Vasopressin-like Immunoreactive Research Articles

VIP-like Immunoreactive and Vasopressin-like Immunoreactive Neuronal Elements in the Suprachiasmatic Nucleus of Japanese Monkey (Macaca fuscata): Light and Electron Microscopic Immunocytochemical Study

VIP-like immunoreactive (VIP-LIR) and vasopressin-like immunoreactive (VP-LIR) neuronal elements located in the monkey suprachiasmatic nucleus (SCN) were investigated by light and electron microscopic immunocytochemistry. Both populations of neurons in the monkey SCN show a similar distribution to those of the rat except for some subtle differences. In addition to the ordinary distribution of VIP-LIR and VP-LIR neurons, VIP-LIR neurons of similar or some smaller size to those in the ventrolateral portion of the nucleus were also observed in the periventricular nucleus adjacent to the third ventricle. VP-LIR neurons with similar size and either strong or weak immunoreactivity compared with those located in the dorsomedial area were detected in the periventricular nucleus in which magnocellular VP-LIR neurons with strong immunoreactivity were distributed. Both VIP-LIR and VP-LIR neuronal perikarya were found to contain well developed cell organelles such as rER and mitochondria as well as immunoreactive dense granules. VIP-LIR neuronal perikarya were generally surrounded by astroglial processes. However, non-immunoreactive axons were frequently found to make synaptic contact with VIP-LIR dendrites or dendritic spines. Thus the VIP-LIR neurons are thought to receive neuronal inputs from other parts of CNS mainly from the dendrites or dendritic spines. VIP-LIR and VP-LIR axons were seen to form synapses on non-immunoreactive dendrites. VIP-LIR axons were also found to form axo-axonic synapses on non-immunoreactive axons. Synaptic contacts between VIP-LIR neuronal elements were also occasionally observed. The present study has demonstrated that both the VIP-LIR and VP-LIR neurons are at least partly intrinsic neurons in the SCN, and also projecting elements to the other neurons of the CNS.

Differential regulation of a homologous peptidergic neuron in grasshoppers: evolution of a neural circuit

The vasopressin-like immunoreactive (VPLI) neurons of grasshoppers have paired cell bodies in the suboesophageal ganglion and both anterior and posterior running axons. In non-oedipodine grasshopper species (e.g. Schistocerca gregaria), most of their arborisations are distributed in dorsal and lateral neuropil, while in oedipodine species (e.g. Locusta migratoria), the neurons have additional extensive axonal projections in both the optic lobes and proximal portions of the ganglionic peripheral nerves. This study demonstrates that these morphological differences correlate with their physiology. In L. migratoria, VPLI neuron activity is regulated primarily via a spontaneously active interneuron which descends from the brain. This descending interneuron is inhibited by a light-activated brain extraocular photoreceptor. Regulation of VPLI neuron activity by an extraocular photoreceptor is also seen in the other oedipodine grasshopper investigated. In the four non-oedipodines examined (from two subfamilies), we find no extraocular photoreceptor regulation of VPLI neuron activity. Despite this, VPLI neuron in S.␣gregaria does appear to be driven by a descending interneuron homologous to that in L. migratoria. The descending interneuron in both species receives similar mechanosensory input and excites the VPLI neuron via cholinergic synapses. Histamine injection into the medial protocerebrum of both species causes strong inhibition of the descending interneuron. The evolution of the neural circuitry, by which an extraocular photoreceptor comes to regulate the descending interneuron in oedipodine species, is discussed.

Evidence that histamine is a neurotransmitter in an insect extraocular photoreceptor pathway

The pair of vasopressin-like immunoreactive (VPLI) neurones of the locust Locusta migratoria have cell bodies in the suboesophageal ganglion and extensive arborization throughout the central nervous sytem. The activity of the VPLI neurone is regulated by a spontaneously active excitatory descending interneurone (DI) that is, in turn, inhibited by an uncharacterised extraocular photoreceptor (EOP) system located in the brain. Light directed at the brain results in inhibition of DI activity, which thereby deprives the VPLI neurone of its major synaptic input. We present evidence that histamine plays an important role in the EOP­DI­VPLI pathway. Histamine mimics the EOP-mediated inhibition of the DI, and the H2-specific histamine antagonists cimetidine and ranitidine block its inhibitory action. Histamine application to various areas of the brain localises the area where histaminergic inhibition occurs; this region is confined to the medial protocerebrum. At least six bilaterally paired histamine-like immunoreactive neurones send axonal projections into this area. Depolarisation of the brain region containing the soma of these neurones with high-K+ saline deactivates the VPLI neurone through the removal of the DI excitatory synaptic input.

