Suppression of Conditioned Fear by Administration of CCKB Receptor Antisense Oligodeoxynucleotide into the Lateral Ventricle

We investigated the role of CCK in the development of anxiety by determining whether CCKB receptor antisense suppressed intracellular Ca²⁺ concentration in vitro or suppressed conditioned fear stress in vivo. First, for the in vitro studies, we used rat pituitary tumor GH3 cells since these cells have CCKB receptors. GH3 cells were stimulated by 10 µM CCK-4; intracellular Ca²⁺ concentration was measured. The CCKB receptor antisense at 1 or 10 µM reduced the subsequent response to 10 µM CCK-4 in a time-dependent manner. Second, for the in vivo studies, the CCKB receptor antisense, sense, random sense, or saline was infused at a constant rate for 6 days into rat lateral ventricles via mini-osmotic pumps. Individual rats were then subjected to 30 min of inescapable electric footshock in a chamber with a grid floor. Twenty-four hours later, the rat was again placed in the chamber and observed for 5 min without shocks. This study showed that CCKB receptor antisense significantly suppressed intracellular Ca²⁺ concentration in GH3 cells and significantly reduced freezing behavior in rats, indicating that the CCKB receptor plays an important role in anxiety.

References

• 1 Akabayashi A, Wahlestedt C, Alexander J T, Leibowitz S F. Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion.  Mol Brain Res. 1994;  21 55-61
• 2 Akiyoshi J, Isogawa K, Tsutsumi T, Kasturagi S, Kohno Y, Furuta M, Yamamoto Y, Yamada K, Fujii I. Cholecystokin in tetrapeptide-induced calcium mobilization in T cells of patients with panic disorder, major depression, or schizophrenia.  Biol Psychiatry. 1997;  42 151-154
• 3 Akiyoshi J, Moriyama T, Isogawa K, Miyamoto M, Sasaki I, Kuga K, Yamamoto H, Yamada K, Fujiii I. CCK-4-induced calcium mobilization in T cells is enhanced in panic disorder.  J Neurochem. 1996;  66 1610-1616
• 4 Akiyoshi J, Nishizono A, Yamada K, Nagayama H, Mifune K, Fujii I. Rapid desensitization of serotonin 5-HT2C receptors-stimulated intracellular calcium mobilizationin CHO cells transfected with cloned human 5-HT2C receptors.  J Neurochem. 1995;  64 2473-2479
• 5 Ballaz S, Barber A, Fortuno A, Del Rio J, Martin-Martinez M, Gomez-Monterrey I, Herranz R, Gonzalez-Muniz R, Garcia-Lopez M. Pharmacological evaluation of IQM-95, 333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models.  Br J Pharmacol. 1997;  121 759-767
• 6 Bourin M. From inducers of panic attack to neurobiology of panic disorder.  Encephale. 1996;  22 35-41
• 7 Bradwejn J, Koszycki D, Couetoux du Tertre A, van Megen H, den Boer J, Westenberg H. The panicogenic effects of cholecystokinin-tetrapeptide are antagonized by L-365,260, a central cholecystokinin receptor antagonist, in patients with panic disorder.  Arch Gen Psychiatry. 1994;  51 486-493
• 8 Bradwejn J, Koszycki D, Shriqui C. Enhanced sensitivity to cholecystokinin tetrapeptide in panic disorder.  Arch Gen Psychiatry. 1991;  48 603-610
• 9 Bremner J D, Koszycki J H, Southwick S M, Charney D S. Noradrenergic mechanisms in stress and anxiety.  Synapse. 1996;  23 28-38
• 10 Cohen H, Kotler M. Anti-sense in medicine and central nervous system disorder.  Harefuah. 1996;  130 500-502
