[The study of biologically active conformation of cholecystokinin-4 dipeptide analog GB-115].

The conformational analysis with 1H NMR spectroscopy method in solution and the structure-activity relationship study of a series sterically restricted analogs allowed to detect the possible biologically active conformation of N-(6-phenylhexanoyl)glycyl-tryptophan amide (GB-115), a highly active dipeptide cholecystokinin-4 analog with anxiolytic activity. The structure-activity relationship study of GB-115 and the series of its' glycine- and proline-containing analogs with different C-terminal substitute detected the anxiolytic activity of compounds with beta-turn like conformation and inactivity of compounds with gamma-turn like conformation. So, the GB-115 biologically active conformation is beta-turn. The results of nuclear Overhauser effect study permitted to qualify the betaII-turn conformation as GB-115 biologically active conformation. The following synthesis of sterically restricted GB-115 analogs (2S)-2-{(3R)-3-[(6-phenylhexanoyl)amino]-2-oxopyrrolidin-1-yl}-3-(1H-indol-3-yl)propionic acid ethyl ester, N-(6-phenylhexanoyl)glycyl-N(alpha)(methyl)-tryptophan ethyl ester, (2S)-2-[10,11-dihydro-5H-dibenzo[b,f] azepin-5-carbonyl)-amino]-3-(1H-indol-3-yl)propionic acid methyl ester and (2S)-2-[({3-[(ethoxycarbonyl)amino]-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl}carbonyl)amino]-3-(1H-indol-3-yl)propionic acid methyl ester confirmed the estimated type of GB-115 biologically active conformation.

Cholecystokinin 1 receptor activation restores normal mTORC1 signaling and is protective to Purkinje cells of SCA mice.

Spinocerebellar ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia and neurodegeneration, often in cerebellar Purkinje neurons. A SCA1 mouse model, Pcp2-ATXN1[30Q]D776, has severe ataxia in absence of progressive Purkinje neuron degeneration and death. Previous RNA-seq analyses identify cerebellar upregulation of the peptide hormone cholecystokinin (Cck) in Pcp2-ATXN1[30Q]D776 mice. Importantly, absence of Cck1 receptor (Cck1R) in Pcp2-ATXN1[30Q]D776 mice confers a progressive disease with Purkinje neuron death. Administration of a Cck1R agonist, A71623, to Pcp2-ATXN1[30Q]D776;Cck-/- and Pcp2-AXTN1[82Q] mice dampens Purkinje neuron pathology and associated deficits in motor performance. In addition, A71623 administration improves motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Moreover, the Cck1R agonist A71623 corrects mTORC1 signaling and improves expression of calbindin in cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results indicate that manipulation of the Cck-Cck1R pathway is a potential therapeutic target for treatment of diseases involving Purkinje neuron degeneration.

Acquisition of analgesic properties by the cholecystokinin (CCK)/CCK2 receptor system within the amygdala in a persistent inflammatory pain condition.

Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.

Stimulation of growth of azaserine-induced putative preneoplastic lesions in rat pancreas is mediated specifically by way of cholecystokinin-A receptors.

Cholecystokinin (CCK) has been shown to stimulate the growth of both normal pancreas and azaserine-induced putative preneoplastic pancreatic lesions in the rat. The present study was performed to determine whether these effects are mediated by way of CCK-A receptors, CCK-B receptors, or both. Sixteen-day-old male Lewis rats were given i.p. injections of azaserine at 30 mg/kg body weight. Starting on day 21, rats were given s.c. injections, 5 days/week for 16 consecutive weeks, of either (a) CCK octapeptide (nonselective CCK agonist) (2.50 micrograms/kg body weight, n = 17), (b) tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl++ +)-Asp- (N-methyl)-Phe-NH2 (highly selective CCK-A agonist) (1.84 micrograms/kg body weight, n = 18), (c) [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 (highly selective CCK-B agonist) (2.40 micrograms/kg body weight, n = 18), or (d) normal saline solution (control, n = 17). Rats were subsequently sacrificed, pancreatic weights were determined, and quantitative morphometric analysis of atypical acinar cell foci and nodules was performed. Both CCK octapeptide and the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 stimulated pancreatic growth and the development of acidophilic atypical acinar cell foci and nodules. Furthermore, the effect produced by the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 was greater than that produced by CCK octapeptide. In contrast, the selective CCK-B agonist [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 had no effect. These findings suggest that the growth of putative preneoplastic lesions (acidophilic atypical acinar cell foci and nodules) in the rat pancreas during the early stages of azaserine-induced pancreatic carcinogenesis is mediated specifically by way of CCK-A receptors.