Opposite changes in the content of oxytocin- and vasopressin-like immunoreactive peptides in the rat thymus during aging

The content of oxytocin- and vasopressin-like immunoreactive (IR) peptides was measured in the thymic extract of 2, 5, 10, 15 and 20 month-old rats by radioimmunoassay before or after fractionation by high-pressure liquid chromatography. In both cases the content of the oxytocin-like IR peptide, which behaved like authentic oxytocin in the chromatography column, increased during aging. Compared to 2 month-old rats a significant 30% increase was observed in 5 month-old rats, whereas the maximal increase (200%) was found in 20 month-old rats. In contrast, the content of the vasopressin-like IR peptide, which behaved like authentic arg 8-vasopressin in the chromatography column, decreased during aging. The decrease (30%) was evident in 5 month-old rats, and was maximal (80%) in 15 month-old rats. The present results suggest that the mechanisms regulating the content of oxytocin- and vasopressin-like IR peptides in the rat thymus undergo differential changes during aging. These processes might be linked to thymic involution.

Oxytocin-producing and vasopressin-producing eosinophils in the mouse spleen: immunohistochemical, immuno-electron-microscopic and in situ hybridization studies.

Oxytocin-like and vasopressin-like immunoreactive cells, and the cells expressing mRNAs for these peptides in the spleen of the C57BL/6 mouse were studied by immunohistochemistry, immuno-electron microscopy and in situ hybridization. Immunoreactive cells were distributed mainly in the splenic cord and marginal zone, whereas there were few in the lymphocyte-packed periarteriolar-lymphoid sheath, lymphoid follicle and germinal center. More numerous vasopressin-positive cells were seen in the splenic cord. The colocalization of oxytocin-like and vasopressin-like immunoreactivity in the same cells was identified by the investigation of mirror sections. By the pre-embedding immuno-electron-microscopic method using antisera against oxytocin and vasopressin, immunopositive reaction products were localized in the matrix around the specific granules, small clear vesicles and mitochondrial membrane of the eosinophils. No immunoreactivity to these peptides was found within the specific granules of the eosinophils. In situ hybridization with synthetic oligonucleotide probes labeled with 32P revealed the presence of mRNAs for oxytocin and vasopressin in the cells of the spleen, the distribution of the mRNAs for these peptides being the same as that of immunopositive cells. These observations suggest that eosinophils synthesize both oxytocin and vasopressin and store them in the matrix. Possible differences in the mechanism of synthesis and storage of these peptides between peripheral eosinophils and hypothalamic neurons are discussed.

Analysis of the peptide content of the locust vasopressin-like immunoreactive (VPLI) neurons.

Isolated cell bodies of the locust vasopressin-like immunoreactive (VPLI) neurons, analyzed by HPLC separation and radioimmune assay, contain three arginine vasopressin-like peptides: a previously identified monomer (Fl, Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2) and its antiparallel homodimer (F2), but also the previously unreported parallel homodimer (PDm). VPLI neuron activity significantly reduces the level of cAMP in the CNS. Of the three synthetic peptides, only the monomer (F1, 10(-8) and 10(-6) M) is capable of inhibiting a forskolin-stimulated increase in cAMP in isolated neural membranes. The antiparallel (F2) and parallel dimers (PDm) of this peptide have no effect on this second messenger.

