The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects. It can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups. In the blowfly Phormia terraenovae, tissue extracts were tested in a bioassay: extracts of blowfly brains exhibit PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. Using standard immunocytochemical techniques, a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroach Leucophaea maderae could be demonstrated. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies, which also send processes into the medulla. These neurons share many features in their lamina-medulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier. It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies can be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina have been found to be very similar to the PDH-IR neurons. Hence, it is tempting to speculate that in both species the PDH- and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system (Nassel, 1991).
Pigment-dispersing hormone immunoreactive (PDHIR) neurons could be detected in the brain and ventral ganglia of the blowfly Phormia terraenovae. PDHIR neurons are found in the optic lobe. Their processes supply the lamina, medulla and lobula complex bilaterally. Large PDHIR cell bodies in the protocerebrum have processes in the proto- and tritocerebrum and axons to the aorta wall and foregut. Eight pairs of PDHIR neurons are found dorsally and three pairs ventrally in the fused abdominal neuromeres; one pair is located ventrally in each of the thoracic neuromeres. The ventral abdominal PDHIR neurons are efferents that innervate the hindgut. PDHIR neurons may play different functional roles as neurohormones or neuromodulators in different parts of the nervous system and its peripheral targets (Shiga, 1993).
An antiserum against the crustacean neuropeptide pigment-dispersing hormone stains a small set of neurons in the optic lobes of several hemimetabolous and holometabolous insects. These cells, the primary branches of which in the optic lobe lie in the accessory medulla, fulfill several criteria predicted for neurons of the circadian clock. For example, in fruit flies they express timeless and period, which are two molecular components of the circadian pacemaker. To test whether pigment-dispersing hormone fulfills a circadian function in the cockroach Leucophaea maderae, 150 fmol of synthetic peptide was injected into the vicinity of the accessory medulla. This results in a stable phase-dependent resetting of the phase of the circadian locomotor activity rhythm, which depends on the amount of pigment-dispersing hormone injected. The resulting phase-response curve differs from that obtained with light pulses, suggesting that pigment-dispersing hormone-immunoreactive neurons are not part of the visual input pathway to the pacemaker but an integral part of it and/or part of a nonphotic input into the clock (Petri, 1997).
The accessory medulla with its associated pigment-dispersing hormone-immunoreactive neurons appears to be the pacemaker that controls the circadian locomotor activity rhythm of the cockroach Leucophaea maderae. To permit studies at the level of individual, identified, pacemaker neurons, specific long-term primary cell cultures of fully differentiated adult neurons of the accessory medulla were developed. As judged from soma diameter distribution, the cultures contain an unbiased representation of apparently all neuronal types of the accessory medulla. The cultured cells survive and grow processes for more than 2 months, with or without additional hemocyte coculturing. However, a strong positive effect on initial outgrowth was observed with hemocyte coculturing. At least six different morphological cell types of the accessory medulla could be distinguished in vitro. Among these, only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiable monopolar type C cells are candidates for circadian pacemaker neurons (Petri, 1999)
Pigment-dispersing factor-immunoreactive circadian pacemaker cells, which arborize in the accessory medulla, control circadian locomotor activity rhythms in Drosophila as well as in the cockroach Leucophaea maderae via unknown mechanisms. Circadian pacemaker candidates of the accessory medulla of the cockroach produce regular interspike intervals. Therefore, the membrane potential of the cells oscillates with ultradian periods. Most or all oscillating cells within the accessory medulla are coupled via synaptic and nonsynaptic mechanisms, forming different assemblies. The cells within an assembly share the same ultradian period (interspike interval) and the same phase (timing of spikes), whereas cells between assemblies differ in phase. Apparently, the majority of these assemblies are formed by inhibitory GABAergic synaptic interactions. Application of pigment-dispersing factor phase locked and thereby synchronized different assemblies. The data suggest that pigment-dispersing factor inhibits GABAergic interneurons, resulting in disinhibition and phase locking of their postsynaptic cells, which previously belonged to different assemblies. These data suggest that phase control of action potential oscillations in the ultradian range is a main task of the circadian pacemaker network. It is hypothesized that neuropeptide-dependent phase control is used to gate circadian outputs to locomotor control centers (Schneider, 2005).
