Distribution of preprocholecystokinin mRNA in motoneurons of the rat brainstem and spinal cord.

The distribution of cholecystokinin (CCK)-immunoreactive neurons in the central nervous system has been questioned because many antisera raised against CCK have been found to cross-react with calcitonin gene-related peptide. The use of in situ hybridization histochemistry has allowed investigators to determine which CCK-immunoreactive cells actually contain the messenger RNA (mRNA) coding for preprocholecystokinin (preproCCK), thus indicating that the peptide is synthesized in these cells. In this study, we report the distribution of preproCCK mRNA in the brainstem and spinal cord of the rat. The main findings of this study are the localization of preproCCK mRNA in motoneurons of cranial nerves IV, V, VI, VII and XII, as well as in motoneurons of the cervical and lumbrosacral levels of the spinal cord. Additionally, cells in lamina III at the cervical and lumbar enlargements contain preproCCK mRNA, suggesting that cells expressing CCK may be important in the processing of sensory information from the appendages.     

Expression of the cholecystokinin gene in rat brain during development

The expression of the gene encoding for cholecystokinin (CCK) was studied pre- and postnatally in rat brain with a preproCCK-specific cDNA probe to quantitate mRNA. In addition, a C-terminal-specific radioimmunoassay was used to measure CCK-immunoreactive peptides. Poly(A)+-enriched RNA from rat brain was analyzed for the presence of CCK mRNA by agarose gel electrophoresis, followed by transfer to nitrocellulose and hybridization to a 32P-labeled CCK cDNA probe. An RNA of approximately 700 nucleotides was observed and there were no obvious CCK mRNA differences between samples taken at the different developmental stages. Brain preproCCK mRNA was detectable by embryonic day 14 and increased continuously until postnatal day 14 when it reached maximal concentrations. In young adult rats, levels were slightly decreased. CCK-immunoreactive peptides began to be detected in the samples from embryonic day 21, but marked development was seen only postnatally with a rapid increase occurring during the first 2 weeks after birth. The highest content of the peptide was present in young adult rats.     

Preprocholecystokinin mRNA in rat brain: regional expression includes thalamus

The regional distribution of forebrain neurons expressing preprocholecystokinin (preproCCK) mRNA was examined using in situ and blot hybridization analysis. We observed a distribution of neurons containing preproCCK mRNA closely parallel to that reported for CCK immunoreactivity, with the exception of the thalamus. In this CCK-immunoreactive cell body-poor structure, we detected relatively abundant RNA: probe hybridization using both our techniques. Control experiments suggested that the hybridizing species in thalamus is authentic preproCCK mRNA. This suggests that there exist forebrain neurons expressing the CCK gene which have gone undetected in immunocytochemical studies.     

Altered cholecystokinin binding site density in the supraoptic nucleus of morphine-tolerant and -dependent rats

The processes underlying the development of neuronal tolerance to and dependence upon opiates are not yet fully understood. To evaluate a possible role for cholecystokinin (CCK) in these processes, quantitative receptor autoradiography and in situ hybridisation histochemistry were used to study both the density and distribution of sulphated CCK octapeptide (CCK8S) binding sites and preproCCK peptide mRNA levels within the dorsal (oxytocin neurone-rich) supraoptic nuclei of rats given an intracerebroventricular (i.c.v.) infusion of morphine over 5 days, which is known to induce tolerance and dependence in mechanisms regulating oxytocin neurones. Specific CCK8S binding was significantly increased in the supraoptic nuclei of both morphine-dependent and salt-loaded (2% sodium chloride to drink for 48 h) rats compared to their respective controls (P<0.05). In situ hybridisation histochemistry revealed no difference in preproCCK mRNA levels within supraoptic neurones of (i.c.v.) morphine-treated compared with either i.c.v. vehicle-treated or untreated control animals. These results suggest that CCK receptor mechanisms involved in the control of magnocellular oxytocin neurone activation are upregulated during chronic morphine treatment, and this may favour increased sensitivity to CCK, thereby offsetting the inhibitory actions of morphine, contributing to tolerance and perhaps to the withdrawal excitation characteristic of dependence.     

