This is a summary of the virtual presentation given at the 2021 meeting of the Society for Research on the Cerebellum and Ataxias, https://www.meetings.be/SRCA2021/ , where the therapeutic potential of the CCK-CCK1R pathway for treating diseases involving Purkinje cell degeneration was presented. Spinocerebellar ataxia type 1 (SCA1) is one of a group of almost 50 genetic diseases characterized by the degeneration of cerebellar Purkinje cells. The SCA1 Pcp2-ATXN1[30Q]D776 mouse model displays ataxia, i.e. Purkinje cell dysfunction, but lacks progressive Purkinje cell degeneration. RNA-seq revealed increased expression of cholecystokinin (CCK) in cerebella of Pcp2-ATXN1[30Q]D776 mice. Importantly, the absence of Cck1 receptor (CCK1R) in Pcp2-ATXN1[30Q]D776 mice conferred a progressive degenerative disease with Purkinje cell loss. Administration of a CCK1R agonist to Pcp2-AXTN1[82Q] mice reduced Purkinje cell pathology and associated deficits in motor performance. In addition, administration of the CCK1R agonist improved motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Furthermore, CCK1R activation corrected mTORC1 signaling and improved the expression of calbindin in the cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results support the Cck-Cck1R pathway is a potential therapeutic target for the treatment of diseases involving Purkinje neuron degeneration.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the μ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed.

Cholecystokinin (CCK) is a peptide hormone that induces bile release into the intestinal lumen which in turn aids in fat digestion and absorption in the intestine. While excretion of bile acids and cholesterol into the feces eliminates cholesterol from the body, this report examined the effect of CCK on increasing plasma cholesterol and triglycerides in mice. Our data demonstrated that intravenous injection of [Thr28, Nle31]-CCK at a dose of 50 ng/kg significantly increased plasma triglyceride and cholesterol levels by 22 and 31%, respectively, in fasting low-density lipoprotein receptor knockout (LDLR(-/-)) mice. The same dose of [Thr28, Nle31]-CCK induced 6 and 13% increases in plasma triglyceride and cholesterol, respectively, in wild-type mice. However, these particular before and after CCK treatment values did not achieve statistical significance. Oral feeding of olive oil further elevated plasma triglycerides, but did not alter plasma cholesterol levels in CCK-treated mice. The increased plasma cholesterol in CCK-treated mice was distributed in very-low, low and high density lipoproteins (VLDL, LDL and HDL) with less of an increase in HDL. Correspondingly, the plasma apolipoprotein (apo) B48, B100, apoE and apoAI levels were significantly higher in the CCK-treated mice than in untreated control mice. Ligation of the bile duct, blocking CCK receptors with proglumide or inhibition of Niemann-Pick C1 Like 1 transporter with ezetimibe reduced the hypercholesterolemic effect of [Thr28, Nle31]-CCK in LDLR(-/-) mice. These findings suggest that CCK-increased plasma cholesterol and triglycerides as a result of the reabsorption of biliary lipids from the intestine.

Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega (Ω) turn. We demonstrate in vivo NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18 or CKR-1 or specific knockdown of CKR-1 in SMD or AIB neurons leads to shallower turns, hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)'s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. In vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin peptidergic signaling modulates an escape circuit to generate robust escape steering.

The objective of this study was to determine the sources of cholecystokinin within the neostriatum of the cat. Cholecystokinin-immunoreactive cells and fibers were detected by means of the peroxidase antiperoxidase immunohistochemical technique. This method was combined with intrastriatal injections of the retrograde marker horseradish peroxidase conjugated with wheat-germ agglutinin to survey the possible afferent sources of cholecystokinin to the feline neostriatum. Intrinsic, apparently aspiny cholecystokinin-immunoreactive neurons organized in a pericapsular pattern were found within both the caudate and putamen of the cat. In addition, both thalamostriatal and mesostriatal projections containing cholecystokinin were observed. These results indicate that cholecystokinin within the neostriatum of the cat arises from both intrinsic and extrinsic sites.

Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCKAR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity.

Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R(-/-),-2R(-/-)) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R(-/-) mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R(-/-),-2R(-/-) mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R(-/-) bone marrow-derived cells developed more albuminuria than diabetic CCK-1R(-/-) mice with WT bone marrow-derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy.

Expression of cholecystokinin is found in myocardial tissues as a gastrointestinal hormone and may be involved in cardiovascular regulation. However, it is unclear whether there is an increase in cholecystokinin expression in myocardial hypertrophy progression induced by abdominal aortic constriction. The study is aimed at exploring the relationship between cholecystokinin expression and myocardial hypertrophy.

