Cholecystokinin-like Immunoreactivity Research Articles

Advantages of Using Egg Yolk Antibodies in the Life Sciences: The Results of Five Studies

It has been known for over a century that specific antibodies can be extracted from the eggs of immunised chickens. However, it was only when animal welfare became a subject of public debate that the chicken was considered as an alternative source of antibodies due to the possibility of non-invasive antibody sampling. Unfortunately, the welfare of animals alone is not sufficient to attract the interest of scientists; it is therefore important to demonstrate to potential users that avian antibodies can be used successfully in a variety of scientific investigations. The particular specificity of avian antibodies would appear to be due to the phylogenetic difference between Mammalia and Aves as well as to differences between the molecular structures of avian immunoglobulin (IgY) and mammalian immunoglobulin (IgG). The use of avian antibodies has additional advantages, as a considerable quantity of antibodies can be obtained from one chicken, and because the specificity of avian antibodies often markedly differs from that of comparable mammalian antibodies. This paper aims to demonstrate the advantages of using avian antibodies by presenting the results of five separate studies. In the first study, coordinated by Rüdiger Schade, the visualisation of cholecystokinin-like immuno-reactivity in the substantia nigra of rats by using anti-cholecystokinin antibody, without the pre-treatment of colchicine, is described. The second study, headed by Albrecht Zott, describes the use of avian antibodies in the identification of modern acellular pertussis vaccines by using rocket immunoelectrophoresis. The identification of unknown vaccine batches and the comparison with reference vaccines is a prerequisite for reducing the number of animal experiments necessary for vaccine control. The third study, coordinated by Martin Gerl, investigates the specificity of antibodies directed against the N-terminal propeptide of procollagen type III (PIIINP). Among the antibodies originating from different species (rabbit, mouse and chicken), only the chicken antibody was able to respond to the PIIINP in both human and rat sera. Thus, a direct comparison between human serum samples (alcoholic liver) and serum samples derived from corresponding animal models was possible. The fourth study, coordinated by Michael Erhard, shows that egg yolk antibodies can be successfully used to manage infectious diarrhoea in young agricultural animals. The final study, led by Andreas Hlinak, describes the successful production of anti-bovine leukaemia virus antibody. This antibody could be used in several diagnostic systems (for example, enzyme immunoassays and cytology). The five studies demonstrate that avian antibodies are an attractive alternative to mammalian antibodies, not only with respect to the welfare of animals, but also with respect to scientific and economic considerations.

Distribution and characteristics of cholecystokinin-like immunoreactivity in the olfactory bulb of the cat

The distribution and characteristics of cholecystokinin (CCK)-like immunoreactive (LIR) nerve fibers was examined in the cat olfactory bulb using immunohistochemistry. CCK-LIR cell bodies were not found; fine varicose CCK-LIR fibers were observed in the most layers. In the main olfactory bulb, no staining was seen in the olfactory nerve layer and white matter. The directions of CCK-LIR fibers in the glomerular, external plexiform and mitral cell layers were generally perpendicular to the bulbar surface; those in the deep granule cell layer were parallel; and those in the internal plexiform and superficial granule cell layers were mixed. In the accessory olfactory bulb, CCK-LIR fibers were localized only in the granule cell layer. The presence of CCK-LIR fibers of the cat olfactory bulb may be involved in the modulation of olfactory transmission.

Galanin and cholecystokinin in cultured magnocellular neurons isolated from adult rat supraoptic nuclei: a correlative light and scanning electron microscopical study.

Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.

Simultaneous determination of cholecystokinin-like immunoreactivity and dopamine release after treatment with veratrine, NMDA, scopolamine and SCH23390 in rat medial frontal cortex: a brain microdialysis study.

