NEUROENDOCRINE PEPTIDES IN THE PATHOGENESIS OF COLORECTAL CARCINOMA.

Colorectal carcinoma (CRC) is the third most frequent neoplasm worldwide and the second leading cause of mortality. Neuroendocrine peptides such as glucagon, bombesin, somatostatin, cholecystokinin, and gastrin as well as growth factors such as platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, and fibroblast growth factor have been postulated as being involved in carcinogenesis. The fact that these neuroendocrine peptides are involved in the development of CRC through the activation of growth factors that stimulate a series of molecular pathways that activate oncogenic signaling mechanisms is emphasized in this review. Peptides such as CCK1, serotonin, and bombesin have been found to be over-expressed in human tumor tissues. Meanwhile, the expression of peptides such as GLP2 has been seen mainly in murine models. The information contained in this review provides a better understanding of the role these peptides play in the pathogenesis of CRC for basic and clinical science studies.

Neonatal overnutrition, but not neonatal undernutrition, disrupts CCK-induced hypophagia and neuron activation of the nucleus of the solitary tract and paraventricular nucleus of hypothalamus of male Wistar rats.

Metabolic programming may be induced by reduction or enhancement of litter size, which lead to neonatal over or undernutrition, respectively. Changes in neonatal nutrition can challenge some regulatory processes in adulthood, such as the hypophagic effect of cholecystokinin (CCK). In order to investigate the effects of nutritional programming on the anorexigenic function of CCK in adulthood, pups were raised in small (SL, 3 pups per dam), normal (NL, 10 pups per dam), or large litters (LL, 16 pups per dam), and on postnatal day 60, male rats were treated with vehicle or CCK (10 µg/Kg) for the evaluation of food intake and c-Fos expression in the area postrema (AP), nucleus of solitary tract (NTS), and paraventricular (PVN), arcuate (ARC), ventromedial (VMH), and dorsomedial (DMH) nuclei of the hypothalamus. Overnourished rats showed increased body weight gain that was inversely correlated with neuronal activation of PaPo, VMH, and DMH neurons, whereas undernourished rats had lower body weight gain, inversely correlated with increased neuronal activation of PaPo only. SL rats showed no anorexigenic response and lower neuron activation in the NTS and PVN induced by CCK. LL exhibited preserved hypophagia and neuron activation in the AP, NTS, and PVN in response to CCK. CCK showed no effect in c-Fos immunoreactivity in the ARC, VMH, and DMH in any litter. These results indicate that anorexigenic actions, associated with neuron activation in the NTS and PVN, induced by CCK were impaired by neonatal overnutrition. However, these responses were not disrupted by neonatal undernutrition. Thus, data suggest that an excess or poor supply of nutrients during lactation display divergent effects on programming CCK satiation signaling in male adult rats.

Modulation of hypothalamic AMPK and hypothalamic neuropeptides in the control of eating behavior: A systematic review.

Eating behavior is regulated by central and peripheral signals, which interact to modulate the response to nutrient intake. Central control is mediated by the hypothalamus through neuropeptides that activate the orexigenic and anorexigenic pathways. Energy homeostasis depends on the efficiency of these regulatory mechanisms. This neuroendocrine regulation of hunger and appetite can be modulated by nutritional sensors such as adenosine monophosphate-activated protein kinase (AMPK). Thus, this systematic review discusses the literature on correlations between AMPK and hypothalamic neuropeptides regarding control of eating behavior. Lilacs, PubMed/Medline, ScienceDirect, and Web of Science were searched for articles published from 2009 to 2021 containing combinations of the following descriptors: "eating behavior," "hypothalamus," "neuropeptide," and "AMPK." Of the 1330 articles found initially, 27 were selected after application of the inclusion and exclusion criteria. Of the selected articles, 15 reported decreased AMPK activity, due to interventions using angiotensin II infusion, fructose, glucose, cholecystokinin, leptin, or lipopolysaccharide (LPS) injection; dietary control through a low-protein diet or a high-fat diet (60 % fat); induction of hyperthyroidism; or injection of AMPK inhibitors. Seven studies showed a decrease in neuropeptide Y (NPY) through CV4 AICAR administration; fructose, glucose, leptin, or angiotensin II injections; or infusion of LPS from Escherichia coli and liver kinase B1 (LKB1) overexpression. Eleven studies reported a decrease in food consumption due to a decrease in AMPK activity and/or hypothalamic neuropeptides such as NPY. The results indicate that there is a relationship between AMPK and the control of eating behavior: a decrease in AMPK activity due to a dietary or non-dietary stimulus is associated with a consequent decrease in food intake. Furthermore, AMPK activity can be modulated by glucose, thyroid hormones, estradiol, leptin, and ghrelin.

