Inhibition of Anxiety in Rats by Antisense to Cholecystokinin Precursor Protein Hagit Cohen, Zeev Kaplan, and Moshe Kotler Background: Cholecystokinin (CCK) and its analogs generates anxiety in humans and measurable anxiety-like behaviors in rats. CCK receptor blockers have had mixed results as a treatment approach for anxiety disorders. Since CCK is a peptide, we explored another strategy to reduce CCK levels in brain by antisense oligodeoxynucle- otide inhibition of DNA transcription or messenger RNA (mRNA) translation for CCK precursor protein. Methods: Antisense oligodeoxynucleotide complementary to the start coding region of rat CCK-precursor was intracerebroventricularly (icv) infused into rats three times at 24-hour intervals. Control groups received infu- sions of either a scramble sequence oligodeoxynucleotide or vehicle. On the fourth day, rats were assessed in the elevated plus maze paradigm. Results: Compared to vehicle and scramble sequence oligodeoxynucleotide control, icv CCK-antisense exoge- nous administration for 3 days significantly diminished anxiety behavior in rats. Conclusions: Antisense inhibition of CCK-mediated anx- iety could have therapeutic potential in human anxiety disorders. Biol Psychiatry 1998;44:915–917 © 1998 So- ciety of Biological Psychiatry Key Words: Cholecystokinin, anxiety, antisense oligode- oxynucleotide, animal model Introduction D ata have accumulated over the past decade supporting the hypothesis that cholecystokinin (CCK) plays a role in the neurobiology of anxiety and panic attacks (Crawley 1994); however, intensive efforts to develop CCK receptor antagonists as human therapeutic agents have not yet proved successful (Bourin et al 1996; Kramer et al 1995). An antisense oligonucleotide (oligo) is a short piece of single strand synthetic RNA or DNA with a nucleotide sequence that is the reverse of and complementary to part of a messenger RNA (mRNA) or DNA. It therefore binds (hybridizes) to the target (mRNA or DNA) under suitable conditions, according to the rules of the genetic code. This hybridization prevents translation or transcription of the mRNA or DNA and interrupts the expression of a partic- ular gene. In other words, the encoded protein cannot be synthesized (Helene and Toulme 1990). Synthetic antisense oligos complementary to specific mRNAs or DNA have been used successfully to block protein translation or transcription in a variety of cell culture systems (Eck and Nabel 1991; Uhlmann and Peyman 1990), and more recently have been demonstrated to modulate behavior (McCarthy et al 1993; Wahlestedt et al 1993) after administration into brain. We report use of CCK-antisense to block anxiety in rats by intracerebroventricular (ICV) administration of DNA antisense to CCK. Preliminary results have already been presented (Cohen et al 1996). Methods and Materials Sixty-six adult male Sprague-Dawley rats weighing 200 –250 g were used. Animals habituated to the housing conditions for at least 10 days. During that time rats were handled once daily, consisting of picking the rats up with a gloved hand. The animals were housed 4 per cage in an animal room with stable temper- ature and reversed 12-hour light/dark cycle with ad libitum food and water. All testing was performed during the dark phase using a dim light. All procedures were approved by the animal care and use committee of Ben-Gurion University. Rats were implanted with guide cannulae in the lateral ventricle using standard stereotaxic procedures under pentobar- bital anesthesia. Coordinates for the cannulae placement are 0.8 mm posterior to bregma, 1.4 mm lateral to midline, and 5 mm below skull surface. Injections were ICV directly into the lateral ventricle, according to methods previously described (Kofman et al 1993). A volume of 10 mL was manually injected over a period of 2 min. At least 1 week after recovery from surgery the rats were randomly divided into three groups: vehicle injected [cere- brospinal fluid (CSF)], antisense oligo, and scramble oligo. The 18-mer (59 GCT TGG CGG TTT CCA ACG 39) and scrambled control (59 TCG CTA GAT GGG CTT CGC 39) From the Ministry of Health Mental Health Center, Faculty of Health Sciences, Anxiety and Stress Research Unit, Ben Gurion University of the Negev, Beer-Sheva, Israel. Address reprint requests to Hagit Cohen, PhD, Beer-Sheva Mental Health Center, Anxiety & Stress Research Unit, Ben-Gurion University of the Negev, P.O. Box 4600, Beer-Sheva, Israel. Received April 11, 1997; revised October 27, 1997; accepted October 31, 1997. © 1998 Society of Biological Psychiatry 0006-3223/98/$19.00 PII S0006-3223(98)00010-9