The release of cholecystokinin (CCK) and dopamine (DA) was measured simultaneously in the medial frontal cortex of the rat using an in vivo microdialysis technique coupled to either enzyme immunoassay or high performance liquid chromatography with electrochemical detection method. Basal levels of CCK-like immunoreactivity (CCK-LI) and DA in the dialysates were 0.29 +/- 0.05 pg/50 microliters and 4.98 +/- 0.56 fmol/20 microliters respectively. After perfusion of 100 micrograms/ml veratrine into rat medial frontal cortex through the microdialysis probe, the release of those neurotransmitters was significantly enhanced. Perfusion of 10 mM NMDA induced significant increases of CCK-LI and DA levels. Co-perfusion of 1 mM SCH23390, a D1 receptor antagonist, suppressed NMDA-evoked CCK-LI release and slightly raised NMDA-evoked DA release. Treatment with scopolamine, a muscarinic receptor antagonist, suppressed veratrine-induced CCK-LI and DA release, but did not change NMDA-evoked CCK-LI and DA release. These results suggest that NMDA may regulate CCK-LI release through the activation of D1 receptor existing on the afferent CCK neurons or interneurons in rat medial frontal cortex, and changes of endogenous CCK release may be involved in NMDA receptor-mediated long-term potentiation.

Simultaneous determination of cholecystokinin-like immunoreactivity and dopamine release after treatment with veratrine, NMDA, scopolamine and SCH23390 in rat medial frontal cortex: a brain microdialysis study

The release of cholecystokinin (CCK) and dopamine (DA) was measured simultaneously in the medial frontal cortex of the rat using an in vivo microdialysis technique coupled to either enzyme immunoassay or high performance liquid chromatography with electrochemical detection method. Basal levels of CCK-like immunoreactivity (CCK-LI) and DA in the dialysates were 0.29 ± 0.05 pg/50 μl and 4.98 ± 0.56 fmol/20 μl, respectively. After perfusion of 100 μ/ml veratrine into rat medial frontal cortex through the microdialysis probe, the release of those neurotransmitters was significantly enhanced. Perfusion of 10 mM NMDA induced significant increases of CCK-LI and DA levels. Co-perfusion of 1 mM SCH23390, a D 1 receptor antagonist, suppressed NMDA-evoked CCK-LI release and slightly raised NMDA-evoked DA release. Treatment with scopolamine, a muscarinic receptor antagonist, suppressed veratrine-induced CCK-LI and DA release, but did not change NMDA-evoked CCK-LI and DA release. These results suggest that NMDA may regulate CCK-LI release through the activation of D 1 receptor existing on the afferent CCK neurons or interneurons in rat medial frontal cortex, and changes of endogenous CCK release may be involved in NMDA receptor-mediated long-term potentiation.

Effects of gastric digestive products from casein on CCK release by intestinal cells in rat

We investigated the ability of gastric digestive products from casein to stimulate cholecystokinin release by intestinal cells using the isolated vascularly perfused rat duodenojejunum. Casein digests were prepared with an in vitro system simulating gastric digestion and emptying. The luminal infusion of the digesta emptied from the artificial stomach for the first 10 minutes produced a sharp rise of portal cholecystokinin-like immunoreactivity to 300% of basal, followed by a well-sustained plateau secretion until the end of the infusion. The residual casein fraction of this digest brought about a modest cholecystokinin secretion, while the peptide component was as strong a stimulant as total digest. The peptide responsible for this effect was the glycomacropeptide that is a glycosylated fragment (106–169) of κ-casein. Only the slightly glycosylated forms of the peptide originating from variant A of κ-casein were active. The carbohydrate-free peptide did not alter basal cholecystokinin. The highly glycosylated forms of the peptide and the slightly glycosylated peptide from κ-casein variant B induced only a transient and low rise of portal cholecystokinin. The removal of N-acetylneuraminic acid from the active peptide suppressed its effect, while the infusion of an N-acetylneuraminic acid solution induced only a very low response. It is concluded that the glycomacropeptide released from dietary casein during gastric digestion can stimulate cholecystokinin release by intestinal cells in the rat. A well-defined structure is required for the peptide activity. A part of the peptide chain and some glycosidic chains containing N-acetylneuraminic acid, especially those bound to the amino acid residue threonyl 31 of caseinomacropeptide variant A, would be involved in this structure.