Conformationally constrained CCK4 analogues incorporating IBTM and BTD beta-turn mimetics.

To test whether a turnlike arrangement is involved in the bioactive conformation of CCK4 analogues upon CCK1 receptor recognition, we describe the preparation of two series of CCK4 derivatives, in which the central dipeptide Met-Asp has been replaced by recognized beta-turn mimetics {(2S,5S,11bR)- and (2R,5R,11bS)-2-amino-5-carboxy-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole (IBTM) and beta-turn dipeptide, 2-oxo-7-thio-1-azabicyclo[4.3.0]nonane (BTD)}. The incorporation of the indolizinoindole IBTM type II beta-turn mimetic is preferred over its type II' counterpart for efficient CCK1 receptor recognition, while BTD derivatives were completely inactive. The structure-conformation-activity relationship study in the IBTM series has shown some essential requirement of these CCK4 derivatives to favorably interact with CCK1 receptors: (a) the adoption of turnlike conformations, (b) the presence of an L-Phe residue and a C-terminal carboxamide moiety, and (c) the indole ring of the IBTM skeleton. Moreover, the existence of pi-pi interactions between the phenyl ring of d-Phe residues and the indole ring of IBTM framework is detrimental for binding affinity. A series of potent and selective CCK1 receptor antagonists, exemplified by compounds 8a and 8b, emerges among these IBTM-containing derivatives.

[Biological activity of cholecystokinin-(30-33) tetrapeptide analogs].

Analogues of the endogenous peptide corresponding to the 30-33 sequence of cholecystokinin (Trp-Met-Asp-Phe-NH2) were synthesized, and their biological activity was studied. It was shown that, in rats, the N-succinylated Nle2 analogue of this tetrapeptide exhibits increased anxiolytic properties in the dark-bright chamber test and an enhanced alcohol intake by both the control animals and the long-time alcohol-dependent animals under the conditions of free choice. Introduction of an isopropyl residue into the C-terminal amide of the Nle2 analogue resulted in the appearance of anxiolytic and antialcohol activity and the ability to increase the morphine analgesic effect in the tail-flick test on rats. The two synthesized analogues retained an affinity to cholecystokinin receptors.

Recent advances in the chemistry of cholecystokinin receptor ligands (agonists and antagonists).

During the last few years, cholecystokinin (CCK) has emerged as an important hormone. This polypeptide has been located either in peripheral tissues such as the gastro-intestinal tract and the pancreas as well as in the central nervous system. High affinity CCK receptors are divided in two main subtypes: the CCK-A (A for (A for "alimentary") and the CCK-B (B for "brain") receptors. The latters are currently associated with the gastrin receptors. Since CCK is involved in many different biological processes such as gut function, digestive processes, control of feeding behaviour and neurotransmitter release, the therapeutical potential of cholecystokinin receptor ligands seems to be extremely broad and promising. Several families of CCK receptor ligands (peptides, peptidomimetics, peptoids or non-peptides) were prepared during the last twenty years. The main goal of these researches was to improve agonistic or antagonistic potency but also to find selective compounds for a specific CCK receptor subtype. This review presents the recent developments (since 1995) in the chemistry of CCK receptor ligands.

Effects of BOC-CCK-4 and L 365.260 on cortical 5-HT release in guinea-pigs on exposure to the elevated plus maze.