• 11 Corwin R C, Jorn A, Hardy M, Crawley J N. The CCKB antagonist CI-988 increases dopamine levels in microdialysate from the rat nucleus accumbens via a tetrodotoxin- and calcium-independent mechanism.  J Neurochem. 1995;  65 208-217
• 12 Costall B, Domeney A M, Hughes J, Kelly M E, Naylor R J, Woodruff G N. Anxiolytic effect of CCK-B antagonists.  Neuropeptides. 1991;  19 65-73
• 13 de Montigny C. Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers.  Arch Gen Psychiatr. 1989;  46 511-517
• 14 Flint A J, Koszycki D, Vaccarino F J, Cadieux A, Boulenger J P, Bradwejn J. Effect of aging on cholecystokinin-induced panic.  Am J Psychiatry. 1998;  155 283-285
• 15 Harro J, Lofberg C, Rehfeld J F, Oreland L. Cholecystokinin peptides and receptors in the rat brain during stress.  Naunyn-Schmiedebergs Arch Pharmacol. 1996;  354 59-66
• 16 Harro J, Pold M, Vasar E. Anxiogenic-like action of caerulein, a CCK-8 receptor antagonist, in the mouse: influence of acute and subchronic diazepam atreatment.  Naunyn-Schmiedebergs Arch Pharmacol. 1990;  341 62-67
• 17 Hernando F, Fuentes J A, Ruiz-Gayo H. Impairment of stress adaptive behaviors in rats by the CCKA receptor antagonist, devazepide.  Br J Pharmacol. 1996;  118 400-406
• 18 Holland K L, Popper P, Micevych P E. Infusion of CCK-A receptor mRNA antisense oligodeoxynucleotides inhibitis lordosis behavior.  Physiol Behav. 1997;  62 537-543
• 19 Hughes J, Boden P, Costall B, Domeney A, Kelly E, Horwell D C, Hunter J C, Pinnock R D, Woodruff G N. Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity.  Proc Natl Acad Sci USA. 1990;  87 6728-6732
• 20 Inoue T, Koyama T, Yamashita I. Effect of conditioned fear stress on serotonin metabolism in the Rat brain.  Pharmacol Biochem Behav. 1993;  44 371-374
• 21 Izumi T, Inoue T, Tsuchiya K, Hashimoto S, Ohmori T, Koyama T. Effect of the selective CCKB receptor antagonist LY288513 on conditioned fear stress in rats.  Eur J Pharmacol. 1996;  300 25-31
• 22 Jenck F, Martin J R, Moreau J L. Behavioral effects of CCKB receptor ligands in a validated simulation of panic anxiety in rats.  Eur Neuropsychopharmacol. 1996;  6 291-298
• 23 Joseph C P, Thomas S. Elevated messenger RNA levels after antisense oligodeoxynucleotide treatment in vitro and in vivo.  Biochem Biophysic Res Comm. 1996;  225 861-868
• 24 Kirsch J, Wolters I, Triller A, Betz H. Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons.  Nature. 1993;  366 745-758
• 25 Landgraf R, Gerstberger R, Montkowski A, Probst J C, Wotjak C, Holsboer F, Engelmann M. V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behavior in rats.  J Neurosci. 1995;  15 4250-4258
• 26 Li D, simmons R M, Houben J J, Rasmussen K, Iyengar S. CCKB receptor tonically modulate A10 dopaminergic neurons selectivity: Neurochemical evaluation of LY288513.  Soc Neurosci Abst. 1993;  19 1547
• 27 McCarthy M M, Kleopoulos S P, Mobbs C V, Pfaff D W. Infusion of antisense oligodeoxynucleotides to the oxytocin receptor in the ventromedial hypothalamus reduces estrogen-induced sexual receptivity and oxytocin receptor bindin gin the female rat.  Neuroendocrinol. 1994;  59 432-440
• 28 McCarthy M M, Nielsen D A, Goldman D. Antisense oligodeoxynucleotide inhibition of tryptophan hydroxylase activity in mouse brain.  Regulatoary Peptides. 1995;  59 163-170