Pharmacological analysis of the cholinergic input to the locust VPLI neuron from an extraocular photoreceptor system

1. The brain of the locust contains an extraocular photoreceptor (EOP), which provides the major synaptic excitation to the vasopressin-like immunoreactive (VPLI) interneuron of the suboesophageal ganglion. Although the precise location of the EOP remains unknown, its activity can be determined indirectly by intracellular recording from the VPLI neuron. The excitatory drive to the VPLI neuron occurs only in darkness and is absent in the light. 2. The EOP is preferentially sensitive to light of wavelength 494 +/- 7 (SD) nm (blue-green) and has an absorption spectrum characteristic of a rhodopsin-like photopigment. 3. In the presence of high divalent saline (20 mM Ca2+ and Mg2+), the VPLI neuron receives excitatory input in the light. This indicates that the excitatory input to the VPLI neuron is from a tonically active descending input, which normally is inhibited by the light-induced activation of the presynaptic EOP. 4. Stimulation of the connectives while recording the resultant excitatory postsynaptic potential (EPSP) evoked in VPLI shows that the descending input projects beyond the suboesophageal ganglion, extending as far as the metathoracic ganglion. 5. Pharmacological analysis shows that the descending input to the VPLI neuron is cholinergic: acetylcholine (ACh) strongly depolarizes the neuron and eserine, an ACh esterase inhibitor, markedly potentiates the synaptic excitation of the VPLI neuron. 6. Nicotinic and muscarinic receptor antagonists show that the excitation of VPLI consists of two pharmacologically discrete components. Nicotinic ACh receptors mediate a fast depolarization, whereas muscarinic ACh receptors evoke a more sustained depolarization. Accordingly, both a fast and slow depolarization can be evoked selectively in VPLI by direct application of either nicotine or muscarine. 7. Voltage-clamp analysis shows that the fast EPSP evoked current is similar to that produced by nicotine in that it decreases linearly with membrane depolarization. The current associated with the sustained depolarization is similar to that evoked by muscarine, increasing nonlinearly with membrane depolarization. 8. Activity of the descending input, or application of muscarine, lowers the spike-initiation threshold of the VPLI neuron, thereby increasing its excitability. 9. It is concluded that the presence of two ACh receptor subtypes act synergistically to allow continuous activity of the VPLI neuron for sustained periods (i.e., throughout the hours of darkness).

Morphology of the vasopressin-like immunoreactive (VPLI) neurons in many species of grasshopper.

It has previously been shown that the pair of vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria, have cell bodies on the ventral midline of the suboesophageal ganglion and extensive arborisations in all ganglia of the central nervous system. In the present study, we have stained vasopressin-like immunoreactive neurons in 16 additional species of grasshopper, and consistently find this pair of extensive neurons: we assume these to be interspecies homologues. However, the anatomy of these neurons falls into two morphological types: the first, typified by Schistocerca gregaria, has most of its processes distributed in dorsal and lateral neuropil of all ganglia; the second, typified by Locusta migratoria, is equally extensive in its arborisation, but the distribution of branches is shifted peripherally into the optic lobes and the proximal portions of peripheral nerves. It has been suggested that the peripheral fibres in Locusta migratoria are neurohaemal organs for the release of a vasopressin-like diuretic peptide. Our sample of 17 Acridoid species has deliberately selected animals from very different habitats, but our extensive survey of VPLI anatomy shows that peripheral fibres are only present in species from the subfamily Oedipodinae (of which Locusta migratoria is a member) and that no peripheral fibres are present in any of the species from the 4 other subfamilies of the Acridoidea that we have examined. The presence of peripheral fibres is therefore determined by phylogeny and not by habitat. The absence of peripheral VPLI fibres in most grasshopper species examined in this study probably means that the release of putative diuretic hormone from VPLI to control water homeostasis cannot be a conserved function of this ubiquitous neuron. In contrast, the extensive central arborisations and rare antigenicity, which are highly conserved features of the VPLI neuron in all those grasshoppers we have examined, suggests that any conserved role is more likely to be central. A central role for the VPLI neuron has yet to be determined.

Neuronal interaction between VIP and vasopressin neurones in the rat suprachiasmatic nucleus.

Synaptic interaction between VIP-like immunoreactive (VIP-LI) and vasopressin-like immunoreactive (VASO-LI) neurones both of which are main neuronal components in the SCN was investigated using double labelling immunoelectron microscopy. VIP-LI axons were identified as having synaptic contacts with VASO-LI neuronal perikarya and dendrites. VIP-LI axons also formed synapses on non-immunoreactive dendrites or dendritic spines. In addition to the above findings, VIP-LI neuronal elements also formed synaptic contacts with other VIP immunoreactive structures.