The elevated plus maze is a well-established model of anxiety, with previous results showing that guinea-pigs handled daily from birth exhibit behaviour in this test similar to rats. In the present microdialysis study exposure of the guinea-pig to the elevated plus maze increased extracellular 5-HT in the lateral prefrontal cortex. The CCK-B receptor agonist BOC-CCK-4 (10 micrograms/kg) produced 'anxious' behaviour and potentiated the rise in 5-HT observed on exposure to the X-maze. The basal release of cortical extracellular 5-HT was not affected by BOC-CCK-4. Pretreatment with the selective CCK-B antagonist L 365.260 (100 micrograms/kg) antagonized both the 'anxious' behaviour and the neurochemical changes induced by BOC-CCK-4 while L 365.260 alone produced 'anxiolytic' behaviour, decreased basal extracellular 5-HT and prevented the increase in extracellular 5-HT seen when the guinea-pigs were exposed to the X-maze. Our results show that CCK-B receptor stimulation and blockade induce changes in central extracellular 5-HT levels associated with 'anxious' and 'anxiolytic' behaviour, respectively.

Minor structural differences in Boc-CCK-4 derivatives dictate affinity and selectivity for CCK-A and CCK-B receptors.

We previously reported novel Boc-CCK-4 (Boc-Trp-Met-Asp-Phe-NH2) derivatives possessing the general structure Boc-Trp-Lys[N epsilon-CO-NH-(R-Ph)]-Asp-Phe-NH2 (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842). In contrast to Boc-CCK-4, which is 70-fold selective for the CCK-B receptor, the modified lysine-bearing tetrapeptides were highly potent and selective full agonists at the CCK-A receptor. Further investigation of the structure-activity profile following modification of the substituted phenylurea moiety appended off the lysine revealed that moving certain substituents, e.g. nitro or acetyl, from the 2- or 3-position on the phenyl ring to the 4-position, a relatively minor and subtle structural modification within the tetrapeptide, resulted in loss of CCK-A receptor selectivity and development of a trend toward CCK-B selectivity. These tetrapeptides, e.g. Boc-Trp-Lys[N epsilon-CO-NH-(4-NO2-Ph)]-Asp-Phe-NH2 and Boc-Trp-Lys[N epsilon-CO-NH-(4-Ac-Ph)]-Asp-Phe-NH2, were full agonists relative to CCK-8 in stimulating intracellular calcium mobilization in a cell line that expresses the CCK-B receptor.

Tetrapeptide CCK agonists: structure-activity studies on modifications at the N-terminus.

We had reported earlier on a novel series of potent and selective tetrapeptide cholecystokinin-A (CCK-A) agonists of the general structure Boc-Trp-Lys[epsilon-Y]-Asp-N(R)PheNH2 [Y = amides, ureas; R = H, Me] that were potent anorectic agents in rats. In an effort to optimize the potency, selectivity, stability, and efficacy of our lead candidate A-71623 [R = Me, Y = o-tolylaminocarbonyl; Tac] toward development of a clinical candidate, we have explored a series of analogues in which the N-terminal Boc functionality was systematically replaced with various amides, ureas, carbamates, and sulfonamides of differing size, hydrophobicity, and stereoelectronic properties. In general, these analogues maintained good potency and selectivity for the CCK-A receptor (guinea pig pancreas), as well as potent anorectic activity in rats. Those analogues exhibiting equal or superior activity compared to A-71623 but differing physicochemical properties may represent superior drug candidates.

CCK-A receptor selective antagonists derived from the CCK-A receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623).

Analogs of the CCK-A receptor selective agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623) were prepared in which the lysine residue was replaced with L-4-aminophenylalanine and D-or L-3-aminophenylalanine. These new analogs were moderately potent antagonists of CCK-8 in the isolated guinea pig gallbladder with exceptional CCK-A receptor selectivity as evaluated in membrane preparations from CHO K1 cells stably transfected with human CCK-A and CCK-B receptors.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Synthesis and biological activities of some pseudo-peptide analogues of tetragastrin: the importance of the peptide backbone.

Pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, in which a peptide bond has been replaced by a CH2-NH bond, i.e. (tert-butyloxycarbonyl)-L-tryptophyl-psi (CH2-NH)-L-leucyl-L-aspartyl-L-phenylalanine amide (8), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-psi (CH2-NH)-L-aspartyl-L-phenylalanine amide (13), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-psi (CH2NH)-L-phenylalanine amide (20), were synthesized. The pseudo-peptides 8 and 13 were shown to have the same affinity as (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-L-phenylalanine amide (21) for the gastrin receptor on isolated mucosal cells. The pseudo-peptide 20 exhibited lower affinity (IC50 congruent to 10(-5) M). The biological activity of these pseudo-peptides was studied on acid secretion in the anesthetized rat. Compound 8 stimulated acid secretion, identically with that of 21. Compound 13 did not exhibit any agonist activity but was able to antagonize the action of gastrin (ED50 = 0.3 mg/kg). Compound 20 did not show any agonist activity but was able to inhibit gastrin-induced acid secretion, with lower potency (ED50 = 15 mg/kg). The importance of the peptide bonds in the mode of action of gastrin is discussed, and a hypothetical approach of the mechanism of action is presented.

CCK-A-selective tetrapeptides containing lys(N epsilon)-amide residues: favorable in vivo and in vitro effects of N-methylation at the aspartyl residue.

Previous structure-activity studies on a series of CCK-A selective tetrapeptide agonists, typified by A-71623 (Boc-Trp-Lys(CONH-Ph-o-Me)-Asp-(N-Me)Phe-NH2), have shown that replacement of the Lys(N epsilon-carbamoyl) substituent with N epsilon-acyl substituents resulted in partial agonists with moderate to high affinities for the CCK-A receptor and that replacement of the C-terminal dipeptide with either (N-Me)Asp-Phe or (N-Me)Asp-(N-Me)Phe was highly favorable to in vitro and in vivo CCK activity. The present study demonstrates that although analogues in the epsilon-amide series that are N-methylated at the Phe position are weakly active or inactive in an in vivo rat appetite suppression assay, incorporation of (N-Me)Asp or (N-Me)Asp-(N-Me)Phe modifications in this series results in analogues with markedly improved in vivo activity. In in vitro assays, there is minimal effect of N-methylation pattern on binding affinity, whereas there is a trend toward improved functional activity in the phosphatidylinositol hydrolysis assay in analogues containing (N-Me)Asp.

CCK-B agonist or antagonist activities of structurally hindered and peptidase-resistant Boc-CCK4 derivatives.

Replacement of Met31 by (N-Me)Nle in CCK8 or CCK4 has been shown to improve the affinity and selectivity for CCK-B receptors. In order to obtain molecules with enhanced bioavailability, two novel series of protected tetrapeptides of the general formula Boc-Trp30-X-Asp-Y33 have been developed. Introduction of (N-Me)Nle and the bulky, aromatic naphthylalaninamide (Nal-NH2) in positions X and Y, respectively, does not greatly modify the affinity for guinea pig brain CCK-B receptors. In contrast, incorporation of hindering N-methyl amino acids such as (N-Me)Phe, (N-Me)Phg, or (N-Me)Chg, but not their non-methylated counterparts, in position X induced a large decrease in affinity for the CCK-B binding sites. Among the various peptides synthesized, Boc-[(N-Me)Nle31,1Nal-NH2(33)]CCK4 (2) (KI = 2.8 nM), Boc-[Phg31,1Nal-NH2(33)]CCK4 (15) (KI = 14 nM), and Boc-[Phg31,1Nal-N(CH3)2(33)]CCK4 (17) (KI = 39 nM) displayed good affinities for brain CCK-B receptors and had good selectivity ratios. These pseudopeptides, in which the presence of unnatural and hydrophobic residues is expected to improve their penetration of the central nervous system, were shown to be very resistant to brain peptidases. Interestingly, whereas compounds 2 and 15 proved to be full agonists for rat hippocampal CCK-B receptors when measured in an electrophysiological assay, compound 17 behaved as a potent antagonist in the same test and displayed a good affinity in rat brain KI(CCK-B) = 51 nM as compared to the Merck antagonist L365,260,KI(CCK-B) = 12 nM. This illustrates a simple means to obtain CCK-B antagonists and suggests that the free, CONH2 group plays a critical role in the recognition of the agonist state of brain CCK-B receptors.

