Vasoactive Intestinal Peptide-immunoreactive Neurons Research Articles

Distribution of hypothalamic vasoactive intestinal peptide immunoreactive neurons in the male native Thai chicken

Avian prolactin (PRL) secretion is under stimulatory control by the PRL-releasing factor (PRF), vasoactive intestinal peptide (VIP). The neuroendocrine regulation of the avian reproductive system has been extensively studied in females. However, there are limited data in males. The aim of this study was to elucidate the VIPergic system and its relationship to PRL and testosterone (T) in the male native Thai chicken. The distributions of VIP-immunoreactive (-ir) neurons and fibers were determined by immunohistochemistry. Changes in VIP-ir neurons within the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) areas were compared across the reproductive stages. Plasma levels of PRL and T were determined by enzyme-linked immunosorbent assay and then compared across the reproductive stages. The results revealed that the highest accumulations of VIP-ir neurons were concentrated only within the IH-IN, and VIP-ir neurons were not detected within other hypothalamic nuclei. Within the IH-IN, VIP-ir neurons were low in premature and aging males and markedly increased in mature males. Changes in VIP-ir neurons within the IH-IN were directly mirrored with changes in PRL and T levels across the reproductive stages. These results suggested that VIP neurons in the IH-IN play a regulatory role in year-round reproductive activity in males. The present study also provides additional evidence that VIP is the PRF in non-seasonal, continuously breeding equatorial species.

Changes in Enteric Neurons of Small Intestine in a Rat Model of Irritable Bowel Syndrome with Diarrhea.

Background/AimsPhysical and/or emotional stresses are important factors in the exacerbation of symptoms in irritable bowel syndrome (IBS). Several lines of evidence support that a major impact of stress on the gastrointestinal tract occurs via the enteric nervous system. We aimed to evaluate histological changes in the submucosal plexus (SMP) and myenteric plexus (MP) of the distal ileum in concert with the intestinal motor function in a rat model of IBS with diarrhea.MethodsThe rat model was induced by heterotypic chronic and acute stress (CAS). The intestinal transit was measured by administering powdered carbon by gastric gavage. Double immunohistochemical fluorescence staining with whole-mount preparations of SMP and MP of enteric nervous system was used to assess changes in expression of choline acetyltransferase, vasoactive intestinal peptide, or nitric oxide synthase in relation to the pan neuronal marker, anti-Hu.ResultsThe intestinal transit ratio increased significantly from control values of 50.8% to 60.6% in the CAS group. The numbers of enteric ganglia and neurons in the SMP were increased in the CAS group. The proportions of choline acetyltransferase- and vasoactive intestinal peptide-immunoreactive neurons in the SMP were increased (82.1 ± 4.3% vs. 76.0 ± 5.0%, P = 0.021; 40.5 ± 5.9% vs 28.9 ± 3.7%, P = 0.001), while nitric oxide synthase-immunoreactive neurons in the MP were decreased compared with controls (23.3 ± 4.5% vs 32.4 ± 4.5%, P = 0.002).ConclusionsThese morphological changes in enteric neurons to CAS might contribute to the dysfunction in motility and secretion in IBS with diarrhea.

Vasoactive intestinal peptide and its role in continuous and seasonal reproduction in birds

Native Thai chicken, an equatorial species breeds throughout the year, whereas turkeys are seasonal temperate zone breeder whose reproductive cycle is terminated by the onset of photorefractoriness. This study investigated VIPergic activity throughout a reproductive cycle in both species, hypothesizing that the differential expression of vasoactive intestinal peptide (VIP) would provide an insight into the differing reproductive strategies of the two species. Distribution of VIP neurons in the native Thai chicken and a comparison of VIPergic activity in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) were investigated. VIP immunoreactivity was found throughout the native Thai chicken brain, predominantly located within the IH–IN. The pattern of VIP distribution in the native Thai chicken supports the findings reported in temperate zone species. Unlike the turkey, where there is a dissociation between VIPergic activity and prolactin levels during photorefractoriness, in the native Thai chicken, which do not express photorefractoriness, changes in VIP immunoreactive (VIP-ir) neurons within the IH–IN were directly correlated with prolactin throughout the reproductive cycle. VIPergic activity reached its lowest level after hatching of the chicks in the native Thai chicken, while in the turkey VIPergic activity was lowest only after exposure to a short day photoperiod and the acquisition of photosensitivity. This suggests that VIP neurons in the IH–IN may play a pivotal role in regulating the reproductive cycle and its differential expression following hatching of the young may, in part, account for the difference in reproductive mode between equatorial, continually breeding, non-photoperiodic birds and seasonally breeding, photoperiodic birds.