Feed intake and gene expression of appetite-regulating hormones in Salminus brasiliensis fed diets containing soy protein concentrate.

Dourado (Salminus brasiliensis) is a large carnivorous fish with high commercial value for which sustainable aquaculture relies on the substitution of expensive dietary animal protein sources in aquafeeds, in particular fish meal (FM), by cheaper plant protein, such as soy protein concentrate (SPC). This study aimed at evaluating feed intake and gene expression of appetite- regulating hormones [orexin, cocaine and amphetamine regulated transcript (CART), leptin, cholecystokinin (CCK) and peptide YY (PYY)] in the intestine, pyloric caeca and hypothalamus of juvenile dourado fed diets containing graded levels of SPC and FM as dietary protein sources for a period of three weeks. Increasing dietary plant protein contents reduced daily feed consumption and the expressions of the anorexigenic hormone CCK in the anterior intestine and in pyloric caeca and PYY in pyloric caeca. No changes were detected in the hypothalamic expression of appetite-regulating hormones, suggesting that gastrointestinal hormones are more involved in the decrease in feeding induced by plant protein diets than central appetite-regulating systems.

Neonatal overnutrition programming impairs cholecystokinin effects in adultmale rats.

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.

Distribution of goblet and endocrine cells in the intestine: A comparative study in Amazonian freshwater Tambaqui and hybrid catfish.

Goblet cells (GCs) and endocrine cells (ECs) play an important role in intestine physiology, and few studies currently exist for Amazonian fishes. This study aimed to quantify the distribution of GCs and ECs producing cholecystokinin-8 and neuropeptide Y, assessed by mucin histochemistry and peptides immunohistochemistry, in the intestine of two Amazonian species with different feeding habits Tambaqui (Colossosoma macropomum) and hybrid catfish (Pseudoplatystoma reticulatum × Leiarius marmoratus), an omnivore and carnivore, respectively. A systematic literature review correlating feeding habit and GC and EC distribution was also included to contribute to the comparative study. The results of this study provided novel information about the gut cells of Tambaqui and hybrid catfish. Both, GCs and ECs can be found sweeping the entire intestine of Tambaqui and hybrid catfish although the cells can be more concentrated in certain segments. The GCs and ECs in Tambaqui were more uniformly distributed in the midgut segments (T1, T2, and T3). Unlike, in hybrid catfish GCs were more concentrated in the hindgut (C4) and ECs mainly in the two midgut segments (C1 and C2) of hybrid catfish. Based on the comparison between Tambaqui, hybrid catfish, and other fishes in the literature review, we suggest that cell distribution can be partially explained by feeding habits, carnivorous vs. omnivorous.

Appetite regulating factors in dourado, Salminus brasiliensis: cDNA cloning and effects of fasting and feeding on gene expression.