Second generation "peptoid" CCK-B receptor antagonists: identification and development of N-(adamantyloxycarbonyl)-alpha-methyl-(R)-tryptophan derivative (CI-1015) with an improved pharmacokinetic profile.

We have previously described the design and development of CI-988, a peptoid analogue of CCK-4 with excellent binding affinity and selectivity for the CCK-B receptor. Due to its anxiolytic profile in animal models of anxiety, this compound was developed as a clinical candidate. However, during its development, it was determined that CI-988 had low bioavailability in both rodent and nonrodent species. In the clinic, it was further established that CI-988 had poor bioavailability. Thus, there was a need to identify an analogue with an improved pharmacokinetic (PK) profile. The poor bioavailability was attributed to poor absorption and efficient hepatic extraction. We envisaged that reducing the molecular weight of the parent compound (5, MW = 614) would lead to better absorption. Thus, we synthesized a series of analogues in which the key alpha-methyltryptophan and adamantyloxycarbonyl moieties, required for receptor binding, were kept intact and the C-terminus was extensively modified. This SAR study led to the identification of tricyclo[3.3.1.1(3,7)]dec-2-yl [1S-[1 alpha(S*)2 beta]-[2-[(2-hydroxycyclohexyl)amino]-1-(1H-indol-3- ylmethyl)-1-methyl-2-oxoethyl]carbamate (CI-1015, 31) with binding affinities of 3.0 and 2900 nM for the CCK-B and CCK-A receptors, respectively. The compound showed CCK-B antagonist profile in the rat ventromedial hypothalamus assay with a Ke of 34 nM. It also showed an anxiolytic like profile orally in a standard anxiety paradigm (X-maze) with a minimum effective dose (MED) of 0.1 microgram/kg. Although the compound is less water soluble than CI-988, oral bioavailability in rat was improved nearly 10 times relative to CI-988 when dosed in HP beta CD. The blood-brain permeability of CI-1015 (31) was also enhanced relative to CI-988 (5). On the basis of the overall improved pharmacokinetic profile as well as enhanced brain penetration, CI-1015 (31) was chosen as a development candidate.

Modification of receptor selectivity and functional activity in cholecystokinin peptoid ligands.

Hybrid analogs of the cholecystokinin A (CCK-A) receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (1,A-71623) and the CCK-B receptor selective antagonists PD-135118 (2) and CI-988 (3) were prepared. Incorporation of the Lys(Tac) side chain into 2 produced a moderately potent antagonist of CCK-8 in the isolated guinea pig gallbladder (GPGB). Incorporation of the Lys(Tac) side chain into 3 produced the novel agonist analog 7 (EC50 = 28 nM in the GPGB) with excellent affinity for both human CCK-A (IC50 = 12 nM) and CCK-B (IC50 = 17 nM) receptors. Analog 7 was a full agonist (EC50 = 3.5 nM) for calcium mobilization on CHO-K1 cells expressing hCCK-A receptors but a partial agonist on CHO-K1 cells expressing hCCK-B receptors, eliciting a weak agonist response (EC50 = 2800 nM) and antagonizing CCK-8-induced calcium mobilization (KB = 20 nM). Despite this unusual in vitro profile, analog 7 was a potent anorectic agent in rats (ED50 = 30 nmol/kg) following intraperitoneal administration.

Cholecystokinin-induced activity changes of dorsal lateral geniculate neurons in rats.