Differential responses of VIPergic and nitrergic neurons in paediatric patients with Crohn's disease

Inflammatory bowel disease is a recurrent intestinal inflammatory disorder that in adults has been associated with changes in enteric nervous system neuropeptide expression. The aim of the present study was to determine whether similar changes were observed in paediatric Crohn's disease. The distribution of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) was determined in colonic tissues from children with ileo-colonic (n=4) and colonic (n=3) Crohn's disease. The submucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons (margin, 48% vs. inflamed tissue, 82% of HuC/D positive neurons). The density of submucosal plexus nNOS immunoreactive neurons was too low to be reliably quantified. Using the pan-neuronal marker HuC/D, no significant difference in numbers of HuC/D positive submucosal neurons was evident except where neurons were normalized to length of tissue (margins, 3.6+/-0.7 vs. inflamed tissue, 4.0+/-0.6 neurons/ganglia, p=0.33; margins, 2.7+/-0.4 vs. inflamed tissue, 5.7+/-1.2, neurons/mm, p=0.03). In the myenteric plexus, there was a significant increase in the percent of NOS neurons (38% vs. 82% of HuC/D positive neurons) while there was no significant difference in percent of VIP neurons (4% vs. 8%). No difference in number of HuC/D positive myenteric neurons among margin and inflamed tissues was observed (margin, 12.2+/-3.0 vs. inflamed tissue, 12.5+/-5.1 neurons/ganglia, p=0.50; margins 9.1+/-2.1 vs. inflamed tissue, 13.7+/-2.3 neurons/mm, p=0.11). These data demonstrate that inflammation is associated with a differential expression of VIP and nNOS neuronal subpopulations within the two major enteric plexi, likely due to phenotypic switch. Such changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease.

Characteristics of galanin and vasoactive intestinal peptide immunoreactivity in the rat amygdala complex

Morphological features and morphometric parameters of galanin (GAL) and vasoactive intestinal peptide (VIP) immunoreactive neurons and neuronal fibres were studied in all nuclei of adult male rat amygdala. After perfusion and fixation, rat brains were immunohistochemically stained with antibodies against GAL and VIP and then visualized by avidin-biotin-peroxidase complex. The greatest number of galanin-immunoreactive neurons were identified in the medial part of the central nucleus and in the dorsal part of the medial nucleus. In the first case, most neurons were bipolar (37%), and in the second, they were ovoid (45%). GAL-immunoreactive fibers were identified in the medial nucleus, "bed nucleus" of the accessory olfactory tract, fiontal cortical nucleus, amygdalo-hippocampal area and basolateral nucleus. VIP-immunoreactive neurons were diffusely distributed in more nuclei than the previous, mostly in the lateral, basolateral, and basomedial nucleus. They were mostly ovoid (40%). VIP-immunoreactive fibers were observed in the lateral part oJ'the central nucleus, while long and radially oriented fibers were present in the frontal and dorsal cortical nucleus. By distribution analysis of GAL and VIP immunoreactive neurons and fibers, and according to literature data, it can be assumed that the medial part of the central nucleus receives VIP fibers from other parts of the amygdaloid body, and then sends GAL fibers to the medial nucleus.

The distribution of purine P2X(2) receptors in the guinea-pig enteric nervous system.

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.