Multiple molecular forms of tachykinins in rat spinal cord: a study comparing different extraction methods

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1–9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neurokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.

Cholecystokinin in Cortico‐striatal Neurons in the Rat: Immunohistochemical Studies at the Light and Electron Microscopical Level

Using immunohistochemical techniques we have analysed the occurrence of cholecystokinin-like immunoreactivity (CCK-LI) in the cortex and striatum of the rat. In the cortex few CCK-immunoreactive cell bodies, mainly interneurons, could be visualized in normal brains, and a moderately dense network of CCK fibres was also observed. Injections of colchicine into the striatum led to an accumulation, in the surrounding cortex, of CCK-LI in the initial segment of the axon of numerous cells. In addition, with an antibody to pro-CCK several cell bodies, many of which with pyramidal shape, could be visualized. Furthermore, retrograde staining of cortical cells after unilateral injection of wheat germ agglutinin into the striatum revealed bilaterally in the cortex a number of labelled cells that also contained pro-CCK-LI. In the striatum CCK-LI was diffusely distributed in fine fibres as well as in patches of fibres located in the medial aspects. After decortication followed by callosotomy these patches disappeared on the side ipsilateral to the lesion, while the pattern of immunoreactivity of several other peptides in the striatum was unaffected. No change was observed on the contralateral side. Decortication or callosotomy alone did not affect the pattern of CCK-LI. At the ultrastructural level several CCK-immunoreactive terminals could be observed, mostly with clear, densely packed vesicles and straight asymmetric synaptic contacts with small spines, characteristic for terminals of cortical origin. The results are consistent with the presence of a major, partly crossed, CCK-containing cortico-striatal pathway.

Cholecystokinin corticostriatal pathway in the rat: Evidence for bilateral origin from medial prefrontal cortical areas

The anterograde tracer Phaseolus vulgaris-leucoagglutinin was used to examine the organization of the projections to the striatum from medial prefrontal and frontal cortical areas in the rat with reference to their relation to cholecystokinin-like immunoreactivity in the striatum. Medial prefrontal cortical areas projected bilaterally, with an ipsilateral predominance, to the striatum. Most of the positive fibres were found in medial and ventral areas of the caudate-putamen and in the nucleus accumbens. Labelled fibres formed distinct patch-like arrangements throughout the dorsomedial striatum, whereas more ventrally the fibres were densely packed and spread to lateral areas. Almost no fibres were found in the dorsolateral aspects of the caudate-putamen. Cholecystokinin-like immuno-reactivity in the striatum was diffusely distributed in the medial aspects, in fine punctate elements as well as in patches of fibres. Overlapping of corticostriatal clusters of fibres, from medial prefrontal cortex, with cholecystokinin-immunoreactive patches was found at all rostrocaudal levels studied, but predominantly in rostral areas. The overlap was present both in the ipsilateral and the contralateral side. Often the cluster of corticostriatal fibres was completely and precisely overlaid by a cholecystokinin-immunoreactive patch. At more caudal planes the overlap was only partial and in some instances cholecystokinin-positive patches “avoided” zones of dense corticostriatal fibre terminations. Frontal cortex injections of tracer gave rise to a network of fibres in the lateral aspects of the striatum, sparing the medial areas. No overlap with cholecystokinin-immunoreactive patches was found in these cases. These results suggest that a large number of cholecystokinin-containing striatal fibres originate in medial prefrontal cortical areas.

