Proctolin-like Immunoreactivity Research Articles

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.

Modulation of a rhythmically active crayfish muscle by the neuropeptide proctolin

Whole-animal studies have indicated that the neuromodulators octopamine, serotonin, and proctolin have multiple effects upon the central crustacean ventilatory system. We were interested in determining if these modulators have a complementary peripheral effect in augmenting the muscle power output in a ventilatory muscle. The availability of the biogenic amines and the presence of proctolin-like immunoreactivity in motor axons of the second maxilla indicate that the ventilatory system may be a target for those neuroactive substances. Intracellular recordings and isotonic muscle tension measurements were made on an isolated preparation of a ventilatory muscle. The amines and the peptide were applied by superfusion in normal saline. Of the three substances only protocolin had a modulatory action, which was seen as significant increases in both nerve-evoked and glutamate-evoked muscle contractions. An enhancement of the amplitude of excitatory postsynaptic potentials occurred at a subset of synapses.

Submicroscopic characterization of proctolin-like immunoreactivity in the nervous system of the cockroach Periplaneta americana L.

Proctolin-like immunoreactivity (PLI) was found in different parts of the arthropod central nervous system and in nerve fibers of muscles. In order to examine whether this PLI is related to a uniform type of secretory vesicle, hindgut musculature and frontal and hypocerebral ganglia were examined with the immunogold technique. PLI occurs exclusively within membran-bounded secretory granules. Neither granular ER nor Golgi stacks show PLI. In some cases close relationships between PLI-bearing granules and lysosomes were observed. In presynaptic areas, PLI-reactive granules are associated with numerous clear synaptic vesicles and restricted to an area distinctly separate from the presynaptic membrane. Three types of granules were found, differing in diameter and electron density: (1) dense, 80 nm; (2) dense, 150 nm; (3) low density, 150 nm. The results demonstrate that: (1) the PLI of the produced peptide occurs shortly after its separation from the Golgi stack; (2) the occurrence of PLI in three different granule types could be the morphological expression of the common occurrence of proctolin with other neuroactive substances. However, a possible cross-reactivity with other, hitherto unknown substances must be considered as well.

Effect of intrathecal proctolin administration on the behaviour evoked by the thyrotrophin-releasing hormone )TRH) analogue (RX 77368) and the indoleamine, TRH, substance P and calcitonin gene-related peptide levels and choline acetyltransferase activity in the rat spinal cord

The behavioral effects and the biochemical changes produced following either a single or repeated intrathecal injection respectively, of the insect peptide proctolin (Arg-Tyr-Leu-Pro-Thr-OH) have been compared with the effects of a stable analogue of thyrotrophin-releasing hormone (TRH) in rats. Intrathecal proctolin (1–100 μg) did not produce any marked behavioural effects on its own, while intrathecal TRH analogue (RX 77368, 0.5 μg) administration produced wet-dog shakes and forepaw-licking behaviours. Proctolin (10 μg) significantly attenuated the wet-dog shake and forepaw-licking behaviours evoked by intrathecal RX 77368 administration when it was given 30 min before, but not when given in combination with RX 77368. Repeated intrathecal proctolin administration (10 μg twice daily for 5 days) significantly reduced the 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid and TRH levels in the ventral, but not in the dorsal, horn of the spinal cord nor in the brainstem, and elevated hypothalamic TRH without affecting plasma free thryroxine levels when compared with values in saline-treated controls. Repeated proctolin injection did not alter substance P levels in any brain region examined, nor did it affect the choline acetyltransferase activity or the calcitonin gene-related peptide-like immunoreactive levels in the ventral horn of the spinal cord, both of which are principally located in motoneurones in this cord region. These results demonstrate that intrathecal proctolin antagonizes the behaviours produced by intrathecal TRH analogue injection, and selectively alters the synthesis and/or release of 5-HT and TRH within the ventral spinal cord and the hypothalamus, regions where proctolin-like immunoreactivity has been shown to co-exist with 5-HT and/or TRH in nerve terminals.