Expression of Immunoreactivity to Neurokinin-1 Receptor by Subsets of Cranial Parasympathetic Neurons: Correlation with Neuropeptides, Nitric Oxide Synthase, and Pathways

We examined the patterns of coexistence of immunoreactivity to the neurokinin-1 (NK1) tachykinin receptor, nitric oxide synthase, and neuropeptides in the sphenopalatine and otic ganglia of guinea pigs using a combination of multiple-labeling immunohistochemistry and pathway tracing in vitro. Most neurons had immunoreactivity to vasoactive intestinal peptide (85–96%) and neuropeptide Y (60%). Subpopulations of vasoactive intestinal peptide-immunoreactive neurons also had immunoreactivity to nitric oxide synthase (37–48%) or enkephalin (25–35%), but these formed mutually exclusive populations. Almost all neurons expressing NK1 receptor immunoreactivity contained immunoreactivity to enkephalin, vasoactive intestinal peptide, and neuropeptide Y, but not nitric oxide synthase. Using a combination of retrograde axonal tracing and axonal crushing, we found that most neurons with immunoreactivity to nitric oxide synthase projected along the nasopalatine and ethmoidal nerves to the nasal mucosa. In contrast, most neurons with immunoreactivity to enkephalin followed the zygomatic nerve to the facial skin and lacrimal gland. Based on their peptide content, we conclude that the neurons with immunoreactivity to enkephalin and NK1 receptor projected selectively to the skin. In both the sphenopalatine and the otic ganglia, about half of the neurons with NK1 receptor immunoreactivity were surrounded by varicose nerve fibers with substance P immunoreactivity. Many of these fibers are likely to have originated in the trigeminal ganglion. Taken together, these observations establish a strong anatomical basis for a range of interactions between trigeminal and cranial parasympathetic pathways that may underlie pathophysiological conditions such as trigeminal neuralgia.

Differences in mediator of nonadrenergic, noncholinergic relaxation of the distal colon between Wistar–ST and Sprague–Dawley strains of rats

Participation of nitric oxide and vasoactive intestinal peptide (VIP) in electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle and in balloon distension-induced descending NANC relaxation of circular muscle were studied in the distal colon of Wistar–ST and Sprague–Dawley rats. The extent of the nitric oxide-mediated component was approximately 50% in longitudinal and circular muscle of Sprague–Dawley rats, whereas this component was absent in both muscles of Wistar–ST rats. The extent of the VIP-mediated component was approximately 40% in longitudinal muscle of Wistar–ST rats and circular muscle of Sprague–Dawley rats, whereas this component was absent in circular muscle of Wistar–ST rats and longitudinal muscle of Sprague–Dawley rats. In circular muscle of Sprague–Dawley rats, in which participation of both nitric oxide and VIP in the relaxation was suggested, inhibition of descending relaxation by N G-nitro- l-arginine ( l-NOARG) together with VIP-(10–28) was similar to that by either of the antagonists, and exogenous VIP-induced relaxation was not affected by l-NOARG, but exogenous nitric oxide-induced relaxation was partly inhibited by VIP-(10–28). These results suggest a linkage of the pathways mediated by nitric oxide and VIP. In the immunohistochemical studies, nitric oxide synthase or VIP immunoreactive neurons were seen in the ganglia, primary internodal strands of the myenteric plexus and in the circular muscle layer. However, the overall appearance of immunoreactive cell bodies in the myenteric plexus and the numbers of immunoreactive fibers in the circular muscle layer appeared to be similar in Wistar–ST and Sprague–Dawley rats. These results suggest that mediators of NANC relaxation in the distal colon are different in different strains of rats, i.e., Wistar–ST and Sprague–Dawley, although no such difference was seen in immunohistochemical studies.

Partial and complete sciatic nerve injuries induce similar increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in primary sensory neurons and their central projections

