The 5-HT6 Receptor Antagonist SB-271046 Selectively Enhances Excitatory Neurotransmission in the Rat Frontal Cortex and Hippocampus Lee A. Dawson, Ph.D., Huy Q. Nguyen, B.S., and Ping Li, B.S. Preclinical evidence has suggested a possible role for the 5-HT6 increases in extracellular glutamate levels in both frontal receptor in the treatment of cognitive dysfunction. However, cortex and dorsal hippocampus, respectively. These effects were currently there is little neurochemical evidence suggesting the completely attenuated by infusion of tetrodotoxin but mechanism(s) which may be involved. Using the selective unaffected by the muscarinic antagonist, atropine. Here we 5-HT6 antagonist SB-271046 and in vivo microdialysis, we demonstrate for the first time the selective enhancement of have evaluated the effects of this compound on the modulation excitatory neurotransmission by SB-271046 in those brain of basal neurotransmitter release within multiple brain regions regions implicated in cognitive and memory function, and of the freely moving rat. SB-271046 produced no change in provide mechanistic evidence in support of a possible basal levels of dopamine (DA), norepinephrine (NE) or 5-HT therapeutic role for 5-HT6 receptor antagonists in the in the striatum, frontal cortex, dorsal hippocampus or nucleus treatment of cognitive and memory dysfunction. accumbens. Similarly, this compound had no effect on [Neuropsychopharmacology 25:662–668, 2001] excitatory neurotransmission in the striatum or nucleus © 2001 American College of Neuropsychopharmacology. accumbens. Conversely, SB-271046 produced 3- and 2-fold Published by Elsevier Science Inc. KEY WORDS: 5-HT6 receptor; SB-271046; Microdialysis; sma et al. 1993; Ruat et al. 1993), the 5-HT6 receptor, like Glutamate; Striatum; Frontal cortex; Dorsal hippocampus; 5-HT4 and 5-HT7, is a G protein-linked receptor, which Nucleus accumbens; Cognition stimulates adenylate cyclase via Gs – coupling. In situ The 5-hydroxytryptamine (5-HT, serotonin) receptor hybridization and Northern blot studies have revealed superfamily currently consists of 14 members divided that 5-HT6 receptor mRNA appears to be almost exclu- sively expressed within the brain (Monsma et al. 1993; into seven classes (5-HT1-7) according to structural and functional homologies (for review see Barnes and Sharp Ruat et al. 1993; Ward et al. 1995). Regional analysis of 1999). One of the most recently identified of these is the expression reveals that the highest levels of mRNA are found within the olfactory tubercle, striatum, nucleus 5-HT6 receptor. Initially cloned from rat striatum (Mon- accumbens, cerebral cortex and subfields of the hippo- campus (Monsma et al. 1993; Ruat et al. 1993; Gerard et From Neuroscience Research, Wyeth Ayerst, Princeton, New Jersey. al. 1996). Similarly, Gerard et al. (1997) showed a com- Address correspondence to: Lee A. Dawson, Ph.D., Neuroscience Research, Wyeth Ayerst CN8000, Princeton, NJ 08543-8000, Tel.: parable distribution of protein using polyclonal anti- 732-274-4702, Fax: 732-274-4755, e-mail: [email protected] bodies raised to a presumed unique portion of the C Received February 7, 2001; revised March 28, 2001; accepted terminus of the receptor. These studies also revealed April 3, 2001. Online publication: 4/13/01 at www.acnp.org/citations/Npp that 5-HT6 receptor expression appears to be present 04050199. within 5-HT projection fields and not in 5-HT neurons NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 © 2001 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/01/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(01)00265-2 NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 SB-271046 Enhancement of Glutamate Release 663 of the raphe, indicating a probable postsynaptic role for zothiophenesulfon- amide (SB-271046; Bromidge et al. this receptor (Ward et al. 1995; Gerard et al. 1997). 1999) and in vivo microdialysis, we have evaluated the The exact functional role of the 5-HT6 receptor has effects of this compound on the modulation of basal yet to be ascertained; however, its distribution together 5-HT, dopamine (DA), norepinephrine (NE) and glu- with its high affinity (nM) for many of the therapeuti- tamate (Glu) release within multiple brain regions in cally effective antipsychotic and antidepressant drugs the freely moving rat. suggests possible therapeutic roles in both schizophre- nia and depression (Monsma et al. 1993; Roth et al. 1994). Early experiments showed that administration of METHODS antisense oligonucleotides into the brain induced a be- havioral syndrome, which could be blocked by the Materials muscarinic antagonist, atropine (Bourson et al. 1995). A All chemicals used were analytical grade and were pur- further study showed that antisense oligonucleotide chased from Aldrich & Sigma chemicals (Milwaukee, treatment failed to alter the gross behavior of animals WI, USA). Tetrodotoxin (TTX) was purchased from Ala- during a conditioned fear stress paradigm (a model of mone labs (Jerusalem, Israel). Atropine was purchased anxiety), but an attenuation of anxiety-induced prefron- from Research Biochemical International (Natick, MA, tal cortex 5-HT release was observed (Yoshioka et al. USA). (5-Chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)- 1998). 