The dourado, Salminus brasiliensis (Cuvier, 1816) is a freshwater piscivorous Characin native to South American rivers. Owing to the high quality of its flesh and its fast growth, it is the object of both capture fisheries and fish farming. However, very little is known about the endocrine regulation of feeding and metabolism of dourado. In this study, cDNAs for orexin, CART and CCK were isolated in dourado, and their mRNA tissue distributions examined. In order to assess the role of these peptides in the regulation of feeding of dourado, the effects of fasting and feeding on mRNA expression levels of orexin, CART and CCK in the brain as well as CCK in the intestine were assessed. Whereas orexin and CCK have widespread mRNA distributions in the brain and peripheral organs, CART seems to be mostly limited to the brain. Orexin brain expression increased with fasting and displayed periprandial changes, suggesting it is involved in both long- and short-term regulation of feeding and appetite. CART and CCK hypothalamic expressions were not affected by fasting, but displayed periprandial changes with post-feeding decreases, suggesting roles in short-term satiation. CCK expression in the anterior intestine was not affected by fasting and did not display periprandial changes. Overall, our results suggest that orexin, CART and CCK are involved in the physiology of feeding of dourado.

Supplementation with a new trypsin inhibitor from peanut is associated with reduced fasting glucose, weight control, and increased plasma CCK secretion in an animal model.

Ingestion of peanuts may have a beneficial effect on weight control, possibly due to the satietogenic action of trypsin inhibitors. The aim of this study was to isolate a new trypsin inhibitor in a typical Brazilian peanut sweet (paçoca) and evaluate its effect in biochemical parameters, weight gain and food intake in male Wistar rats. The trypsin inhibitor in peanut paçoca (AHTI) was isolated. Experimental diets were prepared with AIN-93G supplemented with AHTI. Animals had their weight and food intake monitored. Animals were anesthetized, euthanized, and their bloods collected by cardiac puncture for dosage of cholecystokinin (CCK) and other biochemical parameters. Supplementation with AHTI significantly decreased fasting glucose, body weight gain, and food intake. These effects may be attributed to increased satiety, once supplemented animals showed no evidence of impaired nutritional status and also because AHTI increased CCK production. Thus, our results indicate that AHTI, besides reducing fasting glucose, can reduce weight gain via food intake reduction.

Effects of fasting and feeding on the brain mRNA expressions of orexin, tyrosine hydroxylase (TH), PYY and CCK in the Mexican blind cavefish (Astyanax fasciatus mexicanus).

The effects of fasting and feeding on the brain expression of orexin (OX), tyrosine hydroxylase (TH), peptide Y (PY) and cholecystokinin (CCK) were examined in the blind cavefish Astyanax fasciatus mexicanus. A 10-days fasting period induced increases in both OX and TH brain mRNA expression but had no effect on PYY and CCK expression. Periprandial changes in expression were seen for OX, TH and PYY but not for CCK. OX brain expression peaked 1h prior to a scheduled meal and decreased 1h post feeding in fed fish. A peak in TH expression was seen 1h post feeding in unfed fish whereas a peak in PYY expression was seen 1h post feeding in fed fish. Our result indicates that brain OX, TH and PYY might be involved in the central regulation of feeding of blind cavefish.

Neuroendocrine system of the digestive tract in Rhamdia quelen juvenile: an immunohistochemical study.

In this work, an immunohistochemical study was performed to determine the distribution and relative frequencies of some neuromodulators of the digestive tract of silver catfish (Rhamdia quelen). The digestive tract of silver catfish was divided into six portions; the oesophagus, stomach, intestine (ascendant, descendant and convoluted segments), and rectum. Immunohistochemical method using a pool of specific antisera against-gastrin, -cholecystokinin-8, -leu-enkephalin, -neuropeptide Y, -calcitonin gene-related peptide (CGRP), and -vasoactive intestinal peptide (VIP) was employed. Immunoreactivity to all antisera was identified in neuroendocrine cells (NECs) localized in the gut epithelium, although no reaction was observed in the oesophagus or stomach. The morphology of NECs immunopositive to each antibody was similar. They were slender in shape, with basally located nucleus, and their main axis perpendicular to the basement membrane. The number of NECs immunoreactive to all antisera was higher in the ascendant and descendant intestine, exhibiting a decreasing trend toward distal segments of the gut. In addition, immunoreactivity to CGRP and VIP was observed in the myenteric plexus and nerve fibers distributed in the mucosal, submucosal and muscular layers. The higher number of immunopositive NECs in the ascendant and descendant intestine may indicate the primary role of these segments in the control of food intake by means of orexigenic and anorexigenic peripheral signals.