Partial nerve injury is more likely to cause neuropathic pain than complete nerve injury. We have compared the changes in neuropeptide expression in primary sensory neurons which follow complete and partial injuries to determine if these might be involved. Since more neurons are damaged by complete injury, we expected that complete sciatic nerve injury would simply cause greater increases in neuropeptide Y and vasoactive intestinal peptide than partial injury. We examined neuropeptide Y and vasoactive intestinal peptide immunoreactivities in L4 and L5 dorsal root ganglia, the dorsal horn of L4–L5 spinal cord, and the gracile nuclei of rats killed 14 days after unilateral complete sciatic nerve transection, partial sciatic nerve transection and chronic constriction injury of the sciatic nerves. In all three groups of rats, neuropeptide Y- and vasoactive intestinal peptide-immunoreactive neurons were increased in the ipsilateral L4 and L5 dorsal root ganglion when compared with the contralateral side. Most neuropeptide Y-immunoreactive neurons were of medium and large size, but a few were small. Neuropeptide Y-immunoreactive axonal fibers were increased from laminae I to IV, and vasoactive intestinal peptide-immunoreactive axonal fibers were increased in laminae I and II, of the ipsilateral dorsal horn of L4–L5 spinal cord. The increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in the dorsal horn were similar among the three groups. However, only after constriction injury were some vasoactive intestinal peptide-immunoreactive neurons seen in the deeper laminae of the ipsilateral dorsal horn. Robust neuropeptide Y-immunoreactive axonal fibers and some neuropeptide Y-immunoreactive cells were seen in the ipsilateral gracile nuclei of all three groups of animals, but neuropeptide Y-immunoreactive cells were more prominent after constriction injury. Contrary to our expectations, partial and complete sciatic nerve injuries induced similar increases in neuropeptide Y and vasoactive intestinal peptide in lumbar dorsal root ganglion neurons and their central projections in the dorsal horn and the gracile nuclei two weeks after injury. Some neurons whose axons were spared by partial injury may also increase neuropeptide Y or vasoactive intestinal peptide expression. Altered neuropeptide release from these functional sensory neurons may play a role in neuropathic pain.

Vasoactive intestinal peptide expression in enteric neurons is upregulated by both colchicine and axotomy

Axotomy is known to induce changes in neuropeptide expression in several types of neurons. Colchicine blocks the axonal transport and may mimic axotomy. The effects of colchicine-treatment and axotomy (local nerve crush by clamping of the gut) on enteric neurons expressing vasoactive intestinal peptide, neuropeptide Y and nitric oxide synthase were studied in rat small intestine by immunocytochemistry and in situ hybridization. Colchicine treatment significantly increased the number of submucous and myenteric neurons expressing vasoactive intestinal peptide and its mRNA. In contrast, an increase in the number of neuropeptide Y or nitric oxide synthase expressing neurons could not be detected. Axotomy markedly increased the number of myenteric vasoactive intestinal peptide-immunoreactive neurons in the segment located orally to the lesion, but not in the segment anally to the lesion, whereas that of nitric oxide synthase and neuropeptide Y expressing neurons was not affected. Double immunostaining revealed that the myenteric neurons containing nitric oxide synthase were induced by colchicine and axotomy to express vasoactive intestinal peptide. The present data indicate that colchicine and axotomy may induce marked changes in the neuropeptide expression of enteric neurons.

Ontogeny of radial glia, astrocytes and vasoactive intestinal peptide immunoreactive neurons in hamster suprachiasmatic nucleus

Circadian rhythmicity of rodents is a property of the suprachiasmatic nucleus (SCN). Such rhythmicity can be demonstrated in the prenatal SCN, yet there is little information about the cells in which rhythmicity is generated. The present study was performed to discover the developmental relationships of SCN glial cells and a class of identifiable SCN neurons. Toward this end, vimentin- (VIM), glial fibrillary acidic protein- (GFAP) and vasoactive intestinal peptide- (VIP) immunoreactivity were investigated in SCN radial glia, astrocytes and neurons, respectively. VIP-IR first appears at embryonic day 13 (E13) and is clearly identifiable in neurons at E14. Substantial axon extension begins at E15 and the postnatal day 10 (P10) SCN is adult-like. VIM-IR radial glia fill the SCN region at E13, but by P0, most are absent. On P3, the remaining processes are beaded suggesting degeneration. The first GFAP-IR elements are visible on E15 with a few clear astrocytes present at P0. The number of astrocytes lateral to and in the SCN continues to increase during the postnatal period achieving an adult-like appearance by P21. The data do not support the view that prenatal circadian rhythmicity is derived from astrocytes. VIP-IR neurons are apparently present sufficiently early to be part of the rhythm generating mechanism. These issues are discussed in the context of development of the SCN.