CGRP-like immunoreactivity in A11 dopamine neurons projecting to the spinal cord and a note on CGRP-CCK cross-reactivity

Using the indirect immunofluorescence technique and double labelling procedures combined awith retrograde tracing it could be demonstrated that the A11 dopamine cell group, located at the border between the diencephalon and mesencephalon of the rat brain and some of which project to the spinal cord, contains calcitonin gene-related peptide (CGRP)-like immunoreactivity. Thus, another catecholamine group in the rat brain has been shown to have a coexisting peptide. One of the CGRP antisera used in the present study also stained cholecystokinin (CCK) containing neurons in various brain areas. Absorption and displacement experiments using immunohistochemistry and radioimmunoassay showed that this cross-reactivity was confined to the C-terminal portion of the peptide molecule. Therefore, the present results suggest that CGRP antisera used for immunohistochemistry and radioimmunoassay should be tested for possible cross-reactivity with CCK.     

Excitatory effects of cholecystokinin in rat hippocampus: pharmacological response compatible with 'central'- or B-type CCK receptors

The effects of the sulfated octapeptide of cholecystokinin (CCK8-S), the non-sulfated homolog (CCK8-NS) as well as the C-terminal di-, tri- and tetrapeptidic fragments (respectively CCK2, CCK3 and CCK4) were studied in vitro in rat hippocampal slices by extracellular recording of the spontaneous action potential discharge frequency of neurons located in the CA1 stratum pyramidalis. Bath-applied CCK8-S concentration-dependently increased the action potential discharge frequency of hippocampal CA1-neurons in concentrations ranging from 0.05 to above 1 microM. Both CCK8-NS and CCK4 exhibited reversible and concentration-dependent excitatory effects. They were 4 and 10 times less potent than CCK8-S, respectively, as concentrations of 2 microM CCK8-NS and 5 microM CCK4 were needed to evoke the same excitation as that induced for a given neuron by 0.5 microM CCK8-S. In contrast, none of the shorter fragments (CCK2 and CCK3) were effective in altering spontaneous discharge of CCK8-S-sensitive neurons even at concentrations of 100 microM. The pharmacologic profile of the excitatory response observed in the rat hippocampus follows the same pattern as the binding profile observed on brain membrane preparations. It is therefore concluded that the CCK receptors involved in this response seem to be related more to the 'central'- or B-type CCK receptors rather than to the 'peripheral'- or A-type CCK receptors.     

Accelerated expression of cholecystokinin gene in the brain of rats rendered tolerant to morphine.

Results from behavioural studies have shown that central cholecystokinin octapeptide (CCK-8) is a powerful antagonist against opioid effects and that an increased release of central CCK-8 plays an important role in the mechanisms of tolerance to morphine analgesia. No information is available concerning the rate of biosynthesis of CCK-8 in response to chronic morphine administration. Blot hybridization experiments made in the present study revealed a marked increase in preproCCK mRNA in the brain of rats receiving chronic morphine treatment for 1, 3 and 6 days, showing an increment of 52% (p < 0.05), 62% (p < 0.05) and 97% (p < 0.01) respectively. The results suggest that an acceleration of the expression of the CCK gene during long-term morphine treatment might constitute one of the mechanisms for morphine tolerance.     

Autoradiographic localization of cholecystokinin receptors in rodent brain

Cholecystokinin (CCK) receptor binding sites have been localized by autoradiography in the guinea pig and rat central nervous system. [125I]CCK-triacontatriapeptide labeled the sites in brain slices with an observed association constant equal to 0.041 min-1 and a dissociation constant equal to 0.008 min-1. CCK-triacontatriapeptide (CCK-33) and the C-terminal octapeptide of CCK-33 (CCK-8) potently inhibited [125I]CCK-33 binding with Ki's of 2 nM, whereas desulfated CCK-8 (CCK8-ds) and the C-terminal tetrapeptide of CCK-33 (CCK-4) were much weaker. Receptors were concentrated in the olfactory bulb, in the superficial laminae of the primary olfactory cortex, in the deep laminae of the cerebral cortex, and in the pretectal area. Substantial numbers of sites were also found in the basal ganglia, in the amygdala, and in the hippocampal formation. [125I]CCK-33 binding sites appear to be located on fibers of the optic tract and probably on olfactory tract fibers as well. These results are discussed in terms of physiological functions associated with CCK, presynaptic receptors, and axonal flow of CCK receptors.     