Cholecystokinin and hypothalamic corticotrophin-releasing factor participate in endotoxin-induced hypophagia.

Cholecystokinin (CCK) provides a meal-related signal that activates brainstem neurons, which have reciprocal interconnections with the hypothalamic paraventricular nucleus. Neurons that express corticotrophin-releasing factor (CRF) in the hypothalamus possess anorexigenic effects and are activated during endotoxaemia. This study investigated the effects of CCK(1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia and hypothalamic CRF neuronal activation. Male Wistar rats were pretreated with a specific CCK(1) receptor antagonist (devazepide; 1 mg kg(-1); i.p.) or vehicle; 30 min later they received LPS (100 µg kg(-1); i.p.) or saline injection. Food intake, corticosterone responses and Fos-CRF and Fos-α-melanocyte-stimulating hormone (α-MSH) immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase immunoreactivity in the nucleus of the solitary tract (NTS) were evaluated. In comparison with saline treatment, LPS administration decreased food intake and increased plasma corticosterone levels, as well as the number of Fos-CRF and Fos- tyrosine hydroxylase double-labelled neurons in vehicle-pretreated rats; no change in Fos-α-MSH immunoreactivity was observed after LPS injection. In saline-treated animals, devazepide pretreatment increased food intake, but it did not modify other parameters compared with vehicle-pretreated rats. Devazepide pretreatment partly reversed LPS-induced hypophagia and Fos-CRF and brainstem neuronal activation. Devazepide did not modify the corticosterone and Fos-α-MSH responses in rats treated with LPS. In conclusion, the present data suggest that LPS-induced hypophagia is mediated at least in part by CCK effects, via CCK(1) receptor, on NTS and hypothalamic CRF neurons.

Chronic stress sensitizes rats to pancreatitis induced by cerulein: role of TNF-α.

AIM: To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer. METHODS: Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 µg/kg per hour) cerulein stimulation on chronically stressed rats. RESULTS: In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases' activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases'activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues' ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats. CONCLUSION: In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.

Distributions of the endocrine cells in the gastrointestinal tract of nectarivorous and sanguivorous bats: a comparative immunocytochemical study.

The present study was conducted to clarify the influence of feeding habits on regional distribution and relative frequency of endocrine cells secreting cholecystokinin (CCK), gastrin (GAS), serotonin (5-HT) and enteroglucagon (GLUC) in the nectarivorous Anoura geoffroyi and Glossophaga soricina and the sanguivorous Desmodus rotundus bats of the Phyllostomidae family, by specific immunohistochemical methods. The regional distribution and frequency of the different types of endocrine cells varied according to their location in the GIT. 5-HT immunoreactive cells (IR), detected throughout the GIT of three bats, were the most predominant gastrointestinal endocrine cells. GAS-IR cells in A. geoffroyi were found at the base of the pyloric gland, while in G. soricina they could also be observed in the middle to basal portions of the gland. GLUC-IR cells were located in the fundic region of A. geoffroyi, G. soricina and D. rotundus. These endocrine cells were more abundant in the sanguivorous bat. In nectarivorous bats were compared to sanguivorous bat, which differ in dietary habits, difference in the distribution and relative frequency of gut endocrine cells would be predicted. The absence of some, and decrease in frequency of other, gastrointestinal endocrine cells may reflect, in part, its interspecies differences or dietary habits.

Wiring and volume transmission in rat amygdala. Implications for fear and anxiety.

The amygdala plays a key role in anxiety. Information from the environment reaches the amygdaloid basolateral nucleus and after its processing is relayed to the amygdaloid central nucleus where a proper anxiogenic response is implemented. Experimental evidence indicates that in this information transfer a GABAergic interface controls the trafficking of impulses between the two nuclei. Recent work indicates that interneuronal communication can take place by classical synaptic transmission (wiring transmission) and by volume transmission in which the neurotransmitter diffuses and flows through the extracellular space from its site of release and binds to extrasynaptic receptors at various distances from the source. Based on evidence from our laboratory the concept is introduced that neurotransmitters in the amygdala can modulate anxiety involving changes in fear learning and memories by effects on receptor mosaics in the fear circuits through wiring and volume transmission modes of communication.