Inhibition of axoplasmic transport in the rat vagus nerve alters the numbers of neuropeptide and tyrosine hydroxylase messenger RNA-containing and immunoreactive visceral afferent neurons of the nodose ganglion.

Previous work showed that axotomy-induced deafferentation of the placode-derived visceral afferent neurons of the nodose ganglion altered their expression of some neuropeptides and tyrosine hydroxylase. The present studies were designed to selectively evaluate the loss of axonal transport on the numbers of vasoactive intestinal polypeptide, tyrosine hydroxylase, and calcitonin gene-related peptide mRNA-containing and immunoreactive neurons in the nodose ganglion of the adult rat. Vinblastine (0.15 mM) application to the cervical vagus nerve was used to block axonal transport between ganglionic perikarya and peripheral targets. In situ hybridization histochemistry with 35S-labeled oligonucleotide probes was used to both quantify the number of mRNA-containing neurons and to assess the density of mRNA expression per neuron, and immunocytochemistry was used to visualize the number of immunoreactive neurons. The efficacy of vinblastine to inhibit axonal transport was verified by evaluating the build-up of calcitonin gene-related peptide immunoreactive in the vagus nerve immediately rostral to the site of drug application. The absence of vinblastine-induced neuronal damage was verified by the relative absence of degenerating nerves in the vagus nerve caudal to the site of drug application. Vinblastine treatment of the vagus nerve increased the numbers of vasoactive intestinal peptide mRNA-containing neurons and vasoactive intestinal peptide-immunoreactive neurons in the nodose ganglion at three, seven and 14 days, and increased the numbers of calcitonin gene-related peptide mRNA-containing and calcitonin gene-related peptide-immunoreactive neurons in the nodose ganglion at one, three and seven days. The average labeling density of vasoactive intestinal peptide mRNA-containing neurons was also increased following vinblastine treatment. Vinblastine treatment of the cervical vagus nerve, however, led to the appearance of low-labeling density calcitonin gene-related peptide mRNA-neurons and resulted in reduction of the average labeling density for calcitonin gene-related peptide mRNA-containing neurons. In contrast, application of vinblastine to the cervical vagus nerve, decreased the number of tyrosine hydroxylase mRNA-containing and tyrosine hydroxylase-immunoreactive neurons in the nodose ganglion. In summary, inhibition of the axoplasmic transport between the periphery and the visceral sensory perikarya appeared to alter vasoactive intestinal peptide, calcitonin gene-related peptide, and tyrosine hydroxylase expression and content in visceral sensory neurons of the nodose ganglion. These data suggest the presence of an axonally transported influence on the regulation of neuropeptide and neurotransmitter enzyme synthesis in mature placode-derived visceral sensory neurons.

Plasticity of tyrosine hydroxylase and vasoactive intestinal peptide messenger RNAs in visceral afferent neurons of the nodose ganglion upon axotomy-induced deafferentation

The nodose ganglion contains placode-derived visceral sensory neurons of the vagus nerve. Previous study showed that axotomy-induced deafferentation reduced the number of tyrosine hydroxylaseimmunoreactive and increased the number of vasoactive intestinal peptide-immunoreactive neurons in the ganglion. The present study was conducted to determine whether the changes in neuropeptide/neurotransmitter enzyme content are associated with changes in the expression of tyrosine hydroxylase and vasoactive intestinal peptide messenger RNAs in the nodose ganglion. We used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes for tyrosine hydroxylase and vasoactive intestinal peptide precursor messenger RNAs. Peripheral axotomy of visceral afferent inputs reduced tyrosine hydroxylase messenger RNA and increased vasoactive intestinal peptide messenger RNA expression in neurons of the nodose ganglion of the rat. The number of tyrosine hydroxylase messenger RNA-containing neurons was significantly reduced at three, seven and 14 days after axotomy-induced deafferentation compared with intact and sham-operated controls. Labeling density of tyrosine hydroxylase messenger RNA-containing neurons was significantly reduced at three and seven days. Conversely, the number of vasoactive intestinal peptide messenger RNA-containing neurons increased significantly at three, seven and 14 days, while the labeling density of vasoactive intestinal peptide messenger RNA-containing neurons also increased at one, three, seven and 14 days. The results of the present study indicate that the axotomy-induced down-regulation of tyrosine hydroxylase and up-regulation of vasoactive intestinal peptide in the neurons of the nodose ganglion are associated with changes in their messenger RNAs in response to axotomy-induced deafferentation.