The pigment-dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light-adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, cDNA encoding PDH in the crayfish Orconectes limosus was cloned and sequenced. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor-related peptide (PPRP) and the highly conserved PDH peptide at the carboxy-terminal end. In situ hybridization in combination with immunocytochemistry reveal four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stain both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stain with the PDH antiserum, suggesting the presence of a PDH-like peptide in the LG (de Kleijn, 1993).
A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. Screening resulted in the isolation of a clone [497 bp excluding poly(A) tail] that encodes a beta-PDH previously found in several crustacean species. It displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, indicating the close phylogenetic relationship of both species. A second clone [414 bp exclusive of the poly(A) tail] encodes a novel beta-PDH analog that displays 400-fold less potency in crab bioassays. Both cDNAs encode open reading frames of 234 bp for the prepropeptides, consisting of signal peptides, PDH-precursor-related peptides, and PDH sequences (Klein, 1994).
Neurons immunoreactive with antisera against the crustacean peptide beta-pigment dispersing hormone fulfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations an examination was carried out to see whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlate with the presence of circadian locomotor activity. No rhythm was detected in any animal for at least 12 days after severing both optic stalks. Within the same time period, pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches that regained rhythmic behavior, pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches, either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlates with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects (Stengl, 1994).
The cDNAs encoding the precursors of a chromactive crustacean hormone, Pigment Dispersing Hormone (PDH) of the shrimp Penaeus vannamei, were studied by PCR and molecular cloning. Three different cDNAs were isolated and sequenced. The PDH precursor consists of a putative 22- or 23-amino acid signal peptide, a 34-amino acid PDH-Precursor Related Peptide (PPRP) of unknown function, and the 18-amino acid mature PDH. The deduced mature PDH amino acid sequences are identical except the change of a Leucine by an Isoleucine in one variant and are very similar to those of other species. The signal peptides appear highly variable. The variability between the PPRP sequences is low between the different species, suggesting that this peptide may have a physiological role (Desmoucelles-Carette, 1996).
Three chromatophorotropic neuropeptide hormones were purified from an aqueous extract of the sinus glands of the kuruma prawn Penaeus japonicus by two steps of reverse-phase HPLC and their amino acid sequences were determined. One of them has been found to show pigment concentrating activity and to have an amino acid sequence identical to that of the known red pigment concentrating hormone (RPCH); therefore, it was named Pej-RPCH. The other two peptides show pigment dispersing hormone (PDH) activity and have been named Pej-PDH-I and -II. They both consist of 18 amino acid residues with a free amino-terminus and an amidated carboxyl-terminus, the sequences of Pej-PDH-I and -II being NSELINSLLGIPKVMTDAamide and NSELINSLLGLPKFMIDAamide, respectively. Three amino acid residues at positions 11, 14, and 16 differ between the two PDHs. Pej-PDH-II is about 5-, 7-, and 10-fold more potent than Pej-PDH-I for erythrophores, xanthophores, and melanophores, respectively. The major reason for the difference in potency between the two PDHs can be attributed to differences in residues at position 16. Additionally, each is produced by single individuals. The order of sensitivity of the four types of chromatophores to Pej-RPCH and both PDHs has been found to be erythrophores = xanthophores, which are greater than melanophores, which, in turn, are greater than leukophores (Yang, 1999).
By using an antiserum raised against a crustacean beta-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons has been investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system is large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons show immunoreactivity. The immunostained neurons in Helix are characterized by rich arborizations in all central ganglia and reveal massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex are also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea (Elikes, 1999).
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