5-HT knockout mice have also been reported 6 3-methyl-2-benzothiophenesulfonamide (SB-271046) was and these animals do not appear to have any marked synthesized by Chemical Sciences, Wyeth Ayerst Re- phenotypic abnormalities, but do display some increase search (Princeton, NJ, USA). in anxiety in the elevated zero-maze (Tecott et al. 1998). More recently, Bentley et al. (1999) and Bourson et al. (1998) have demonstrated that the selective antagonist Ro Animals 04-6790 can induce stretching and inhibit 6-OHDPAT lesion-induced rotational behaviors. Both these phe- Male Sprague-Dawley rats (280–350 g, Charles River nomena can be blocked by the application of muscar- Laboratories, Wilmington, MA) were used in all experi- ϭ inic antagonists. Routledge et al. (1999) observed a po- ments (n 8–14 per study group). Animals were group tentiation of physostigmine-induced chewing behavior housed in cages with food and water available ad libitum. by SB-271046 and the same compound has been shown Following surgery, the animals were singly housed in ϫ ϫ to be effective in enhancing cognitive function in mod- Plexiglass cages (45 45 30 cm) with food and water els of learning and memory (Rogers et al. 1999). Taken available ad libitum. All animal studies were carried out in accordance with the Guide for the Care and Use of together these data indicate that the 5-HT6 receptors may be involved in the modulation of cholinergic func- Laboratory Animals as adopted and promulgated by tion, suggesting a possible therapeutic utility in the the National Institute of Health. treatment of memory and cognitive dysfunction. Although learning and memory has been suggested Surgical procedure as a therapeutic target, very little neurochemical data has been reported in support of this hypothesis. A re- Following induction of anesthesia, with gaseous ad- cent abstract communication demonstrated increases in ministration of halothane (3%) (Fluothane, Zeneca, extracellular levels of acetylcholine within both the cor- Cheshire, UK), the animals were secured in a stereo- tex and hippocampus following administration of the taxic frame with ear and incisor bars. Anesthesia was selective antagonist Ro 65-7199, at doses shown to be ef- maintained by continuous administration of halothane fective in behavioral models of memory deficit (Sleight (1–2%). A microdialysis probe guide cannula (CMA/ et al. 1999). Furthermore, we have recently reported Microdialysis, Stockholm, Sweden) was implanted into preliminary observations demonstrating increases in either the striatum (RC ϩ0.2, L–3, V–3), frontal cortex extracellular levels of glutamate within the frontal cor- (RCϩ3.5, L-3.2, V-1.5), dorsal hippocampus (RC-4.3, tex by SB-271046 (Dawson et al. 2000). Therefore, to L-2.6, V-2.1) or nucleus accumbens (RCϩ2.2, L-1.4, more fully elucidate the neurochemical mechanism re- V-6.0). Co-ordinates were taken according to Paxinos sponsible for the observed cognitive enhancement, we and Watson (1986) with reference points taken from have examined the role of the 5-HT6 receptor in the bregma and vertical from the skull. A subcutaneous modulation of multiple neurotransmitters in those cannula (s.c.) was also implanted at this time between brain regions shown to have the highest receptor ex- the animal’s shoulders. Both cannula were secured to pression levels (Monsma et al. 1993; Ruat et al. 1993; the skull using dental acrylic (Plastics One, Roanoke, Ward et al. 1995; Gerard et al. 1997). Using the potent, VA, USA). The wound was sutured and the animals left selective and bioavailable 5-HT6 antagonist 5-Chloro- to recover for 18–24 h in their home cages with free ac- N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-ben- cess to food and water. 664 L. A. Dawson et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 5 Microdialysis according to Dawson et al. (1997) in fused silica capil- laries (75 ␮m id, 375 ␮m od, 47 cm; Polymicro technolo- A pre-equilibrated microdialysis probe was inserted gies, NM, USA) with an applied voltage of 0.6 kV/cm. into the guide cannula, in either the striatum (O.D. 0.5 Samples (5 nl) were applied to the capillary via a high mm, membrane length 4 mm; CMA/Microdialysis, pressure injection system. Separations used 30 mM bo- Sweden), frontal cortex, dorsal hippocampus or nucleus ric acid pH 9.5 (pH adjusted using 1 M NaOH). The accumbens (O.D. 0.5 mm, membrane length 2 mm; capillary was rinsed with 0.1 M NaOH (1.5 min) and CMA/Microdialysis, Sweden) of the unrestrained rat, running buffer (1.5 min) between analyses. post surgery. The probe was perfused with artificial All data were acquired using the Atlas software cerebrospinal fluid (aCSF; NaCl 125 mM, KCl 3.0 mM, package (Thermo Labsystems, Gulph Mills, PA) for MgSO 0.75 mM, CaCl 1.2 mM and 0.1 M phosphate 4 2 the PC.