Cholecystokinin (CCK), a hormone or neuropeptide, is secreted in response to intraluminal nutrients by enteroendocrine I-cells of the intestine and has important physiological actions related to appetite regulation and satiety. The stimulation on CCK secretion from the intestine is of potential relevance for body weight management. Naringenin (4',5,7-trihydroxyflavanone) and its glycoside naringin (naringenin 7-rhamnoglucoside) have been reported to have many biological functions. In the current study, we investigated the question of whether naringenin and naringin could stimulate CCK secretion and then examined the mechanisms involved in CCK release.

Associating contexts with rewards depends on hippocampal circuits, with local inhibitory interneurons positioned to play an important role in shaping activity. Here, we demonstrate that the encoding of context-reward memory requires a ventral hippocampus (vHPC) to nucleus accumbens (NAc) circuit that is gated by cholecystokinin (CCK) interneurons. In a sucrose conditioned place preference (CPP) task, optogenetically inhibiting vHPC-NAc terminals impaired the acquisition of place preference. Transsynaptic rabies tracing revealed vHPC-NAc neurons were monosynaptically innervated by CCK interneurons. Using intersectional genetic targeting of CCK interneurons, ex vivo optogenetic activation of CCK interneurons increased GABAergic transmission onto vHPC-NAc neurons, while in vivo optogenetic inhibition of CCK interneurons increased cFos in these projection neurons. Notably, CCK interneuron inhibition during sucrose CPP learning increased time spent in the sucrose-associated location, suggesting enhanced place-reward memory. Our findings reveal a previously unknown hippocampal microcircuit crucial for modulating the strength of contextual reward learning.

The cholecystokinin 2 receptor (CCK2R) is expressed in the central nervous system and peripheral tissues, playing an important role in higher nervous and gastrointestinal functions, pain sensation, and cancer growth. CCK2R is reversibly activated by cholecystokinin or gastrin, but whether it can be activated permanently is not known. In this work, we found that CCK2R expressed ectopically in CHO-K1 cells was permanently activated in the dark by sulfonated aluminum phthalocyanine (SALPC / AlPcS4, 10-1,000 nM), as monitored by Fura-2 fluorescent calcium imaging. Permanent CCK2R activation was also observed with AlPcS2, but not PcS4. CCK2R previously exposed to SALPC (3 and 10 nM) was sensitized by subsequent light irradiation (> 580 nm, 31.5 mW·cm-2). After the genetically encoded protein photosensitizer mini singlet oxygen generator (miniSOG) was fused to the N-terminus of CCK2R and expressed in CHO-K1 cells, light irradiation (450 nm, 85 mW·cm-2) activated in-frame CCK2R (miniSOG-CCK2R), permanently triggering persistent calcium oscillations blocked by the CCK2R antagonist YM 022 (30 nM). From these data, it is concluded that SALPC is a long-lasting CCK2R agonist in the dark, and CCK2R is photogenetically activated permanently with miniSOG as photosensitizer. These properties of SALPC and CCK2R could be used to study CCK2R physiology and possibly for pain and cancer therapies.

The participation of cholinergic mechanisms in cholecystokinin octapeptide (CCKOP) action on canine gall bladder was studied in vivo and in vitro, using three different experimental conditions. In vitro the responses of canine gall bladder smooth muscle to CCKOP (0.01 to 10 nm) were insensitive to atropine (1 to 10 microM) and tetrodotoxin (3 microM). When gall bladder muscle preparations were contracted by field electrical stimulation (0.7 ms, 40 Hz) CCKOP (0.001 to 0.1 nM) enhanced these contractions while atropine (1 microM) abolished them. This suggests that CCKOP is able to influence acetylcholine (ACH)-release from intrinsic cholinergic nerve terminals. In vivo the responses of canine gall bladder smooth muscle to CCKOP (1 to 10 ng/kg i.v.) were reduced and even abolished by atropine (10 to 50 micrograms/kg i.v.) and hexamethonium (0.5 to 3 mg/kg i.v.). The results suggest the participation of at least two mechanisms in CCKOP action on canine gall bladder motility: a direct action on smooth muscle cells, insensitive to atropine or tetrodotoxin, and an indirect action, which is dependent on pre- and post ganglionic cholinergic pathways.

