Neuroscience Letters, 167 (1994) 55-58 55 © 1994ElsevierScienceIreland Ltd. All rights reserved0304-39401941507.00 NSL 10199 Ventral tegmental area (VTA) injections of tyrosine hydroxylase phosphorothioate antisense oligonucleotide suppress operant behavior in rats Thomas Skutella*, Joseph Christopher Probst, Gustav F. Jirikowski, Florian Holsboer, Rainer Spanagel Ma.v-Planck irt~titute /br Psychiatrl; Clinical Institute. Kraepelinstr. 2. 80804 Munh'h. Germany (Received 8 August 1993: Revisedversion received24 November 1993;Accepted26 November 1993) Key word.~: Tyrosine hydroxylase: Operant behavior: Mesolimbic dopaminesystem:Antisensetargeting DopaminergicA 10 neuronsare knownto be the essential part of the brain reinforcement system.An antisense oligonucleotide correspondingto the start coding region of rat tyrosinehydroxylase (TH) mRNA. the transcriptionalmessageof the rate limiting enzymein the metabolicpathway leading to catecholaminesynthesis,was constructed and injectedinto the ventral tegmentalarea (VTA). 36 h after injectionoperant behavior was markedly reduced,and this suppression was fullyreversed within 5 days following the antisense injection.Accordingly, TH immunoreactivity in the VTA was reduced in comparison to control experimentsusing mixed bases oligonucleotides. Our results demonstralethat highlyspecificinhibition of TH expressioncan be accomplished in the intact mesolimbic systemby antisensetreatment,thus providing a noveltool for studieson motivational processes in vix.o. Research in behavioral neuroscience has generated a substantial body of evidence indicating that the mesolim- bic dopaminergic system seems to be critically involved in the initiation and mediation of reinforcement proc- esses [2,23]. The major body of evidence indicating that the mesolimbic dopamine (DA) system is involved in reinforcement comes from studies of intracranial self- stimulation in regions containing catecholamine or en- dorphin cellbodies or pathways [I 5] and of drug self-ad- ministration (e.g. opioids, cocaine) [12] in the VTA or nucleus accumbens (NAC). at doses below those neces- sary to produce changes in appetite or general motor activity [6]. Neurochemical evidence for increased meso- limbic DA expression/release associated with positively reinforced behavior has been produced by post mortem studies [8] and with in vivo voltammetry [10] and micro- dialysis [9]. The hypothesis that the mesolimbic dopam- ine system has reinforcement-related functions has been further investigated in vivo by destructive processes initi- ated in DA neurons by intraparenchymal injection of the neurotoxin 6-hydroxydopamine (6-OHDA) [17] and by pathway lesions of VTA-NAC fibers [11]. Further dis- ruption of DA synthesis after ¢x-methyl-p-tyrosine [4] and pharmacological blockade of DA D I and/or DAD 2 re- ceptors [5,22] attenuated responding in conditioned rein- forcement. The NAC represents the main projection area *Corresponding author. Fax: (49) (89) 306-22200. of the mesolimbic DA system. The fibers of this system originate in the VTA and have been linked to operant responding for natural reinforcing stimuli, which seem to be mediated by activation of DA neurotransmission within the NAC [20]. Given the above neurobiological data concerning the involvement of the mesolimbic DA system in the initia- tion and maintainance of operant behavior, we studied whether blocking of the transcriptional message of TH, the rate limiting enzyme in catecholamine synthesis, might suppress operant behavior in a physiological and highly specific way. Thus far, antisense technology in the brain has been used in vivo to reduce neuropeptide and neurotransmitter receptors [18,19] and to block the ex- pression of neuropeptides [23] and early onset genes [3,16]. Accordingly our aim was to use a TH antisense oligonucleotide which was protected from degradation with a phosphorothioate backbone and injected into the VTA. On the basis of the sequence of TH [7] we designed and synthezised a 19-base phosphorothioate antisense oligonucleotide that corresponded to the initiation codon of the TH mRNA (5" GGT GGG CAT AGT GCA AGC TG 3'). Mixed bases phosphorothioate ol- igonucleotides were used as controls. Male Sprague-Dawley rats (Charles Rivers, Sulzfeld/ Germany) weighing 250-270 g were housed 2 per cage with free access to water. Body weight was 80% of their SSDI 0304-3940(93)E0823-E