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

The murine neuroendocrine cell line, STC-1, was found to contain 296.8 ± 1.8 fmol of cholecystokinin-like immunoreactivity (CCK-LI) per mg cell protein. Immunocytochemical stain of STC-1 cells maintained in monolayer culture indicated that CCK-LI activity was present in 93% of the cells. Analysis by reverse-phase high-performance liquid chromatography indicated that STC-1 cells contained CCK-8 and an unidentified form as the predominant storage form. However, only CCK-8 was released into the culture medium upon stimulation by various secretagogues. The release of CCK-LI from STC-1 cells was stimulated by dibutyryl cAMP, forskolin, KCl, A23187, 4β-phorbol 12-myristate 13-acetate and luminal stimulants, e.g., sodium oleate, l-tryptophan, camostat and plaunotol. The release of CCK-LI from STC-1 cells was also stimulated by a neuropeptide, bombesin. The stimulatory effects of most of these agents were dose dependent. The stimulatory effects of dibutyryl cAMP, forskolin, and plaunotol were potentiated by 3-isobutyl-1-methyl xanthine, while that of camostat was not. The results obtained in this study indicate that the release of CCK from STC-1 cells shares the same characteristics of CCK release as from the CCK-secreting cells of the intestinal mucosa observed both in the dog and the rat in vitro and in vivo. Thus, the cellular mechanism of CCK release which appears to be cAMP- and Ca 2+-dependent may be modulated by cellular protein kinase C activity. The STC-1 cell appears to be a suitable model for studying the mechanism of CCK release.

Kainic acid seizures cause enhanced expression of cholecystokinin-octapeptide in the cortex and hippocampus of the rat.

Immunocytochemistry and in situ hybridization techniques were used for investigating changes in cholecystokinin immunoreactivity and mRNA in the cerebral cortex and hippocampus after kainic acid-induced limbic seizures in the rat. Marked increases in cholecystokinin mRNA concentrations were observed in layers II/III and V/VI of the cerebral cortex, in CA1 pyramidal neurons of the hippocampus, and in presumptive basket cells of the dentate gyrus 1 and 2 days after the acute seizures. Whereas cholecystokinin mRNA contents returned to normal in the cerebral cortex and the CA1 sector at later intervals, high concentrations were observed in basket cells even 2 months after the initial seizures. Accordingly, cholecystokinin-like immunoreactivity was intensified in the cerebral cortex, CA1 sector and in presumed basket cells of the hippocampus 30 days after kainic acid. Besides its high content in basket cells, cholecystokinin-like immunoreactivity was primarily present in neuronal fibers or diffusely distributed in the respective brain area. In the hippocampus, strongly enhanced staining for cholecystokinin was also observed in the alveus, the stratum lacunosum moleculare, and in the inner molecular layer, suggesting increased concentrations of the peptide in afferent and efferent fibers of the hippocampus. The present experiments suggest a strong activation of cholecystokinin systems in the brain after kainic acid-induced limbic seizures in the rat. This is indicated by pronounced increases in cholecystokinin mRNA in the cortex and individual cell types of the hippocampus (basket cells, granule cells, and CA1 pyramidal neurons). The subsequent increases in cholecystokin immunoreactivity even surpass those in mRNA. The observed changes may be part of the self-defense mechanisms that protect the animals during subsequent epileptic episodes.

Release of cholecystokinin-like immunoreactivity in the frontal cortex of conscious rats as assessed by transcerebral microdialysis: effects of different depolarizing stimuli.