Cholecystokinin 8 (CCK8) is an entero-octapeptide that participates in crosstalk with components of intestinal immunity via the CCK receptor (CCKR), but its role in modulation of the IgA response is not fully known under physiological conditions. Male eight-week-old BALB/c mice each were intraperitoneally injected once during 7 days with CCK8, devazapide (CCKR1 antagonist), L365,260 (CCKR2 antagonist) or vehicle (sham group). In intestinal lavages, total and secretory IgA (SIgA) were determined by ELISA; in lamina propria, IgA+ B lymphocytes and IgA+ plasma cells were analyzed by flow cytometry; mRNA levels of polymeric immunoglobulin receptor (pIgR) in epithelial cells and α chain, interleukins (ILs) in lamina propria cells were assessed by qRTPCR. Regarding the sham conditions, IgA+ plasma-cell percentage and IL-2, IL-5, IL-10 and transforming growth factor-β (TGF-β) mRNA levels were either increased by CCK8 or decreased by both CCKR antagonists. For IgA/SIgA responses, IL-4/IL-6 mRNA levels were decreased by all drugs and pIgR mRNA was increased by CCK8 and reduced by L365,260. IgA+ B cell percentage and α chain mRNA levels were elicited by CCK8 and L365,260. Data suggested a presumable differential role of CCK/CCKR on the IgA-response; outcome of L365,260 on the elicitation of IgA+ B cells and α chain mRNA needs further examination.

The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain-gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. CCK1R couples to Gs and Gq, whereas CCK2R primarily couples to Gq. Here we report the cryo-electron microscopy (cryo-EM) structures of CCK1R-Gs signaling complexes liganded either by sulfated cholecystokinin octapeptide (CCK-8) or a CCK1R-selective small-molecule SR146131, and CCK2R-Gq complexes stabilized by either sulfated CCK-8 or a CCK2R-selective ligand gastrin-17. Our structures reveal a location-conserved yet charge-distinct pocket discriminating the effects of ligand PTM states on receptor subtype preference, the unique pocket topology underlying selectivity of SR146131 and gastrin-17, the conformational changes in receptor activation, and key residues contributing to G protein subtype specificity, providing multiple structural templates for drug design targeting the brain-gut axis.

Altered GABA-mediated inhibition is proposed to play a role in the pathogenesis of epilepsy. Previous studies have demonstrated a loss of somatostatin-containing GABAergic interneurons innervating granule cells in epileptic animals. However, the reorganization of synapses between interneurons and granule cells has not been investigated. We studied synapse organization in an animal model of temporal lobe epilepsy (TLE) using continuous hippocampal stimulation. The distribution of axon terminals and inhibitory synapses on granule cell dendrites was studied using a combination of immunohistochemistry and pre-embedding electron microscopy techniques. A whole-cell patch-clamp technique was applied to study the functional changes in GABAergic input from different interneurons. In epileptic animals, the density of cholecystokinin (CCK)-immunoreactive (IR) fibers and α2 subunit containing GABAA receptors in the inner molecular layer of the dentate gyrus was reduced. Quantitative immuno-electron microscopy study revealed that the ratio of CCK-containing symmetric synapses to the total symmetric synapses was reduced. The frequency of GABAergic synaptic currents (sIPSC) was decreased and their amplitude was increased. The inhibitory effect of the activation of cannabinoid 1 (CB1) receptors was also reduced in epileptic animals. Isolation of CCK- and parvalbumin (PV)-containing GABAergic inputs by N- and P/Q-type calcium channel blockers respectively suggested that GABA release from CCK-containing interneurons was selectively reduced in epileptic rats. This study found that there was a loss of CCK-containing GABAergic synapses to granule cells both morphologically and functionally. These studies add to our understanding of the mechanisms that contribute to altering GABAergic inhibition of granule cells in TLE.

Hesperetin (3',5,7-trihydroxy 4'-methoxyflavanone) and its glycoside hesperidin (hesperetin 7-rhamnoglucoside) in oranges have been reported to possess pharmacological effects related to anti-obesity. However, hesperetin and hesperidin have not been studied on suppressive effects on appetite. This study examined that hesperetin and hesperidin can stimulate the release of cholecystokinin (CCK), one of appetite-regulating hormones, from the enteroendocrine STC-1 cells, and then examined the mechanisms involved in the CCK release. Hesperetin significantly and dose-dependently stimulated CCK secretion with an EC50 of 0.050 mM and increased the intracellular Ca(2+) concentrations ([Ca(2+)]i) compared to the untreated control. The stimulatory effect by hesperetin was mediated via the entry of extracellular Ca(2+) and the activation of TRP channels including TRPA1. These results suggest that hesperetin can be a candidate biomolecule for the suppression of appetite and eventually for the therapeutics of obesity.