• 29 Mclean H A, Champagnat J, Denavit-Saubie M. A Cholecystokinin-B receptor antagonist potentiates GABAergic and Glycinergic Inhibition in the nucleus of the solitary tract of the rat.  Eur J Neurosci. 1996;  8 1078-1084
• 30 Osen-Sand A, Catsicas M, Staple J K, Jones K A, Ayala G, Knowles J, Grenningloh G, Catsicas S. Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo.  Nature. 1993;  364 445-448
• 31 Singh L, Field M J, Hill D R, Horwell D C, McKnight A T, Roberts E, Tang K W, Woodruff G N. Peptide CCK receptor antagonists: Pharmacological evaluation of CCKA, CCKB and mixed CCKA/B receptor antagonists.  Eur J Pharmacol. 1995;  286 185-191
• 32 Singh L, Field M J, Hughes J, Menzies R, Oles R J, Vass C A, Woodruff G N. The behavioural properties of CI-988, a selective cholecystokinin B receptor antagonist.  Br J Pharmacol. 1991;  104 239-245
• 33 Singh L, Lewis A S, Field M J, Hughes J, Woodruff G N. Evidence for an involvement of the brain cholecystokinin B receptor in anxiety.  Proc Natl Acad Sci USA. 1991;  88 1130-1133
• 34 Skutella T, Montkowski A, Stohr T, Probst J C, Landgraf R, Holsboer F, Jirikowski G F. Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide treatment attenuates social defeat-induced anxiety in rats.  Cell Mol Neurobiol. 1994;  14 579-588
• 35 Skutella T, Probst J C, Engelmann M, Wotjak C T, Landgraf R, Jirikowski G F. Vasopressin antisense oligodeoxynucleotide induces temporary diabetes insipidus in rats.  J Neuroendocrinol. 1994;  6 121-125
• 36 Standifer K M, Chien C C, Wahlestedt C, Brown G P, Pasternak G W. Selective loss of δ opioid analgesia and binding by antisense oligodeoxynucleotides to a δ opioid receptor.  Neuron. 1994;  12 805-810
• 37 Tsutsumi T, Isogawa K, Kohno Y, Hikichi T, Nagayama H, Akiyoshi J. Suppression of conditioned fear by administration of CCKB receptor antagonist PD 135158.  Neuropeptides. 1999;  33 483-486
• 38 Vasar E, Lang A, Harro J, Bourin M, Bradwejn J. Evidence for potentiation by CCK antagonists of the effect of cholecystokinin octapeptide in the elevated plus-maze.  Neuropharmacol. 1994;  33 729-735
• 39 van Megen H J, Westenberg H G, den Boer J A, Kahn J A, Kahn R S. Cholecystokinin in anxiety.  Eur Neuropsychopharmacol. 1996;  6 263-280
• 40 Wank S A, Pisegna J R, De Weerth A. Cholecystokinin receptor family. Molecular cloning, structure, and function expression in rat, guinea pig, and human.  Ann NY Acad Sci. 1994;  713 49-66
• 41 Woodruff G N, Hughes J. Cholecystokinin antagonist.  Ann rev pharmacol toxicol. 1991;  31 469-501
• 42 Xu B, Sahu A, Kalra P S, Crowley W R, Kalra S P. Disinhibition from opioid influence augments hypothalamic neuropeptide Y (NPY) gene expression and pituitary luteinizing hormone release: effects of NPY messenger ribonucleic acid antisense oligodeoxynucleotides.  Endocrinol. 1996;  137 78-84
• 43 Yoshioka M, Matsumoto M, Togashi H, Saito H. Effects of conditioned fear stress on 5-HT release in the rat prefrontal cortex.  Pharmacol Biochem Behav. 1995;  51 515-519
• 44 Zapata A, Capdevila J L, Tarrason G, Adan J, Martinez J M, Piulats J, Trullas R. Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies.  Brain Res. 1997;  745 114-120