Distribution of cholecystokinin (CCK) in the rat lower brain stem nuclei

The cholecystokinin (CCK) concentration in individual brain stem nuclei of rat was determined using the Palkovits punch method¹⁹ and the CCK RIA³. CCK has a unique distribution in the brain stem, unlike other neuropeptides and biogenic amines^8,19. In general, the CCK levels in the brain stem are 5–20% of rat cerebral cortex. The colliculi, midbrain central gray, nucleus of the solitary tract, and the interpeduncular nucleus had the highest CCK content (2.7-1 ng CCK mg protein).     

Microinjection of cholecystokinin into the rat ventral tegmental area potentiates dopamine-induced hypolocomotion

Cholecystokinin, (CCK) 1-400 ng, significantly potentiated the hypolocomotion induced by dopamine, when simultaneously microinjected bilaterally into the ventral tegmental area (VTA) of rat brain. Within this dose range, CCK had no effect alone on ambulatory locomotion. Topographical analysis indicated that the modulatory effect of CCK was greatest in the central and caudal regions of the VTA, and absent at sites lateral, dorsal, rostral, and caudal to the VTA. Pharmacological analysis indicated that both unsulfated CCK octapeptide (100 ng) and the C-terminal tetrapeptide of CCK (400 ng) potentiated dopamine-induced hypolocomotion in a manner identical with sulfated CCK octapeptide (100 ng). Proglumide, an antagonist of the peripheral-type CCK receptor, did not block the potentiating actions of CCK, at doses of proglumide up to 500 mg/kg i.p., or 100 micrograms into the ventral tegmental area. L-364,718, an antagonist of the peripheral-type CCK receptor with lesser affinity for the central-type CCK receptor, blocked the potentiating actions of CCK at relatively high doses of L-364,718 (1-10 mg/kg i.p.). These findings suggest that CCK acts as a facilitatory modulator of dopamine at a central-type CCK receptor on the A10 cell bodies.     

癫痫易感大鼠脑内CCK—8肽含量,CCK基因结构及CCK—mRNA表达初探

对听源性癫痫易感大鼠P_(77)PMC及对照Wistar大鼠发育中脑内基础CCK-8肽含量、mRNA表达水平及胆囊收缩素(cholecystokinin,CCK)基因结构进行初探。提示:(1)P_(77)PMC在发育中,成年脑内基础CCK-8肽显著高于对照;(2)Northern印迹杂交示P_(77)PMC组脑CCK-mRNA与对照无显著差异;(3)CCK DNA结构在两组大鼠未发现RFLPs不同。证实两系大鼠脑内CCK系统存在差异,其与癫痫易感性的关系尚待进一步研究。     

The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay

The regional distribution of cholecystokinin (CCK) in the rat brain was determined utilizing a radioimmunoassay which detects both gastrin and CCK. CCK concentration is highest in the caudate nucleus (10–14 ng CCK8 equivalents/mg protein), followed by the cerebral cortex. Within the cerebral cortex, CCK is highest in the cingulate, pyriform, and entorhinal areas. There are substantial CCK concentrations in all other brain regions except pons, medulla and cerebellum. CCK is widely distributed in the hypothalamus, where it is highest in the median eminence and ventromedial nucleus. Considerable CCK-like immunoreactivity is also present in the posterior lobe of the pituitary gland, but is not detectable in anterior and intermediate lobes.Though the antisera used in this study cross-react with gastrin the dominant CCK-like material found in rat brain co-elutes with sulfated CCK8 and separates from gastrin on Sephadex G-25 and HPLC chromatography.     

The regional and subcellular development of cholecystokinin immunoreactivity in vertebrate brain

