Pigment‐dispersing factor in the locust abdominal ganglia may have roles as circulating neurohormone and central neuromodulator

Pigment‐dispersing factor (PDF) is a neuropeptide that has been indicated as a likely output signal from the circadian clock neurons in the brain of Drosophila. In addition to these brain neurons, there are PDF‐immunoreactive (PDFI) neurons in the abdominal ganglia of Drosophila and other insects; the function of these neurons is not known. We have analyzed PDFI neurons in the abdominal ganglia of the locust Locusta migratoria. These PDFI neurons can first be detected at about 45% embryonic development and have an adult appearance at about 80%. In each of the abdominal ganglia (A3–A7) there is one pair of lateral PDFI neurons and in each of the A5–A7 ganglia there is additionally a pair of median neurons. The lateral neurons supply varicose branches to neurohemal areas of the lateral heart nerves and perisympathetic organs, whereas the median cells form processes in the terminal abdominal ganglion and supply terminals on the hindgut. Because PDF does not influence hindgut contractility, it is possible that also these median neurons release PDF into the circulation. Release from one or both the PDFI neuron types was confirmed by measurements of PDF‐immunoreactivity in hemolymph by enzyme immunoassay. PDF applied to the terminal abdominal ganglion triggers firing of action potentials in motoneurons with axons in the genital nerves of males and the 8th ventral nerve of females. Because this action is blocked in calcium‐free saline, it is likely that PDF acts via interneurons. Thus, PDF seems to have a modulatory role in central neuronal circuits of the terminal abdominal ganglion that control muscles of genital organs. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 19–41, 2001     

Neural substrate and allatostatin-like innervation of the gut of Locusta migratoria

Allatostatin-like immunoreactivity (ALI) is widely distributed in processes and varicosities on the fore-, mid-, and hindgut of the locust, and within midgut open-type endocrine-like cells. ALI is also observed in cells and processes in all ganglia of the central nervous system (CNS) and the stomatogastric nervous system (SNS). Ventral unpaired median neurons (VUMs) contained ALI within abdominal ganglia IV-VII. Neurobiotin retrograde fills of the branches of the 11th sternal nerve that innervate the hindgut revealed 2-4 VUMs in abdominal ganglia IV-VIIth, which also contain ALI. The VIIIth abdominal ganglion contained three ventral medial groups of neurons that filled with neurobiotin and contained ALI. The co-localization of ALI in the identified neurons suggests that these cells are the source of ALI on the hindgut. A retrograde fill of the nerves of the ingluvial ganglia that innervate the foregut revealed numerous neurons within the frontal ganglion and an extensive neuropile in the hypocerebral ganglion, but there seems to be no apparent co-localization of neurobiotin and ALI in these neurons, indicating the source of ALI on the foregut comes via the brain, through the SNS.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

FMRFamide-expressing efferent neurons in eighth abdominal ganglion innervate hindgut in the silkworm, Bombyx mori

The tetrapeptide FMRFamide is known to affect both neural function and gut contraction in a wide variety of invertebrates and vertebrates, including insect species. This study aimed to find a pattern of innervation of specific FMRFamide-labeled neurons from the abdominal ganglia to the hindgut of the silkworm Bombyx mori using the immunocytochemical method. In the 1st to the 7th abdominal ganglia, labeled efferent neurons that would innervate the hindgut could not be found. However, in the 8th abdominal ganglion, three pairs of labeled specific efferent neurons projected axons into the central neuropil to eventually innervate the hindgut. Both axons of two pairs of labeled cell bodies in the lateral rind and axons of one pair of labeled cell bodies in the posterior rind extended to the central neuropil and formed contralateral tracts of a labeled neural tract with a semi-circular shape. These labeled axons ran out to one pair of bilateral cercal nerves that extended out from the posterior end of the 8th abdominal ganglion and finally to the innervated hindgut. These results provide valuable information for detecting the novel function of FMRFamide-related peptides in metamorphic insect species.     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Location and major postembryonic changes of identified 5-HT-immunoreactive neurones in the terminal ganglion of a cricket (Acheta domesticus)

Summary In the terminal ganglion of the cricket, Acheta domesticus, the somata of certain interneurones and efferent neurones consistently react to 5-HT immunohistochemistry. There are serially homologous pairs of bilateral interneurones seen in the neuromeres of the 7th to the 10th segment and hindgut neurones with their somata located at the posterior median end of the ganglion. In adult crickets, pairs of large efferent neurones with lateral somata supply specific genital muscles in the 8th and the 9th segment of females. In males, only one pair of these efferent neurones supplies genital muscles of the 9th segment only. These identified 5-HT-immunoreactive neurones are not detected in larval crickets before development of the genital apparatus.     

Serotonin immunoreactivity of neurons in the gastropod Aplysia californica

Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation.     