Peripheral participation of cholecystokinin in the morphine-induced peripheral antinociceptive effect in non-diabetic and diabetic rats.

The effects of cholecystokinin (CCK-8) and the CCK receptor antagonist proglumide, on antinociception induced by local peripheral (subcutaneous) injected morphine in non-diabetic (ND) and streptozotocin-induced diabetic (D) rats, were examined by means of the formalin test. Morphine induced dose-dependent antinociception both in ND and D rats. However, in D rats, antinociceptive morphine potency was about twofold less than in ND rats. Pre-treatment with CCK-8 abolished the antinociceptive effect of morphine in a dose-dependent manner in both groups of rats. Additionally, proglumide enhanced the antinociceptive effect induced by all doses of morphine tested. Both CCK-8 and proglumide had no effect on flinching behaviour when given alone to ND rats. Unlike ND rats, in D rats proglumide produced dose-dependent antinociception and CCK-8 enhanced formalin-evoked flinches, as observed during the second phase of the test. In conclusion, our data show a decrease in peripheral antinociceptive potency of morphine when diabetes was present. Additionally, peripheral CCK plays an antagonic role to the peripheral antinociceptive effect of morphine, additional to the well known CCK/morphine interaction at spinal and supraspinal level.

Involvement of dorsolateral periaqueductal gray cholecystokinin-2 receptors in the regulation of a panic-related behavior in rats.

Cholecystokinin (CCK) has been implicated in anxiety disorders. The midbrain periaqueductal gray (PAG), which modulates anxiety and panic reactions, contains CCK-immunoreactive fibers and CCK(2) receptors. The present study investigated the involvement of CCK(2) receptors of the PAG dorsolateral subdivision (dlPAG) in the regulation of inhibitory avoidance and escape, two defensive behaviors that have been related in terms of psychopathology to generalized-anxiety and panic disorders, respectively. Male Wistar rats were microinjected in the dlPAG with the CCK(2) receptor agonist cholecystokinin-tetrapeptide (CCK-4; 0.08-0.32 nmol/0.2 microL), the CCK(2) receptor antagonist LY-225910 (0.05-0.20 nmol/0.2 microL) or LY-225910 prior to CCK-4. Inhibitory avoidance and escape behaviors were evaluated in the elevated T-maze. Whereas CCK-4 facilitated escape, indicating a panic-like action, LY-225910 had the opposite effect. Pretreatment with a non-effective dose of LY-225910 prevented the panic-eliciting action of CCK-4. Neither CCK-4 nor LY-225910 affected inhibitory avoidance acquisition. The present results substantiate the view that dlPAG CCK(2) receptors modulate panic-related behaviors.

Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats.

The ventrolateral periaqueductal gray (PAG) is a key structure for the development of opioid tolerance. An increased activity of 'anti-opioids' like cholecystokinin (CCK) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of PAG-located CCK in the opioid tolerance induced by repeated microinjections of morphine (MOR) into PAG. Male rats were implanted with chronic guide cannulae aimed at the PAG. Microinjection of MOR (0.5 microg in 0.5 microl) into PAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the non-selective CCK receptor antagonist proglumide (PRO), (0.4 microg in 0.5 microl) into the same PAG site, the microinjections of MOR always produced antinociception and did not induce tolerance. If PRO microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single PRO microinjection into the same PAG site was enough to restore the antinociceptive effect of MOR. On the other hand, if CCK (1 ng in 0.5 microl) was microinjected into PAG, then MOR microinjection administered 15 min later into the same PAG site did not elicit antinociception. These results show that CCK has anti-opioid activity in PAG and that tolerance to MOR in PAG can be prevented or reversed if CCK receptors are blocked with PRO. Finally, opioid tolerance induced by repeated systemic MOR injections (5mg/kg intraperitoneal ) was reversed by a single microinjection of PRO into PAG. This emphasizes the central importance of PAG in the MOR/CCK interactions that lead to opioid tolerance.