A comparison of the effects of two putative diuretic hormones from Locusta migratoria on isolated locust malpighian tubules.

Previous work has shown that a peptide related to arginine vasopressin is present in the suboesophageal ganglion of the locust, Locusta migratoria. This peptide was determined to be an anti-parallel dimer of the nonapeptide Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2 and was reported to stimulate cyclic AMP production and fluid secretion in a combined Malpighian tubules and midgut preparation from locusts. For these reasons the peptide has been called the arginine-vasopressin-like insect diuretic hormone (AVP-like IDH). Recently, a second diuretic peptide (Locusta-DP), which is related to corticotropin releasing factor, has been identified: this is a potent stimulant of fluid secretion and cyclic AMP production by isolated locust tubules. Because water balance in insects is likely to be controlled by a cocktail of hormones acting on both Malpighian tubules and hindgut, this study directly compares the activity of these two peptides in fluid secretion and cyclic AMP production bioassays on one target organ, the isolated Malpighian tubule of Locusta migratoria. Locusta-DP was synthesised directly, whereas the dimeric AVP-like IDH was obtained by oxidation of a synthetic nonapeptide monomer. Products were separated by RP-HPLC and their structures unequivocally confirmed by enzymatic digestion, sequence analysis and electrospray mass spectrometry. We show that Locusta-DP causes strong stimulation of fluid secretion and cyclic AMP production, whereas the AVP-like IDH has no effect in either assay. These findings are discussed in the light of recent work on the anatomy and physiology of the vasopressin-like immunoreactive (VPLI) neurones in the suboesophageal ganglion of Locusta migratoria, the proposed source of the AVP-like peptide.

The vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria. I. Anatomy

Antiserum to arginine-vasopressin has been used to characterise the pair of vasopressin-like immunoreactive (VPLI) neurons in the locust. These neurons have cell bodies in the suboesophageal ganglion, each with a bifurcating dorsal lateral axon which gives rise to predominantly dorsal neuropilar branching in every ganglion of the ventral nerve cord. There are extensive beaded fibre plexuses in most peripheral nerves of thoracic and abdominal ganglia, but in the brain, the peripheral plexuses are reduced while neuropilar branching is more extensive, although it generally remains superficial. An array of fibres runs centripetally through the lamina-medulla chiasma in the optic lobes. Lucifer Yellow or cobalt intracellular staining of single VPLI cells in the adult suboesophageal ganglion shows that all immunoreactive processes emanate from these two neurons, but an additional midline arborisation (that was only partially revealed by immunostaining) was also observed. Intracellularly staining VPLI cells in smaller larval instars, which permits dye to reach the thoracic ganglia, confirms that there is no similar region of poorly-immunoreactive midline arborisation in these ganglia. It has been previously suggested that the immunoreactive superficial fibres and peripheral plexuses in ventral cord ganglia serve a neurohaemal function, releasing the locust vasopressin-like diuretic hormone, F2. We suggest that the other major region of VPLI arborisation, the poorly immunoreactive midline fibres in the suboesophageal ganglion, could be a region where VPLI cells receive synaptic input. The function of the centripetal array of fibres within the optic lobe is still unclear.

The vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria. II. Physiology

The two vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria, have cell bodies in the suboesophageal ganglion and extensive arborizations throughout the CNS. One of the two peptides responsible for AVP-like immunoreactivity is a vasopressin-related peptide with putative 'diuretic hormone' properties. These neurons also have FLRF-like immunoreactivity, probably due to the FMRF-amide-related peptide. SchistoFLRF-amide, isolated from Schistocerca gregaria. This peptide has cardioinhibitory activity and a dual potentiation/inhibition of slow motoneuron induced muscle-twitch tension. Although haemolymph AVP-like peptide titre fluctuates under various conditions, the mechanism that regulates neurohaemal release of this peptide is not understood. Very little is known of the release of SchistoFLRF-amide. We have used intracellular recording from VPLI neurons in vivo to reveal synaptic inputs that lead to changes in their level of spiking activity, and probably, release of both the AVP-like peptides and SchistoFLRF-amide. This pair of neurosecretory cells has a major, common excitatory input whose sustained rate of activity is inversely related to light intensity; VPLI spiking activity, driven by this input, is greater in the dark than in light. This input is from a pair of descending brain interneurons. Their light-sensitivity persists after ablation of compound eyes, optic lobes and ocelli, showing them to be part of an extra-ocular photoreceptor system. Attempts to record from, and individually stain, the descending neuron have been unsuccessful, although its axon location and diameter in the circumoesophageal connective have been determined. Possible locations for its cell body have been identified; one region, close to the pars intercerebralis, is known to be photosensitive in some insects. Mechanosensory stimuli also lead to brief increases in VPLI spiking activity via the descending interneuron, though this modality rapidly habituates. We detect no changes in VPLI spiking activity that consistently correlate with the osmolality of perfusion salines; such changes might have been expected from their previously proposed role in water homeostasis. Alternative roles for VPLI cells are discussed.

Electron Microscopic Studies of Medullary Synaptic Inputs to Vasopressin-containing Neurons in the Hypothalamic Paraventricular Nucleus

We have observed the ascending projections from the lower brain stem to the magnocellular vasopressin-like immunoreactive (VP-LI) neurosecretory neurons in the paraventricular nucleus (PVN) using electron microscopy. The tissues were prepared by a double labeling technique combining anterograde tracing after iontophoretic injection of wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) in the A1 group (lateral reticular nucleus in the medulla oblongata) with VP immunocytochemistry. Both the WGA-HRP-labeled and the unlabeled axon terminals made synaptic contacts with VP-LI cell bodies and processes in the PVN. This indicates a direct synaptic influence of medullary A1 group on the secretory activity of the VP-containing neurons in the hypothalamic paraventricular nucleus.

Vasopressin‐ and proctolin‐like immunoreactive efferent neurons in blowfly abdominal ganglia: Development and ultrastructure

In the neural sheath of the fused thoracicoabdominal ganglia of the blowfly Calliphora erythrocephala, extensive neurohaemal areas can be seen in the electron microscope. A separate set of neurohaemal areas located in the sheath of the lateral abdominal nerve roots contain neural terminals of at least three morphological types. To determine which bioactive substances are stored and possibly released from the neurons supplying these neurohaemal areas, we applied a large number of antisera raised against different neuropeptides of invertebrate and mammalian type. Antisera to two types of neuropeptides react with neurons innervating the sheath of the abdominal nerve roots: antisera to lysine-vasopressin and proctolin. There are only 14-24 vasopressin-like immunoreactive (VPLI) neurons in the entire nervous system of Calliphora. These are all restricted to a bilateral cluster in the fused abdominal ganglia. From this cluster, the neurohaemal areas in abdominal nerve roots are supplied. Proctolin-like immunoreactivity (PLI) can be seen in a large number of neurons in the nervous system of blowflies. The supply of PLI terminals to the abdominal nerve roots is from 12 to 14 neurons in a bilateral cluster of abdominal PLI neurons. It is clear from light- and electron-microscopic immunocytochemistry that the two antisera label two separate populations of neurons that form overlapping terminals in the neural sheath. The immunoreactive terminals are located just below the permeable acellular basal lamina of the neural sheath. Hence, it is likely that at least two different bioactive peptides can be released neurohormonally into the circulation. An additional set of four efferent PLI neurons send axons into the medial abdominal nerve. These do not form neurohaemal terminals in the nerve root, but may innervate the hindgut. Also in the larval nervous system, VPLI and PLI neurons can be recognized. In the larva, the peptide-containing neurons are segmentally arranged. The 14 larval VPLI neurons supply segmental abdominal nerves with axons that run inside the nerves to their targets. During metamorphosis, the segmental nerves fuse and the VPLI axons invade the neural sheath where they arborize and form varicose terminals. About the same number of PLI neurons could be detected in the abdominal ganglia of larval and adult flies. Only for a set of four caudal PLI neurons could efferent axons be traced in the larva. These axons run inside the medial abdominal nerves. The same four PLI neurons, with the same axonal projections, can be recognized in the adults.