ANXIOGENIC-LIKE EFFECTS AND REDUCED STEREOLOGICAL COUNTING OF IMMUNOLABELLED 5-HYDROXYTRYPTAMINE 6 RECEPTORS IN RAT NUCLEUS ACCUMBENS BY ANTISENSE OLIGONUCLEOTIDES A. OTANO,* D. FRECHILLA,* A. COBREROS,* L. M. CRUZ-ORIVE,† A. INSAUSTI,† R. INSAUSTI,‡ M. HAMON§ and J. DEL RIO*k *Department of Pharmacology, University of Navarra Medical School, 31008 Pamplona, Spain †Department of Mathematics, University of Cantabria, Santander, Spain ‡Department of Anatomy, School of Medicine, University of Castilla-La Mancha, Albacete, Spain §INSERM U288, School of Medicine Pitie ´-Salpe ˆtrie `re, 75634 Paris, Cedex 13, France Abstract —The physiological role of 5-hydroxytryptamine 6 receptors in the central nervous system has not yet been elucidated. The high affinity of various psychotropic drugs for 5-hydroxytryptamine 6 recep- tors has led to the suggestion that this receptor type may be a novel target in neuropsychiatry. We have found that continuous intracerebroventricular administration of a 5-hydroxytryptamine 6 receptor antisense oligonucleotide, but not of a missense oligonucleotide, produced an anxiogenic-like response in rats using two different models of anxiety, the social interaction test and the elevated plus-maze. Neither oligo- nucleotide treatment modified locomotor activity, rectal temperature or food intake, suggesting a low or null neurotoxicity. The effectiveness of the treatment with the designed antisense oligonucleotide to block the synthesis of the protein encoded by the target mRNA was assessed by immunolabelling 5-hydroxy- tryptamine 6 receptors in the nucleus accumbens, where this receptor is highly expressed, using previously characterized specific antibodies. The density of the immunostaining was quantified by means of an unbiased three-dimensional stereologic procedure, which revealed a significant reduction (-25%) in the number of immunolabelled neuronal elements. These results suggest that, in addition to other 5-hydroxytryptamine receptor subtypes, 5-hydroxytryp- tamine 6 receptors in the nucleus accumbens may participate in anxiety-related neurobiological mechan- isms.  1999 IBRO. Published by Elsevier Science Ltd. Key words: anxiety, 5-ht 6 receptor, antisense oligonucleotide, nucleus accumbens, stereology. Serotonin (5-hydroxytryptamine, 5-HT) plays an important and complex role in behaviour and appears to be involved in the etiology of several mental diseases. Indeed, pharmacological treatments modifiying the serotonergic transmission are exten- sively used in the treatment of depression, obsessive compulsive disorders, anxiety and schizophre- nia. 15,35 In the brain, serotonin is synthesized in restricted populations of neurons, located in the raphe nuclei, which project to numerous brain regions. 6 The multiple effects of 5-HT are mediated by its interaction with different receptor types that were initially classified into four families according to ligand binding experiments, second messenger coupling and functional responses. More recently, molecular biology studies confirmed the existence of these four receptor families and also led to the identification of novel 5-HT receptors. Serotonin receptors may be grouped at present into seven families based upon cDNA deduced primary sequences, signal transduction mechanisms and pharmacological profile. 7 With the exception of the 5-HT 3 receptor, which forms a ligand-gated ionic channel, all other 5-HT receptors belong to the superfamily of G-protein-coupled receptors. However, the physiological roles of the recently discovered 5-ht 5 , 5-ht 6 and of 5-HT 7 receptors (for nomenclature see Hoyer and Martin 26 ) remains to be elucidated. Interestingly, it was reported that several antidepressant and antipsychotic agents show high affinity for the 5-ht 6 receptor transfected in mamma- lian cells. 21,33,39 Consequently, much attention has been paid to the possible implication of this 5-HT receptor as a novel target for some psychotropic drugs. The cloning of both the rat and the human 5-ht 6 receptor gene revealed a sequence distant from other 5-HT receptor genes. 29,33,40 The gene contains two introns in its coding region and the predicted protein shows a short third intracellular loop and a long carboxy-terminal tail, as expected for a receptor positively coupled to adenylyl cyclase. 42,43 In situ 1001 Neuroscience Vol. 92, No. 3, pp. 1001–1009, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00066-4 Pergamon kTo whom correspondence should be addressed. Abbreviations: AO, antisense oligonucleotide; 5-HT, 5-hydro- xytryptamine, serotonin; PBS, phosphate-buffered saline.