Organotypic slice culture of the rat suprachiasmatic nucleus: Sustenance of cellular architecture and circadian rhythm

The suprachiasmatic nucleus of the mammalian brain is thought to be the anatomical locus of circadian rhythms. To examine the functional organization of the suprachiasmatic nucleus in vitro with intact intercellular connections for a prolonged period, we have established an organotypic slice culture system using a roller-tube technique. Brain slices (400 μm in thickness) containing the bilateral suprachiasmatic nuclei, were obtained from newborn rats at four to seven days old and were maintained in vitro for more than three weeks. During this three-week period, the slices flattened to one to three cell layers and two tightly packed neuronal cell-masses (neuronal zones), with diameters of about 1 mm were formed, which were surrounded by a peripheral glial cell-dispersed zone. In the neuronal cell zones, peptides and their messenger RNAs were found cytochemically with characteristic patterns similar to the suprachiasmatic nucleus in the brain. In situ hybridization and immunocytochemistry showed that vasoactive intestinal peptide messenger RNA expressing and vasoactive intestinal peptide-immunoreactive neurons were detected predominantly in the ventrolateral part of the neuronal zones in the suprachiasmatic nucleus slice culture. Vasopressin messenger RNA-expressing and vasopressin-immunoreactive cells were localized in the dorsomedial neuronal zones near the ependymal cell zone. The distribution of cell bodies and fibers containing these neuropeptides and their messenger RNAs in the neuronal zones of suprachiasmatic nucleus organotypic slice culture were similar to that of the suprachiasmatic nucleus in vivo. This suggests that the suprachiasmatic nucleus in these organotypic slice cultures retains the biological characteristics of these cells in vivo as the cells did develop, form compact neuronal masses and did establish connections. To examine the possibility that suprachiasmatic nucleus neurons in slice cultures show a persistent rhythmic activity, we also measured the amount of vasopressin released into the culture medium. Sampling at 4-h intervals combined with enzyme immunoassay revealed that vasopressin concentration in the medium embracing suprachiasmatic nucleus slice cultures fluctuated with a period of approximately 24 h. The present findings suggest that the intranuclear neuronal networks of the suprachiasmatic nucleus are maintained in vitro for a long duration and that organotypic cultures of the suprachiasmatic nucleus produce and release bioactive substances in an oscillating manner. The suprachiasmatic nucleus in slice cultures may be useful for future analysis of circadian rhythms in vitro.

Neuronal subtypes and anatomic asymmetry: changes in neuronal number and cell-packing density

The combined volume of an asymmetric cytoarchitectonic area is smaller than that of symmetric homologs. Asymmetry reflects fewer numbers of neurons in the smaller of the two sides. In this study we examined two types of neurons to check whether lateral differences in neuronal numbers affect different types of neurons comparably in the neocortex of the rat. As with overall neuronal numbers, both parvalbumin-immunoreactive (mostly long projection) neurons, and vasoactive intestinal peptide-immunoreactive interneurons increase in number in the larger of the two sides. Moreover, the concentration of parvalbumin- but not vasoactive intestinal peptide-immunoreactive neurons also increases on the larger side. Thus, there may be qualitative as well as quantitative differences in the connectivity of the larger side of an asymmetric architectonic region.

The suprachiasmatic nucleus of the mink (Mustela vison): apparent absence of vasopressin-immunoreactive neurons.