The release of cholecystokinin-like immunoreactivity (CCK-LI) from the frontal cortex of freely moving rats has been studied using a transcerebral microdialysis technique coupled to a radioimmunoassay procedure. Basal levels of CCK-LI in the dialysate were above detection limits (2.4 +/- 0.7 pg/20 min; n = 8). High-K+ media evoked CCK-LI overflow in a concentration-dependent manner. The threshold concentration was 50 mM KCl. The peak overflow evoked by 100 mM K+ amounted to 42.7 +/- 2.8 pg/20 min (n = 6); it was totally Ca2+ dependent but insensitive to 1 microM tetrodotoxin. Infusion of 4-aminopyridine (1 mM; 20 min) evoked an overflow of CCK-LI (32 +/- 2.3 pg/20 min; n = 4), which was totally Ca2+ dependent and tetrodotoxin sensitive. Depolarization with 100 micrograms/ml of veratrine (20 min) provoked a CCK-LI overflow (62.2 +/- 10 pg/20 min; n = 6), which was also blocked by tetrodotoxin or by the absence of Ca2+ ions. The CCK-LI material collected under basal conditions or during veratrine infusion consisted essentially of CCK octapeptide sulfate. The veratrine-induced CCK-LI overflow did not change significantly when the infusion time was prolonged to 100 min. A second 20-min stimulus with 100 micrograms/ml of veratrine applied 200 min after a first 20-min stimulus evoked a barely significant CCK-LI overflow. These data suggest that one single 20-min stimulus with 100 micrograms/ml of veratrine may be sufficient to deplete the CCK-LI releasable stores and that > 200 min are required to replenish the depleted CCK-containing vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical analyses of the human olfactory bulb

These studies explore the distribution of putative neuroactive peptides in the human olfactory bulb. Localization of synaptophysin-, serotonin-, cholecystokinin-, substance P-, and somatostatin-like staining was examined by immunocytochemical protocols. The results provide new insights into the composition and laminar segregation of subpopulations of neurons and neuronal processes in the human olfactory bulb. The prominent synaptophysin-like immunoreactivity observed in the glomeruli of the human olfactory bulb is consistent with the notion that the density of synapses, and hence the density of synaptic vesicles, is highest in the glomeruli. Serotonin-like immunoreactivity suggested a variable innervation of glomeruli ranging from a dense tangled ball of fibers within the glomerulus to a sparse innervation by a single immunoreactive fiber. There was no evidence of serotonin-like immunoreactive cell bodies in either the olfactory bulb proper, anterior olfactory nucleus, or proximal regions of the lateral olfactory tract. Cholecystokinin-like immunoreactivity was limited to fibers found largely in the juxtaglomerular region of the glomerular layer. In the deeper layers of the olfactory bulb, cholecystokinin-like immunoreactive fibers did not show any of branching or arborization that was evident in the juxtaglomerular region. Substance P-like immunoreactivity was seen in varicose fibers distributed in all of the human olfactory bulb laminae. In addition, stained multipolar neurons were found in the area of the anterior olfactory nucleus. Somatostatin-like immunoreactivity was similar to that of substance P in that a plexus of stained fibers was found in all laminae of the olfactory bulb. Also, somatostatin-like immunoreactive cell bodies were found in the area of the anterior olfactory nucleus. However, as compared to substance P, somatostatin had a less dense plexus of immunoreactive fibers in the olfactory bulb. These results increase our understanding of the fundamental organization of the human olfactory system. The current data, coupled with prior studies, provide a foundation from which to study the cellular pathology of diseases with known olfactory system sequelae such as Alzheimer's, Parkinson's, and schizophrenia.

Effect of peripheral nerve cut on neuropeptides in dorsal root ganglia and the spinal cord of monkey with special reference to galanin

