Modulation of the Extracellular 5-Hydroxytryptamine Brain Concentrations by the Serotonin and Noradrenaline Reuptake Inhibitor, Milnacipran Microdialysis Studies in Rats Núria Bel, Ph.D. and Francesc Artigas, Ph.D. We examined the effects of the administration of antagonist WAY-100635 caused a small potentiation of the milnacipran, a dual inhibitor of serotonin (5-hydroxy- effects of milnacipran. The baseline 5-HT output was tryptamine, 5-HT) and noradrenaline uptake on the 5-HT unaffected by 2-week treatments with milnacipran (30 and output in rat brain. Local milnacipran administration 60 mg/kg?day). The distinct regional profile and the lack of increased the 5-HT output in frontal cortex and the enhancement of its effects by WAY-100635 and prolonged midbrain raphe nuclei 7- and 10-fold by a Ca21- and treatment suggest that milnacipran does not exert its tetrodotoxin-dependent mechanism. However, the antidepressant action through an enhancement of the subcutaneous administration of milnacipran (1–60 mg/kg serotonergic function. [Neuropsychopharmacology SC) elevated the 5-HT output much less in these areas 21:745–754, 1999] © 1999 American College of (200–230% of baseline at 60 mg/kg). In hypothalamus, 10 Neuropsychopharmacology. Published by Elsevier mg/kg SC raised 5-HT levels to 170%. The 5-HT1A Science Inc. KEY WORDS: 5-HT uptake; 5-HT1A receptors; et al. 1995; Malagié et al. 1995). In all instances, these el- Antidepressants; Frontal cortex; Hypothalamus; Raphe; evations are superior to those produced in frontal cor- Selective serotonin reuptake inhibitors tex or, when examined, in hippocampus. The excess The administration of selective 5-HT reuptake inhibi- 5-HT in the extracellular space of the midbrain raphe tors (SSRIs), monoamine oxidase inhibitors (MAOIs), activates 5-HT1A autoreceptors in the soma and den- and clomipramine markedly increases the extracellular drites of serotonergic neurones and reduces the neu- concentration of 5-HT in the raphe nuclei of the mid- ronal activity and release of 5-HT by nerve terminals in brain (Adell and Artigas 1991; Bel and Artigas 1992; In- forebrain (see Artigas et al. 1996 for review). vernizzi et al. 1992; Celada and Artigas 1993; Gartside Selective serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline reuptake inhibitors (SNRIs) are a new class of antidepressant drugs. These agents inhibit in From the Department of Neurochemistry, Instituto de Investiga- ciones Biomédicas de Barcelona (CSIC), IDIBAPS, Barcelona, Spain. vitro the neuronal uptake of 5-HT and noradrenaline Address correspondence to: Dr. F. Artigas, Instituto de Investiga- without exhibiting significant activity at the dopamine ciones Biomédicas de Barcelona, IDIBAPS, Department of Neuro- transporter or aminergic receptors (see Artigas 1995; chemistry, Rosselló 161, 08036 Barcelona, Spain. Received January 17, 1999; revised May 21, 1999; accepted June Briley 1998 for review). Consequently, they are devoid 18, 1999. of the unwanted side effects of classic antidepressant NEUROPSYCHOPHARMACOLOGY 1999–VOL. 21, NO. 6 © 1999 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(99)00076-7 746 N. Bel and F. Artigas NEUROPSYCHOPHARMACOLOGY 1999–VOL. 21, NO. 6 drugs, mainly derived from their affinity for a-adrener- 1 mmol/l milnacipran (1 hour, restabilization period) gic, cholinergic, and histamine receptors (Richelson and 1 mmol/l milnacipran plus 1 mmol/l TTX (1 addi- 1978; Richelson and Nelson 1984). Milnacipran is one tional hour). Acute systemic administrations of mil- such drug that blocks in vitro and in vivo the reuptake nacipran were carried out SC or IP. For chronic admin- of 5-HT and noradrenaline, although it displays a istrations to rats, osmotic minipumps (Alzet 2002) were slightly higher affinity for the latter (Moret et al. 1985; used. Two different doses were used, 30 and 60 mg/ Stenger et al. 1987). kg?day. Given the weight gain, these corresponded to Despite the potential advantages of SNRIs, the infor- the 7th day of the treatment. The minipumps of control mation on their in vivo effects on serotonergic transmis- animals were filled with saline. Minipumps were im- sion is scarce (e.g., Engleman et al. 1995; 1996). In partic- planted SC under light ether anesthesia. Animals were ular, a comparison of their effects in the somatodendritic housed one per cage. On the 14th day of treatment (1 region in the raphe nuclei and in forebrain has not been day after probe implant), rats received a challenge dose performed. Data obtained with the microdialysis tech- of 30 mg/kg SC milnacipran. After killing the rats, the nique suggest that the dual blockade of the 5-HT and minipumps were removed, cut with a surgical blade, noradrenaline transporters elevates the extracellular and checked by visual inspection that they had deliv- 5-HT concentration in rat brain, with a regional profile ered their content. different from that of