Puerperal blockade of cholecystokinin (CCK1) receptors disrupts maternal behavior in lactating rats.

Blockade of cholecystokinin (CCK) receptors potentiates the morphine-induced disruption of maternal behavior. The present study was undertaken to determine whether treatment with lorglumide, a CCK1 antagonist during late pregnancy and early lactation can influence the maternal behavior during lactation. A possible influence of this treatment on general activity was also assessed. Twenty-seven female Wistar rats were pretreated with lorglumide (1.0mg/kg/day; sc) or saline for seven days, starting on the 17th d of pregnancy. After the withdrawal of this treatment, animals were acutely challenged with saline on day 5 and with morphine sulfate (3.0mg/kg; sc) on days 6,10, and 17 of lactation. Groups were pretreated with saline and challenged with saline (group SS) and morphine (group SM), pretreated with lorglumide and challenged with saline (group LS) and morphine (group LM). Animals were also tested for general activity on days 25 and 33 postpartum after an acute challenge with saline and morphine, respectively. Maternal behavior testing began 30 min after the acute injections at which time pups were placed throughout each mother's cage. Latencies for pup retrieval, grouping, crouching and for full maternal behavior responses were scored. Lorglumide pretreatment inhibited maternal behavior of LS vs SS group and potentiated the morphine-induced disruption of this behavior in all days of test (LM vs SM group). No significant differences were found in general activity on days 25 and 33 postpartum. These data suggest that blockade of CCK1 receptors during puerperal period has long-term implications for maternal behavior.

Coexistence of neuropeptides and their possible relation to neuritic regeneration in primary cultures of magnocellular neurons isolated from adult rat supraoptic nuclei.

The coexistence of vasopressin (VP), oxytocin (OXY), galanin (GAL) and cholecystokinin (CCK) and the synthesis of GAL and CCK during neuritic regeneration was investigated in cultured magnocellular neurons, isolated from adult rat supraoptic nuclei. Double-labelling immunofluorescence was performed after 7 days of culture using primary antibodies for VP, OXY, GAL and CCK (paired in all possible combinations) and secondary antibodies labelled with either fluorescein or rhodamine. Confocal laser scanning microscopy revealed the coexistence of the mentioned peptides in all possible combinations, an unexpected result considering that the only combinations observed in tissue sections are VP-GAL and OXY-CCK. Freshly dispersed cells were devoid of any neuritic processes and showed a very poor immunocytochemical staining reaction for GAL and CCK. In contrast, neurons cultured for 7, 12 and 21 days showed many neurites and a strong immunoreactivity for GAL and CCK indicative of an increased synthesis of both peptides in the regenerating neurons. This increased synthetic activity is consistent with transient upregulation of these peptides observed in situ after hypophysectomy by other authors. The results suggest that the upregulation of GAL and CCK is functionally related to the neuronal regeneration processes observed during culture and that the 'uncommon' coexistences as well as the prolonged sythesis of GAL and CCK may be due to the lack of environmental inputs, which normally regulate the expression and up- and downregulation of these peptides in vivo.

NOS is present in the brain of Triatoma infestans and is colocalized with CCK.

Immunohistochemistry was used to establish the presence of nitric oxide synthase (NOS) in the central nervous system of Triatoma infestans which is the main vector for Chagas' disease in Argentina and neighbouring countries. In addition, we have investigated the presence of cholecystokinin (CCK) and studied the possible coexistence of these molecules. The results show NOS-like immunoreactivity (LI) in neurones of the soma rind of the protocerebrum, the optic lobe and in the lateral part of the sensory deutocerebrum with a few cells in the suboesophageal and the prothoracic ganglia. The distribution of CCK-LI was similar to that of NOS-LI and in several areas both molecules coexisted in neurones and fibres. The results suggest that nitric oxide may act as a neurotransmitter in the brain of insects.