Using the indirect immunofluorescence method and in situ hybridization, the localization and levels of immunoreactivities and mRNAs for several neuropeptides were studied in lumbar dorsal root ganglia and spinal cord of untreated monkeys (Macaca mulatta) and after unilateral transection of the sciatic nerve. Immunoreactive galanin, calcitonin gene-related peptide, substance P and somatostatin and their mRNAs were found in cell bodies in dorsal root ganglia of untreated monkeys and on the contralateral side of the monkeys with unilateral sciatic nerve lesion. After axotomy there was a marked decrease in the number of calcitonin gene-related peptide-, substance P- and somatostatin-positive neurons in dorsal root ganglia ipsilateral to the lesion, whereas the number of galanin positive cells strongly increased. A few neuropeptide tyrosine-positive cells were seen in after axotomy, whereas no such neurons were found in controls. No vasoactive intestinal polypeptide-, peptide histidine isoleucine-, cholecystokinin-, dynorphin-, enkephalin-, neurotensin- or thyrotrophin releasing hormone-positive cell bodies were seen in dorsal root ganglia of any of the groups studied. In the dorsal horn of the spinal cord all peptide immunoreactivities described above, except thyrotropin releasing hormone, were found in varying numbers of nerve fibres with a similar distribution in untreated monkeys and in the contralateral dorsal horn in monkey with unilateral sciatic nerve lesion. Two cholecystokinin antisera were used directed against the C- and N-terminal portions, respectively, showing a distinctly different distribution pattern in the dorsal horn. Somatostatin- and dynorphin-like immunoreactivities were also observed in small neurons in the dorsal horn. No certain effect of axotomy on these interneurons could be seen. However, marked changes were observed after this type of lesion for some peptide containing fibres in the ipsilateral dorsal horn. Thus, there was a marked increase in galanin-like immunoreactivity, whereas calcitonin gene-related peptide-, substance P-, somatostatin-, peptide histidine isoleucine neurotensin- and cholecystokinin-like immunoreactivities decreased. No changes could be observed in neuropeptide tyrosine or enkephalin-positive fibres. The present results demonstrate marked ganglionic and transganglionic changes in peptide levels after peripheral axotomy. When compared to published results on the effect of axotomy on peptides in dorsal root ganglia and spinal cord of rat, both similarities and differences were encountered.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of cholecystokinin-like and calcitonin-like peptides in infant carotid bodies: a light- and electron-microscopic immunohistochemical study

Previous immunohistochemical studies have identified several regulatory peptides in the carotid body chief cells in both humans and animals. These peptides, together with amines, may be important in the modulation of the chemoreflex by the carotid body. We report the localization and distribution of calcitonin and cholecystokinin-like (CCK) immunoreactivity in chief cells of human infant carotid body by light- and electron-microscopic immunohistochemical techniques. Consecutive sections immunostained with calcitonin and/or CCK antibodies revealed positively stained chief cells, both alone and in clusters, scattered throughout the carotid body lobule. Generally more chief cells were positive for calcitonin than for CCK. This was confirmed by quantitative analysis showing that the ratio of calcitonin to CCK immunoreactive cells was consistently > 2:1 in all cases studied. There was no apparent correlation between the immunoreactivity for the two peptides and the age, sex, or postmortem interval. Calcitonin-like and CCK-like immunoreactivities were localized electron-microscopically over the dense core granules of the chief cells. Calcitonin and CCK-like peptides in carotid body chief cells may act as neurotransmitters or neuromodulators involved in chemoreception.

Inhibition by 5-HT3 receptor antagonists of release of cholecystokinin-like immunoreactivity from the frontal cortex of freely moving rats.

Effects of the 5-HT3 receptor antagonists, ondansetron and tropisetron, on the release of cholecystokinin-like immunoreactivity (CCK-LI) in rat frontal cortex were investigated in conscious, unrestrained rats using intracerebral microdialysis. The release of CCK-LI was augmented by perfusion with 100 micrograms/ml veratrine and was fully Ca(2+)-dependent and tetrodotoxin-sensitive. Ondansetron and tropisetron, each at 0.1-1 mumol/l, decreased concentration-dependently the veratrine-evoked efflux of CCK-LI. The reduction of CCK-LI output was approximately 30% when the antagonists were infused at 0.1 mumol/l. The data suggest that 5-HT3 receptor antagonists prevent the release of CCK evoked by endogenous 5-hydroxytryptamine. These drugs may thus represent a novel therapeutic approach in disease states, like anxiety, in which an inappropriately high release of brain CCK or 5-hydroxytryptamine seems to be involved.