Overfeeding and rapid weight gain during early life are risk factors for the development of obesity in adulthood. This metabolic malprogramming may be mediated by endocrine disturbances during critical periods of development. Cholecystokinin (CCK) acts on the central nervous system by elevating thermogenesis and the activity of anorectic neurons, modulating overall energy balance. Therefore, we tested the hypothesis that postnatal overfeeding impaired CCK effects. Pups were raised in either a litter of three (neonatal overnutrition/small litter group) or 12 (controls/normal litter group) pups per dam to study the effects of postnatal overfeeding on the central and peripheral CCK systems in adulthood. Rats raised in small litters became overweight during lactation and remained overweight as adults, with increased adiposity and plasma levels of lipids, glucose, insulin, and leptin. Neonatally over-nourished rats showed attenuation of gastric emptying and anorexigenic response to CCK, suggesting that offspring from the SL group may present CCK resistance as adult male rats. Consistent with this idea, overweight rats displayed impaired central response in c-Fos immunoreactivity on the nucleus tractus solitarius, area postrema, paraventricular nucleus, central amygdala, arcuate nucleus, and dorsomedial hypothalamus in response to peripheral CCK at adulthood. The small litter group of adult male rats also exhibited reduced norepinephrine- and CCK-stimulated thermogenesis. Unresponsiveness to the effects of CCK may contribute to overweight and metabolic dysfunctions observed in postnatally over-nourished adult rats. Thus, the involvement of an impaired CCK system, among other neurohormonal failures, may contribute to the development of obesity.

The aim of this study was to investigate whether the effects of the neuropeptide cholecystokinin on neuronal firing can be changed by acetylcholine in various structures of the brain. Single unit activity was extracellularly recorded in rats anesthetized with urethane. The neurons were located in several nuclei of the thalamus, the basal ganglia and the cerebral cortex. Neurons responding to the sulfated octapeptide of cholecystokinin (CCK-8S) were mainly activated by the drug [Wilcoxon test (Wt) p < 0.0001, n=113]. Thalamic neurons could also increase the number of burst discharges (Wt p < 0.005, n=39). Iontophoretically administered acetylcholine could reduce the activating effects of CCK-8S on firing and burst discharges. In its presence, even inhibitory effects of CCK-8S predominated (Wt p < 0.0001, n=113). The suppressive action seemed not to depend on the direction of the effect of acetylcholine itself and concerned neurons of all locations studied. Atropine could diminish or block the suppressive action of acetylcholine. In the presence of both drugs, CCK-8S mainly activated the neurons (Wt p < 0.005, n=43). Atropine itself did not significantly change the responses to CCK-8S (Wt p > 0.05). It can be concluded that cholecystokinin may reduce neuronal firing instead of increasing it during activation of the cholinergic system.

Expression of the brain-gut peptide cholecystokinin (CCK) in the developing olfactory-gonadotropin-releasing hormone-1 (GnRH-1) neuroendocrine systems was characterized, and the function of CCK in these systems was analyzed both in vivo and in vitro. We present novel data demonstrating that CCK transcript and protein are expressed in sensory cells in the developing olfactory epithelium and vomeronasal organ, with both ligand and receptors (CCK-1R and CCK-2R) found on olfactory axons throughout prenatal development. In addition, migrating GnRH-1 neurons in nasal regions express CCK-1R but not CCK-2R receptors. The role of CCK in olfactory-GnRH-1 system development was evaluated using nasal explants, after assessing that the in vivo expression of both CCK and CCK receptors was mimicked in this in vitro model. Exogenous application of CCK (10(-7) m) reduced both olfactory axon outgrowth and migration of GnRH-1 cells. This inhibition was mediated by CCK-1R receptors. Moreover, CCK-1R but not CCK-2R antagonism caused a shift in the location of GnRH-1 neurons, increasing the distance that the cells migrated. GnRH-1 neuronal migration in mice carrying a genetic deletion of either CCK-1R or CCK-2R receptor genes was also analyzed. At embryonic day 14.5, the total number of GnRH-1 cells was identical in wild-type and mutant mice; however, the number of GnRH-1 neurons within forebrain was significantly greater in CCK-1R-/- embryos, consistent with an accelerated migratory process. These results indicate that CCK provides an inhibitory influence on GnRH-1 neuronal migration, contributing to the appropriate entrance of these neuroendocrine cells into the brain, and thus represent the first report of a developmental role for CCK.