± 0.07 and 12.13 ± 2.09 fmol ␮l−1 min−1 for NA and DA, respectively. Idazoxan treatment basal values were 0.57 ± 0.14 and 13.76 ± 2.19 fmol ␮l−1 min−1 for NA and DA, respectively. Drugs were dissolved in aCSF and perfused via reverse dialysis into the PFC, at the time indicated by the black bars. Molecular Psychiatry


which competes for the same transport mechanism into NA terminals, for which both amines have the same affinity.26 On the other hand, a much higher proportion of DA released from DA terminals is recaptured from the same terminals. Consistent with this hypothesis, haloperidol has been shown to increase extracellular DOPAC in the PFC,21 the increase in the deaminated DA metabolite reflecting an increase in DA synthesis, release and re-uptake, in line with the ability of haloperidol to stimulate mesocortical DA neurons.21 It is pertinent to recall that DOPAC concentration in the PFC dialysate is about 100-fold higher than that of DA.21 At variance from our results, Westerink et al27 found that haloperidol increased extracellular DA in the medial prefrontal cortex. Their findings suggest that D2-ligand mediated changes in DA release in the cortex could be detected provided that extracellular DA is monitored within a restricted area with dense DA innervation, such as the medial prefrontal cortex. An alternative explanation for the concomitant modifications in DA and NA produced by the ␣2ligands might be that ␣2-receptors controlling DA release are located both on NA and on DA neurons. However, against this hypothesis stands the finding that ␣2-ligands produced the same changes in the extracellular DA in the PFC as in cortical areas where minor or no DA terminals are present, whereas ␣2-ligands modified NA but not DA in the caudate nucleus, an area with dense DA but fewer NA terminals. Conversely, D2-ligands modify extracellular DA in the caudate nucleus.28,29 In spite of the interesting data reported here there are a number of contentions that need to be addressed. Firstly, it is very difficult to directly and unequivocally test the hypothesis that DA is released from NA terminals. Terminal destruction experiments are not trivial and preliminary experiments in this respect are indeed showing a potential complicated interpretation (ie the precise evaluation of the contribution from spared fibers to release and/or uptake). Secondly, in the absence of a complete terminal destruction the corelease of NA and DA may be interpreted only as the net result of the NA and DA fibers output respectively, to the dialysate. An important problem concerns the functional significance of the DA-NA co-release in PFC. NA and DA are considered to play an important role in arousal, anxiety, stress, fear, reward, in the symptomatology of schizophrenia and depression and in the action of psychostimulants, antipsychotics and antidepressants;26,30,31 extracellular DA and NA concentrations have been shown to co-vary with many of the above conditions.5–12 Our results raise the problem of whether DA and NA neurons are distinctly influenced in these conditions or whether the observed co-variations of the two amines may depend on the fact that they are coreleased from the same NA neurons. Moreover, the possibility that DA may be released by NA nerve terminals raises the question of whether such DA acts on D2 or D1-like receptors, whether it is respon-

sible for some of the effects of the drugs causing such release, and whether the co-release of DA and NA might occur in other brain areas, where DA and NA terminals are co-localised.

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Acknowledgements The authors thank Dr MA Casu for the skilful performance of histological analysis, and Laboratorio Farmaceutico CT (Sanremo, IM, Italy) for the generous gift of ␥-butyrolactone. This study was partially supported by a grant from the European Community, the Italian Government and the Regione Sardegna through the POP Sardegna.

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