We have previously demonstrated that the development of CCK in rat brain occurs during the first postnatal month. In order to determine whether the appearance of CCK is associated with specific aspects of brain histogenesis, we examined the development of brain CCK immunoreactivity in both precocial and altricial mammals and birds. The two precocial species (guinea pig and chicken) were found to achieve adult CCK concentrations prenatally, while the altricial species (zebra finch and rat) manifested adult brain CCK concentrations only after several weeks of postnatal development. In adulthood, both mammals showed relatively high forebrain CCK concentrations, while the two species of birds manifested much lower forebrain levels. Brainstem levels of CCK were similar in all species studied. In each species, the development of CCK followed a common time course across all major brain areas, although adult brainstem levels of CCK were generally attained shortly before adult forebrain levels. Correlation of our comparative ontogenetic data with known patterns of brain histogenesis indicated that CCK development follows regional neuroblastic proliferation, migration and differentiation, and occurs during or soon after local synaptogenesis. In the rapidly developing precocial chicken brain, CCK production precedes the postnatal gliogenic and myelinogenic increases in brain weight, suggesting that neurogenic production of CCK occurs independently of these non-neuronal maturation events. Subcellular fractionation of developing chicken brain revealed that a substantial fraction of brain CCK is localized in synaptosomes relatively early in embryogenesis; this synaptosomal localization becomes even more pronounced with further brain maturation. This early appearance of CCK in synaptic terminals indicates a correspondingly precocial maturation for the intraneuronal mechanisms subserving peptide cleavage, axonal transport and vesicular insertion, and suggests that CCK may be available for neurotransmission quite early in development. In an analysis of the molecular forms of CCK, gel filtration disclosed no differences between species or different brain areas in the form of CCK present. CCK-8 always predominated in brain, with smaller void volume (pro-CCK) peaks, and negligible amounts of CCK-33. Finally, duodenal CCK (largely CCK-33) appeared much earlier than brain CCK in all species examined, suggesting that the gut and brain CCK systems develop independently of one another.     

The neurochemical effects of anxiolytic drugs are dependent on rearing conditions in Fawn-Hooded rats

There is a vast literature examining the neurochemical effects of anxiolytics throughout the rat brain; however, although the behavioural actions of anxiolytic drugs are routinely assessed in animal models of anxiety, the majority of neurochemical studies have been performed in rats with relatively 'normal' behavioural phenotypes. Since there is significant evidence that an anxious phenotype is associated with numerous neurochemical alterations, it is feasible that the central effects of anxiolytics may vary depending on the underlying behavioural state (and corresponding neuropathology) of the experimental animal. For this reason, the aim of the present study was to examine the effect of chronic anxiolytic drug administration on the central CCK and dopamine systems in anxious (isolated from weaning) and nonanxious (group-housed) Fawn-Hooded (FH) rats. It is important to note that these studies were performed in rats with continued access to ethanol, which may affect the responses to anxiolytic treatment. Chronic anxiolytic treatment with the selective CCK-B (CCK(2)) receptor antagonist, Ci-988 (0.3 mg/kg/day ip) or diazepam (2 mg/kg/day ip), induced numerous effects throughout the central nervous system (CNS), with Ci-988 inducing significant changes in the density of dopamine D(2) receptors, and diazepam producing marked changes in both dopamine D(2) and CCK-B receptor binding density as well as preproCCK mRNA expression. Interestingly, the neurochemical effects of these anxiolytic drugs varied significantly depending on the rearing conditions of the rats, demonstrating the importance of using adequate animal models when correlating the behavioural and central effects of drugs acting throughout the CNS.     

Cholecystokinin peptides in the brain and pituitary of the bullfrog Rana catesbeiana: distribution and characterization

The distribution of CCK peptides in the bullfrog brain was determined with a CCK radioimmunoassay. Frog brain CCK distribution resembles rat porcine and human brain in that CCK concentration is moderate to high in hypothalamus, diencephalon, and medulla (3 18.4 ng/mg protein) and low in cerebellum (0.6 ng/mg protein). However, unlike all mammalian species examined, the CCK content of frog cerebral cortex, hippocampus and olfactory lobe is quite low (0.03 0.23 ng/mg protein). The elution of CCK-like peptides in frog brain extracts was determined on two HPLC systems. On both systems the bulk of the CCK-like material eluted with CCK 8 sulfate and separated from gastrin and other CCK peptides. These data suggest that though the chemical structure of CCK appears to be the same in the brains of frogs and mammals, the distribution of CCK in the brain appears to have shifted during the course of evolution, becoming a cortical, hippocampal, and olfactory system peptide only in more evolved organisms.     

Cholecystokinin peptides in the brain and pituitary of the bullfrog Rana catesbiana: distribution and characterization

The distribution of CCK peptides in the bullfrog brain was determined with a CCK radioimmunoassay. Frog brain CCK distribution resembles rat, porcine, and human brain in that CCK concentration is moderate to high in hypothalamus, diencephalon, and medulla (3–18.4 ng /protein) and low in cerebellum (0.6 ng/mg protein). However, unlike all mammalian species examined, the CCK content of frog cerebral cortex, hippocampus and olfactory lobe is quite low (0.03–0.23 ng/mg protein).The elution of CCK-like peptides in frog brain extracts was determined on two HPLC systems. On both systems the bulk of the CCK-like material eluted with CCK8 sulfate and separated from gastrin and other CCK peptides.These data suggest that though the chemical structure of CCK appears to be the same in the brains of frogs and mammals, the distribution of CCK in the brain appears to have shifted during the course of evolution, becoming a cortical, hippocampal, and olfactory system peptide only in more evolved organisms.     