Pattern of substance P- and cholecystokinin-like immunoreactivity during regeneration of the neural complex in the ascidian Ciona intestinalis.

The neural ganglion of ascidians exhibits a novel and rapid pattern of regeneration whereby within approximately 28-35 days of total ablation an entirely new neural complex is formed. In normal adults, neuronal cell bodies expressing substance P- (SP-Li), neurokinin A-(NKA-Li), CCK/gastrin- (CCK-Li), and insulin-like immunoreactivity exhibit a clearly defined pattern of localization in the cortical rind of the ganglion with characteristic long processes arising from the perikarya running throughout the neuropile. CCK-Li cell bodies are particularly concentrated close to the points of exit of the main nerve trunks. We have used antisera raised against these peptides to monitor the process of regeneration up to postoperative (pa) day 35. Only SP and CCK antisera produced positive staining in the regenerating tissue. Immunoreactive cell bodies first appear following 14 days pa. At this time CCK-Li neurons are more abundant than SP-Li neurons and in contrast to the pattern found in the normal adult ganglion, immunoreactive cell bodies are located both peripherally and centrally in the core of the ganglion and processes were rarely seen. Later stages exhibited an increasing number of SP-Li neurons and at 35 days pa SP-Li cell bodies clearly predominate. CCK-Li neurons typically become clustered close to the points of emergence of the anterior nerve roots. The early expression of CCK-Li and SP-Li molecules during regeneration is considered in terms of their potential role in development and cell proliferation in the newly forming ganglion.

Short‐term restraint stress and s.c. saline injection alter the tissue levels of substance P and cholecystokinin in the peri‐aqueductal grey and limbic regions of rat brain

Rats were exposed to short-term restraint (held by the tail for 1 min), injected s.c. with saline or subjected to the combination of these treatments. Fifteen and 30 min after these treatments the means serum corticosterone level was significantly increased by more than four times, compared to rats taken directly from their home cages, indicating a stress response. In the peri-aqueductal grey, the level of substance P-like immunoreactivity was increased by 45% (P < 0.01) and 65% (P < 0.01) 30 and 60 min after the combined treatment, respectively. Significant increases of the level of substance P-like immunoreactivity in the peri-aqueductal grey were also found after restraint only and after a s.c. saline injection. Similar, but less marked, changes in the level of cholecystokinin-like immunoreactivity in the PAG were also seen. In the accumbens a significantly decreased level of substance P-like immunoreactivity was encountered at 15 and 30 min after treatment, while the levels of cholecystokinin- and neuropeptide Y-like immunoreactivity were not significantly changed. In other regions studied, no effects on peptide levels were seen. The changes in peptide levels had a time course similar to that of the increase in serum corticosterone. Also the successive removal of rats from a common cage was found to increase significantly the serum corticosterone and the substance P-like immunoreactivity in the peri-aqueductal grey in the animals that were taken late in sequence from the cage.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus.

To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.

Increased calcitonin gene-related peptide- and cholecystokinin-like immunoreactivities in spinal motoneurones after dorsal rhizotomy.

Possible changes in neuropeptides within the ventral horn of the spinal cord were investigated after unilateral dorsal rhizotomy at the lumbar level (L1-L6) in adult rats. Ten days after the surgery, immunohistochemical observations and radioimmunological determinations confirmed a marked loss of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities within the superficial layers of the deafferented dorsal horn, as expected from the degeneration of primary afferent fibres containing these peptides. A concomitant increase in immunohistochemical staining and levels of CGRP (+296%) and CCK (+71%)-like immunoreactivities was observed in the ipsilateral ventral horn where both peptides are located in motoneurones. In contrast, substance P-like immunoreactivity that is confined to fibres and terminals within the ventral horn, was not altered by dorsal rhizotomy. These data indicate that the expression of neuropeptides in spinal motoneurones can be influenced by primary afferent inputs.