The effects of iontophoretically applied cholecystokinin fragments and cholecystokinin antagonists on neurons of the dorsal lateral geniculate nucleus were investigated with extracellular recordings in rats anesthetized with urethane. The peptide cholecystokinin-8S, which has affinity for both cholecystokinin-A and -B receptors, altered the baseline firing as well as the responses to visual stimuli of about one half of the investigated neurons (90 out of 190). Excitatory effects predominated (P < 0.01, Wilcoxon test), although inhibitory effects were also observed. The effects of cholecystokinin-8S were dose-dependent. Neurons sensitive to cholecystokinin-8S could be found in all regions of the dorsal lateral geniculate nucleus, but they differed in their susceptibility to cholecystokinin in relation to their location. The B-agonist, BOC-cholecystokinin-4, also changed the baseline firing as well as the light-evoked activity of dorsal lateral geniculate nucleus neurons. The effects were either excitatory or inhibitory. Changes induced by cholecystokinin-8S could be effectively blocked by the cholecystokinin-B antagonist, CAM 1028 (19 out of 22 cholecystokinin-sensitive neurons tested). The cholecystokinin-A antagonist, Ge 410, blocked cholecystokinin-induced effects in 10 out of 16 neurons. These results indicate that the modulation of geniculate cell firing by cholecystokinin is mediated by both A-and B-receptor types.

Differential startle reactivity following central CCK-8S and systemic Boc CCK-4 administration in mice: antecedent stressor history and testing condition.

The influence of intraventricular cholecystokinin-8S (CCK-8S) and systemic N-t-Boc-Trp-Met-Asp-Phe-amide (Boc CCK-4) was evaluated in the acoustic and fear-potentiated startle paradigms in CD-1 mice. In the light + tone startle condition. CCK-8S increased startle 168 hr after administration, compared with saline. In the tone startle condition, CCK-8S decreased startle immediately and 24 hr after administration, compared with saline. Among nonshocked mice, CCK-8S increased startle at 48 and 168 hr, compared with saline. In the light + tone condition, 5 microg Boc-CCK-4 did not influence startle, whereas 15 microg Boc CCK-4 decreased startle immediately, 24 hr, and 48 hr following administration. Results demonstrate that antecedent environmental experiences interact with subsequent pharmacological challenges in provoking the temporal expression of alterations in startle magnitude.

Modulation of memory, reinforcement and anxiety parameters by intra-amygdala injection of cholecystokinin-fragments Boc-CCK-4 and CCK-8s.

This series of experiments examined the effects of the cholecystokinin (CCK) fragments Boc-CCK-4 and CCK-8s on memory, reinforcement and anxiety following unilateral injection into the central nucleus of the amygdala (CeA). In experiment 1, rats with chronically implanted cannulae were injected with CCK-8s or Boc-CCK-4 and were tested on a one-trial uphill avoidance task. Post-trial injection of 20 ng Boc-CCK-4 or 1 ng CCK-8s was found to improve the retention performance, whereas lower and higher doses had no effect. The hypermnestic effects of Boc-CCK-4 and CCK-8s were no longer evident when injection was performed 5 h, rather than immediately, after the learning trial. In experiment 2, the elevated plus-maze was used to gauge anxiogenous properties of intra-amygdala injections of Boc-CCK-4 and CCK-8s in memory-enhancing doses. The treatment with 20 ng Boc-CCK-4 and 1 ng CCK-8s did not influence the number of entries into and time spent on the open and enclosed arms of the maze as well as other anxiety-related behaviors. In experiment 3, possible reinforcing effects of the CCK-fragments were examined. After intra-amygdala injection of Boc-CCK-4 or CCK-8s in memory-enhancing doses the rats were placed into one of four restricted quadrants of a circular open field (closed corral) for a single conditioning trial. Subsequent tests for conditioned corral preference revealed no evidence for reinforcing or aversive effects of the CCK-fragments. In sum, these findings indicate that Boc-CCK-4 and CCK-8s facilitate memory processing upon injection into the CeA without exerting reinforcing or anxiogenous effects.