SSRIs (Bel and Artigas 1996). Be- cause an increment of 5-HT in forebrain may be rele- Surgery and Microdialysis Procedures vant for the clinical effects of antidepressant drugs (Blier and de Montigny 1994; Artigas et al. 1996), we Microdialysis procedures in rats were carried out essen- have conducted the present microdialysis study aimed tially as described in Adell and Artigas (1991). Concen- at examining the modifications of extracellular 5-HT in- tric dialysis probes were used, equipped with Cu- duced by milnacipran in rat brain. prophan membranes having a cut-off limit of 6,000 daltons (Gambro, Lund, Sweden). The length of mem- brane exposed to the brain tissue in rats was 3.5 mm for MATERIAL AND METHODS all regions sampled, frontal cortex, raphe nuclei, and hypothalamus (o.d. 0.25 mm). Although the microdial- Animals ysis technique enables separate sampling of the dorsal Male Wistar rats (Iffa Credo, Lyon, France) weighing and median raphe nuclei (e.g., Casanovas and Artigas 280 to 320 g were used. Animals were kept in a con- 1996), we performed this study with longer probes, be- trolled environment (12 h light–dark cycle and 22 6 28C cause this permitted the comparison with previous data room temperature). Food and water were provided ad obtained with the same type of probes using such refer- libitum before and during microdialysis experiments. ence antidepressants as clomipramine (Adell and Arti- Animal care followed the Spanish legislation on “Pro- gas 1991) or imipramine (Bel and Artigas 1996). At the tection of Animals Used in Experimental and Other Sci- stereotaxic coordinates used (see below), these probes entific Purposes,” in agreement with the European sampled the lateral parts of the dorsal and median Union regulations (EEC Council Directive 86/609 of 24 raphe nuclei and a very high density of efferent sero- November 1986). tonergic fibers (Halliday et al. 1995). Before implanta- tion, rats were anesthesized with sodium pentobarbital (60 mg/kg IP) and placed in a David Kopf stereotaxic Drugs and Treatments frame. The stereotaxic coordinates (in mm) for the 5-HT, tetrodotoxin (TTX), and WAY-100635 [N-(2-(4-2- raphe nuclei (AP 2 7.8, DV 2 9.0, L 2 0.5), frontal cor- methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cy- tex (AP 1 3.2, DV 2 6.0, L 1 2.5) and hypothalamus clohexanecarboxamide?3HCl] were obtained from RBI (AP 2 2.8, DV 2 9.5, L 2 0.5) were taken from bregma (Natick, MA). Milnacipran was obtained from Pierre and surface of the skull according to the rat brain atlas Fabre Médicament (Castres, France). Other materials of Paxinos and Watson (1986). and reagents were from local commercial sources. All Animals were allowed to recover from surgery in drug doses are expressed as the base. For local applica- metacrylate dialysis cages (cubic, 40-cm side). On the tions, appropriate amounts of milnacipran and TTX day after surgery (20–24 h after), the probes were per- were dissolved in the artificial CSF used to perfuse the fused with artificial cerebrospinal fluid (CSF) (125 probes (final concentrations of milnacipran: 10, 100, and mmol/l NaCl, 2.5 mmol/l KCl, 1.26 mmol/l CaCl2 and m m 1,000 mol/l; tetrodotoxin was perfused at 1 mol/l). 1.18 mmol/l MgCl2) at 0.5 ml/min using a microinjec- In a separate group of animals, the perfusion fluid (see tion pump (CMA or Harvard) and liquid swivels (In- below) was changed to one devoid of Ca21 ions for 1 stech, Plymouth, MA). Ca21-free perfusion fluid was hour while perfusing milnacipran. Probes were per- made up by isosmotic replacement of CaCl2 with fused again with the normal perfusion fluid containing MgCl2. Sample collection started 60 min after the begin- NEUROPSYCHOPHARMACOLOGY 1999–VOL. 21, NO. 6 Milnacipran and 5-HT Output 747 ning of perfusion. After a 1-h equilibration period, 4 to vidual means of four fractions before drug administra- 5 fractions were collected to obtain basal values before tion. The effects of the local administration of milnacip- local or systemic administration of drugs. For local de- ran are given as the individual means of two dialysate livery of milnacipran and TTX, aliquots of concentrated fractions (1 h) for each concentration. Statistical analysis solutions were dissolved in the artificial CSF used to was performed with the raw (nontransformed) data us- perfuse the probes. When necessary, the pH of concen- ing one- or two-way analysis of variance (ANOVA) for trated drug solutions was adjusted to z7 with independent or repeated measures and post-ANOVA NaHCO3. After collection of baseline fractions, succes- tests for multiple comparisons, where appropriate. The m sive 30 min (15 l) dialysate samples were collected. At EC50 values of the local effects of milnacipran in rat the end of the experiments, the correct placement of the brain were calculated by using the GraphPad Prism probes was checked by infusing methylene blue software (San Diego, CA).