An HPLC and RIA analysis of the cholecystokinin peptides in rat brain

Methanol and boiling water extracts of rat brain were chromatographed on two HPLC columns and the major cholecystokinin (CCK) peptides that eluted were detected by radioimmunoassay. The major CCK-like substance in both extracts co-chromatographed with CCK octapeptide sulfate on both columns, in agreement with previous reports of gel filtration chromatography of extracts of rat (1), porcine (2), human (2), and guinea pig brain (3). Minor immunoreactive components were detected in both extracts which chromatographed with CCK8 desulfate, CCK4 or 5, and variable amounts of partially or completely oxidized CCK8 sulfate and desulfate. Little or no immunoreactivity comigrated with sulfated or desulfated gastrin 17.In boiling water extracts, two additional peaks of immunoreactivity were detected, one which co-elutes with CCK33 and one which precedes CCK33 and has an apparent molecular weight of 40,000–50,000. This “pro-CCK” is not dissociated by treatment with sodium dodecyl sulfate and is considerably less hydrophobic than CCK8 or CCK4. It may be the same as the putative CCK precursor reported by Rehfeld to incorporate ³⁵S methionine in rat cortex (4) which yields CCK8 upon trypsinization (5).     

Immunohistochemical studies of cholecystokininlike peptides and their relation to 5-HT, CGRP, and bombesin immunoreactivities in the brainstem and spinal cord of lampreys

The distribution of cholecystokinin (CCK)-like immunoreactivity in the brainstem and spinal cord of lampreys was studied by using CCK antisera with different properties. In the spinal cord, three separate systems reacted with CCK antisera: (1) A ventral and lateral fiber system descending from a group of neurons in the posterior reticular nucleus of the rhombencephalon was labeled by both a C-terminal-directed CCK antiserum and a monoclonal CCK antibody. (2) A dorsal root-dorsal column system of fibers originating from cell bodies in the dorsal root ganglia was labeled only by the C-terminal CCK antiserum. This CCK immunoreactivity could be abolished by preabsorption with calcitonin-gene-related peptide (CGRP), suggesting that it was due to cross-reactivity with a CGRP-like peptide. This system also contained 5-hydroxytryptamine (5-HT)-, bombesin-, and CGRP-like immunoreactivities. (3) An intraspinal system of 5-HT neurons was labeled with an antiserum to the midportion of CCK-33 but not by the other CCK antisera. The CCK labeling of this system was difficult to reduce by preabsorption with CCK peptide and thus appeared to be nonspecific. Groups of cell bodies in the middle reticular nucleus of the rhombencephalon, the reticular nucleus of the mesencephalon, and the hypothalamus were labeled by both the C-terminal and the monoclonal CCK antisera. The gut contained two types of CCK-like immunoreactivity, one of which appeared to be due to cross-reactivity with CGRP. A biochemical analysis showed that the content of CCK was low in the spinal cord compared to the brain, and these results agreed with the immunohistochemical findings.     

Neuronal localization of cholecystokinin mRNA in the rat brain by using in situ hybridization histochemistry

The distribution of cholecystokinin (CCK) mRNA in the rat brain was determined by means of in situ hybridization histochemistry. Our results demonstrate a widespread distribution of neurons containing CCK mRNA throughout the rat brain. Hybridization-positive neurons were distributed throughout the neocortex, olfactory bulb, claustrum, amygdala, the dentate gyrus and hippocampus proper, and several subnuclei of the thalamus and the hypothalamus. The most abundant and most heavily labeled neurons were found in the endopiriform/piriform cortex, tenia tecta, and the ventral tegmental area. The distribution of neurons positive for CCK mRNA paralleled that of CCK-like immunoreactive neurons. These results detail the distribution of CCK mRNA and clearly identify the existence of CCK-synthesizing neurons in regions such as the paraventricular and supraoptic nuclei of the hypothalamus, where the presence of CCK cell bodies was previously uncertain.