The presence and distribution of proctolin in the blood-feeding bug, Rhodnius prolixus

The distribution of proctolin throughout the central nervous system and visceral structures of Rhodnius prolixus has been analysed using reversed-phase high-performance liquid chromatography (RP-HPLC) together with a sensitive bioassay. Immunohistochemistry has also been used to identify somata and terminals reactive with a proctolin antiserum. The central nervous system of adult females contained the equivalent of about 378 fmol proctolin distributed unequally throughout all of the ganglia. The highest amount was found in the mesothoracic ganglionic mass, the lowest in the brain. Proctolin was found to be associated with the reproductive and digestive systems, with substantial amounts present in the oviduct and high concentrations per mg tissue proteins in ovaries, oviduct and hindgut. The number and diameter of proctolin-immunoreactive neurones present in each ganglion correlates well with the content of proctolin quantified following RP-HPLC. Over 200 proctolin-staining neurones were distributed over dorsal and ventral aspects of the various ganglia. In addition terminals showing proctolin-like immunoreactivity were observed on the oviducts and hindgut.

Peptidergic innervation of insect reproductive tissue: the association of proctolin with oviduct visceral musculature.

The visceral muscles of the oviducts of Locusta migratoria are sensitive to the pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). Amounts of proctolin as low as 2 fmol induce a tonic contraction that is dose-dependent up to 200 fmol. By use of this bioassay we have quantified the amount of material showing proctolinlike bioactivity associated with the oviducts. Reversed-phase high performance liquid chromatography of tissue extracts indicates that material with proctolinlike bioactivity and co-eluting with proctolin is present in the oviducts, the oviducal nerve, and the VIIth abdominal (penultimate) ganglion. The proctolin is present in areas of oviduct that receive extensive innervation. There is tenfold less proctolin in areas of oviduct that receive little or no innervation. Proctolinlike immunoreactivity is present in axons of the oviducal nerve as well as in a number of cell bodies in the VIIth abdominal ganglion. Three of these neurons lie in a position similar to that of the previously described oviduct motoneurons. Neuropilar axons and processes, as well as axons in the median nerve, also show proctolinlike immunoreactivity. The results indicate that proctolin, which has previously been identified as a neurotransmitter of insect hindgut visceral muscle, is also associated with visceral muscle of the reproductive system.

Peptidergic innervation of insect skeletal muscle: Immunochemical observations

Proctolin (Arg-Tyr-Leu-Pro-Thr) is a pentapeptide present in the hindgut or proctodeum of the cockroach Periplaneta americana where it may be a transmitter. Its widespread distribution among peripherally projecting neurons in the CNS (Bishop and O'Shea, '82) suggested that proctolin's motor function is not restricted to the hindgut, but has a variety of peripheral targets. This idea was further supported when proctolin was localized to an identified skeletal motoneuron, the slow coxal depressor, where it acts as a cotransmitter (O'Shea and Bishop, '82; Adams and O'Shea, '83). Our objective was to investigate the proctolinergic innervation of a variety of skeletal muscles of the cockroach Periplaneta americana. We used immunohistochemical and radioimmunochemical methods to map the distribution of proctolin immunoreactivity. This survey revealed that a subpopulation of skeletal muscles are innervated by proctolinergic motoneurons. The anatomical features of the peptidergic innervation and the levels of proctolinlike immunoreactivity of one muscle group, the coxal depressor system, are here described in detail. The source of the proctolin innervation to the metathoracic coxal depressor group is identified as the slow coxal depressor motoneuron. The results of a survey of fast and slow skeletal muscles revealed that proctolin is associated with slow motor function. The functional implications of the association of a peptide with motoneurons are discussed in relationship to the organization of the insect motor pool.

The distribution of a proctolin-like immunoreactive material in the terminal ganglion of the cockroach, Periplaneta americana L.