The hypothalamic suprachiasmatic nucleus is centrally involved in generation of several circadian rhythms. Neurons of the mammalian suprachiasmatic nucleus express a number of neuropeptides including vasopressin. The suprachiasmatic nucleus of the mink (Mustela vison) is easily distinguished from neighbouring hypothalamic areas and the underlying optic chiasm as a small nucleus containing densely packed parvocellular neurons. A dorsal and ventral subdivision were clearly recognized within the midportion and caudal part of the nucleus. Using immunohistochemistry, we have identified vasopressin-, neurophysin-, and vasoactive intestinal peptide-immunoreactive neuronal elements in the hypothalamus of the mink. Vasoactive intestinal peptide-immunoreactive neurons can be observed in the ventral aspect of the suprachiasmatic nucleus, but to our surprise, no vasopressin immunoreactive perikarya are found within the suprachiasmatic nucleus, this absence being independent of the experienced annual cycle. The hypothalamic paraventricular and supraoptic nuclei contain large numbers of vasopressin-, neurophysin- and vasoactive intestinal peptide-immunoreactive magnocellular neurons with extensive projections towards the infundibulum and neurohypophysis. A comparative analysis of the distribution of vasopressin-immunoreactive elements in a number of conventional laboratory animals has demonstrated that, in contrast to the rat, golden hamster and Mongolian gerbil, neither vasopressin-containing perikarya in the suprachiasmatic nucleus nor fine calibered immunoreactive fibres entering the adjacent subparaventricular zone are present in the mink. The mink is a photodependent seasonal breeder, and thus vasopressin-immunoreactive neurons in the suprachiasmatic nuclei may not be essential for the photoperiodic regulation of reproduction and seasonal events experienced by this species.

Vasoactive intestinal peptide immunoreactive neurons in the rat suprachiasmatic nucleus demonstrate diurnal variation

The suprachiasmatic nucleus (SCN) of mammals is considered to be a circadian oscillator and it also demonstrates circadian rhythmicity of its multiple unit activity. A number of neuropeptides have been found in the SCN. Vasoactive intestinal peptide (VIP)- and vasopressin-containing neurons comprise large populations of these cells and have a distinct distribution within the nucleus. Therefore we attempted to examine whether the VIP neurons show a diurnal alteration of their immunoreactivity by combined immunocytochemistry and color image analysis. Our results demonstrate that VIP-like immunoreactive neurons show a diurnal change in the amount of immunoreactivity. Immunoreactivity was most intense in the sections from rats maintained in the cyclic photoperiod and sacrificed at 02.00 h and weakest in teh SCN from animals sacrificed at 14.00 h. We considered that VIP-like immunoreactive neurons showed diurnal variation of VIP synthesis depending strongly on the light from the retina.

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

The gross morphology and growth patterns of substance P, enkephalin-, somatostatin- and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P- and enkephalin-immunoreactive neurons were more abundant than somatostatin- and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.

Antibodies to fragments of provasoactive intestinal peptide reveal subpopulations of vasoactive intestinal peptide containing neurons in the rat gut

The cellular origin of peptides derived from preprovasoactive intestinal peptide has been studied in rat stomach and ileum. Antisera specific for the C-terminal regions of the N-terminal flanking peptide (preprovasoactive intestinal peptide 22–80), bridging peptide (preprovasoactive intestinal peptide 111–124), C-terminal flanking peptide (preprovasoactive intestinal peptide 156–170) and vasoactive intestinal peptide were used in immunohistochemical studies on sections and whole mounts. All four antisera stained nerve fibres and cell bodies in the stomach and intestine. However, there were distinct differences in the pattern of colocalization of peptides derived from provasoactive intestinal peptide. In the sub-mucous plexus of the ileum virtually 100% of neurons reacting with vasoactive intestinal peptide antibodies also reacted with antibodies to the other three peptides. In contrast, in the stomach, while all vasoactive intestinal peptide-immunoreactive neurons of the myenteric plexus contained C-terminal flanking peptide- and bridging peptide-like immunoreactivity, only 50% of these cells reacted with the antiserum to N-terminal flanking peptide. The data indicate that in a population of neurons in the myenteric plexus of the rat stomach, preprovasoactive intestinal peptide is processed in such a way that the antigenic determinant of the N-terminal flanking peptide is not produced. In a second population of enteric neurons in the stomach and in the intestine, it appears that processing of preprovasoactive intestinal peptide results in the production of peptides reacting with antibodies to vasoactive intestinal peptide, the flanking and bridging peptides.