Neurons with proctolin-like immunoreactivity were mapped in the terminal ganglion of Periplaneta americana. The effect of different fixation methods on the variability of immunostaining is described and discussed. The appearance of immunoreactive presynaptic terminals, described here for the first time in insects, points to a function of proctolin as neurotransmitter or neuromodulator in the central nervous system of P. americana besides its known role in the periphery. Proctolin-like immunoreactivity was shown in pre- and postsynaptic profiles. Synaptic contacts are described in detail.

Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone

A radioimmunoassay, immunohistochemical techniques, and high pressure liquid chromatography (HPLC) methods have been developed for the study of the pentapeptide proctolin in the lobster Homarus americanus. Proctolin-like immunoreactivity is present in nearly every portion of the lobster nervous system; immunoreactivity is found in the brain, in each of the ganglia and connectives of the ventral nerve cord, and in many of the nerve roots that emerge from the cord. The greatest amounts are found in the pericardial organs, which are well known neurosecretory structures, and these structures have been selected for more detailed study. The immunoreactive material in the pericardial organs appears to be authentic proctolin. This material co-migrates with synthetic proctolin in two HPLC systems. Furthermore, a peptide that is purified from pericardial organs by HPLC is indistinguishable from synthetic proctolin in high resolution fast atom bombardment mass spectrometry. Cytochemistry reveals that the surface of the pericardial organs is densely covered with immunoreactive varicosities. No cell bodies that stain for proctolin are found in the pericardial organs, and the cells that give rise to the varicosities have not yet been located. The nerve endings in pericardial organs are capable of releasing proctolin-like material when depolarized in the presence of Ca++. These findings suggest that proctolin is a neurohormone in the lobster.

Neuropeptide proctolin (H-Arg-Try-Leu-Pro-Thr-OH): immunocytochemical mapping of neurons in the central nervous system of the cockroach.

Proctolinlike immunoreactivity was mapped in the central nervous system of the cockroach, Periplaneta americana, by using whole-ganglion immunoreacted preparations. The procedure for this immunohistochemical staining of whole-mounts is described. Immunoreactivity was confined to neuronal cell bodies and and processes. These were found in all ganglia of the CNS. The cells varied in the consistency and intensity of their staining. The occurrence and variability of staining is described in detail. Cell bodies were found in the dorsal, ventral, and lateral regions of the ganglia. The highest number of cell bodies was found in the terminal ganglion and the lowest number in the cerebral ganglion. Those in the cerebral ganglion occurred mainly in the tritocerebral lobes. The distribution of immunoreactive cell bodies correlated with results previously obtained by radioimmunoassay. Immunoreactive processes were detected in all interganglionic connectives and many ganglionic nerve roots. Dense ramifications of immunoreactive processes and variocosities were detected in many of the ganglia. The widespread presence of immunoreactivity suggests that proctolin has diverse central and peripheral functions. The mapping immunoreactive neuronal somata provides a valuable step in the identification of putative proctolin-containing neurons suitable for further biochemical, anatomical, and physiological analysis.

Neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH): immunological detection and neuronal localization in insect central nervous system.

Proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is a pentapeptide first extracted from cockroaches. It is known to have many neurohormonal effects and has been associated with specific, identified cockroach neurons. We have produced proctolin antisera and report here on their application in detecting proctolin-like immunoreactivity (PLI) in the cockroach central nervous system. Radioimmunoassay, capable of detectable 50 fmol of proctolin, was used to quantify the distribution of PLI. Highest concentrations were detected in the genital ganglia and lowest in the cerebral ganglia. Immunohistochemistry on the cockroach central nervous system demonstrated that PLI is localized to neurons. Neurons stained by using immunohistochemistry were widespread in the ganglia. Cell bodies were found to be in constant positions from animal to animal and to occur in bilaterally symmetrical pairs. These neurons are potentially identifiable.

Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.).

In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl2 iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.