Serotonin-like immunoreactivity in the ventral nerve cord of the Colorado potato beetle,Leptinotarsa decemlineata: Identification of five different neuron classes

Summary In an immunohistochemical study of the ventral nerve cord of L. decemlineata, five distinct neuron categories were distinguished: 1) Two paired segmental twin interneurons occur in each ganglion or neuromere; their axons distribute processes over almost the entire nerve cord and run to the cerebral ganglion complex. In contrast, other axons are distributed locally. 2) Four large frontal neurosecretory neurons occur in the suboesophageal ganglion (SOG), two of which have axons that run into the mandibular nerves to form a neurohemal plexus on the surface of cerebral nerves. 3) A pair of large caudal neurons occur in the terminal ganglion and innervate the hindgut. 4) Local miniature interneurons occur in the SOG. 5) Terminal neurons are present in the last abdominal ganglion. Segmental twin interneurons appear to be grouped into 3 functional units spanning several ganglia. Their axons run to specific projection areas, which separate the functional units, and which mark the externally visible separation of condensed ganglion complexes. A possible role of the most caudal functional unit might be the synaptic control of caudal neurons innervating the hindgut.     

Crustacean cardioactive peptide in the nervous system of the locust,Locusta migratoria: an immunocytochemical study on the ventral nerve cord and peripheral innervation

Summary Crustacean cardioactive peptide-immunoreactive neurons occur in the entire central nervous system of Locusta migratoria. The present paper focuses on mapping studies in the ventral nerve cord and on peripheral projection sites. Two types of contralaterally projecting neurons occur in all neuromers from the subesophageal to the seventh abdominal ganglia. One type forms terminals at the surface of the thoracic nerves 6 and 1, the distal perisympathetic organs, the lateral heart nerves, and on ventral and dorsal diaphragm muscles. Two large neurons in the anterior part and several neurons of a different type in the posterior part of the terminal ganglion project into the last tergal nerves. In the abdominal neuromers 1–7, two types of ipsilaterally projecting neurons occur, one of which gives rise to neurosecretory terminals in the distal perisympathetic organs, in peripheral areas of the transverse, stigmata and lateral heart nerves. Four subesophageal neurons have putative terminals in the neurilemma of the nervus corporis allati II, and in the corpora allata and cardiaca. In addition, several immunoreactive putative interneurons and other neurons were mapped in the ventral nerve cord. A new in situ whole-mount technique was essential for elucidation of the peripheral pathways and targets of the identified neurons, which suggest a role of the peptide in the control of heartbeat, abdominal ventilatory and visceral muscle activity.Abbreviations AG abdominal ganglia - AM alary muscle - AMN alary muscle nerve - CA corpus allatum - CC corpus cardiacum - dPSO distal perisympathetic organ - LHN lateral heart nerve - LT CCAP-immunoreactive lateral tract - NCA nervus corporis allati - NCC nervus corporis cardiaci - NM neuromer - PMN paramedian nerve - PSO perisympathetic organ - SOG subesophageal ganglion - VDM ventral diaphragm muscles - VNC ventral nerve cord     

AN ANATOMICAL STUDY OF THE ABDOMINAL NERVOUS AND MUSCULAR SYSTEMS OF DRAGONFLY (AESHNIDAE) NYMPHS

The musculature of the fourth to eighth abdominal segments is typically composed of twenty pairs of segmental muscles associated with the body wall. In the first to third and ninth and tenth segments certain modifications to the basic plan occur in association with the abdominal-thoracic junction, the respiratory apparatus and the anal appendages. In some segments there are also paired muscles associated with the alimentary canal. Two large transverse muscles are present in the abdomen. There are eight abdominal ganglia, the first seven of which each give rise to three pairs of lateral nerves, the eighth to five pairs. In addition there are ten median abdominal nerves. The innervation fields of the various nerves are described. The first three pairs of lateral nerves of the last ganglion are homologous with the lateral nerves of the other abdominal ganglia; the fourth pair innervates most of segment nine; and the fifth pair innervates the remainder of segment nine, segment ten and the anal appendages. Certain of the abdominal muscles are innervated by branches from two different nerve roots. In segments six and seven the anterior point of attachment of the longitudinal stretch receptors is normally different from that in the other abdominal segments. This is discussed in the light of the types of movement which involve the abdomen and it seems apparent that these receptors are affected not only by swimming and abdominal flexion, as are the other longitudinal stretch receptors, but also by respiratory movements. Two distinct types of epidermal sensilla are present on the abdomen, spines and hairs. The former are the more numerous on the body, the latter on the anal appendages.     

Periviscerokinin-like immunoreactivity in the nervous system of the American cockroach

A highly specific polyclonal antiserum has been raised against periviscerokinin, the first neuropeptide isolated from the perisympathetic organs of insects (Predel et al. 1995). In this study, two different neuronal systems with periviscerokinin-like immunoreactivity were distinguished in the central nervous system of the American cockroach: (1) An intrinsic neuronal network, restricted to the head-thoracic region, was formed by intersegmental projecting neurons of the brain, suboesophageal ganglion and metathoracic ganglion. In addition, groups of local interneurons occurred in the proto- and tritocerebrum. (2) A typical neurohormonal system was stained exclusively in the abdomen; it was represented by abdominal perisympathetic organs which were supplied by three cell clusters located in each unfused abdominal ganglion. As revealed by nickel backfills, most neurons with axons entering the perisympathetic organs contained a periviscerokinin-like peptide. Immunoreactive fibres left the perisympathetic organs peripherally, innervated the hyperneural muscle and ran via the link nerves/segmental nerves to the heart and segmental vessels. All visceral muscles innervated by periviscerokinin-immunoreactive fibres were shown to be sensitive to periviscerokinin, whereas the hindgut gave no specific response to this peptide.     

Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicina third instar larvae

The stomatogastric nervous system (SNS) associated with the foregut was studied in 3rd instar larvae of Drosophila melanogaster and Calliphora vicina (blowfly). In both species, the foregut comprises pharynx, esophagus, and proventriculus. Only in Calliphora does the esophagus form a crop. The position of nerves and neurons was investigated with neuronal tracers in both species and GFP expression in Drosophila. The SNS is nearly identical in both species. Neurons are located in the proventricular and the hypocerebral ganglion (HCG), which are connected to each other by the proventricular nerve. Motor neurons for pharyngeal muscles are located in the brain not, as in other insect groups, in the frontal ganglion. The position of the frontal ganglion is taken by a nerve junction devoid of neurons. The junction is composed of four nerves: the frontal connectives that fuse with the antennal nerves (ANs), the frontal nerve innervating the cibarial dilator muscles and the recurrent nerve that innervates the esophagus and projects to the HCG. Differences in the SNS are restricted to a crop nerve only present in Calliphora and an esophageal ganglion that only exists in Drosophila. The ganglia of the dorsal organs give rise to the ANs, which project to the brain. The extensive conformity of the SNS of both species suggests functional parallels. Future electrophysiological studies of the motor circuits in the SNS of Drosophila will profit from parallel studies of the homologous but more accessible structures in Calliphora.     

Tracheation of abdominal ganglia and cerci in the house cricket Acheta domesticus (Orthoptera,Gryllidae)

Patterns of tracheation in the abdominal central nervous system and the cerci of Acheta domesticus are described from whole mounts, and light and electron microscopy. The tracheal supply of the ganglia is derived from ventral longitudinal tracheal trunks which have segmental connections to the spiracels. Each abdominal ganglion is served by a single pair of tracheal trunks, except the terminal ganglion, which has two pairs. Within the ganglia, tracheoles occur principally in association with glia-rich areas of the neuropile. We suggest that the respiratory exchange may be concentrated in the cell bodies of neurons and glia. Each cercus has a tracheal supply in paralle with a large air sac which, it is suggested, serves to lighten the cercus, functions as a resonator for sound reception, or facilitates tidal flow of hemolymph and postecdysial expansion of the cercus. No tracheae run continuously between ganglia or between the terminal ganglion and the cerci, and they do not appear to have a potential role as a contact guidance pathway for cercal nerve growth.     

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

Summary 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 laminamedulla 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.Abbreviations AVP arginine vasopressin - DIT dorsal intermediate tract - FLRF Phe-Leu-Arg-Phe - FMRF-amide Phe-Met-Arg-Phe-amide - LDT lateral dorsal tract - LVP lysine vasopressin - MDT median dorsal tract - MVT median ventral tract - SEM scanning electron microscopy - SOG suboesophageal ganglion - VIT ventral intermediate tract - VNC ventral nerve cord - VPLI vasopressin-like immunoreactive     

Structure of identified neurons innervating the lateral cardiac nerve cords in the migratory locust,Locusta migratoria migratorioides (Reiche and Fairmaire) (Orthoptera,Acrididae)

Our knowledge about the morphology of neurons innervating the lateral cardiac nerve cords (LCNCs) in migratory locusts, Locusta migratoria migratorioides (R. and F.) (Orthoptera, Acrididae) has increased considerably during recent years, mainly owing to immunocytochemical studies using antisera directed against members of insect neuropeptide families. In principle, there are three morphological types of neurons located within the CNS, which innervate the LCNCs in locusts: abdominal ganglia contain (i) bilaterally projecting, possibly unpaired neurons (BPNs) and (ii) paired, unilaterally projecting neurons. In addition, (iii) the LCNCs receive innervation from a pair of neurons, which is located within the suboesophageal ganglion. The axons of all three types of neurons project into the LCNCs via the segmental heart nerves, the most distal extensions of the dorsal segemental nerves of abdominal ganglia. When estimating the number of axons contained in one segmental heart nerve and formed by all central neurons so far identified, this number exceeds the number of axon profiles previously seen using the electron microscope. This indicates that most, or perhaps all central neurons projecting into the LCNCs, have been identified in these insects.     

The origin and possible significance of substance P immunoreactive networks in the prevertebral ganglia and related structures in the guinea-pig

The distribution and origin of substance P immunoreactive nerve elements have been studied in the guinea-pig prevertebral ganglia by the indirect immunohistochemical technique, using a monoclonal antibody to substance P. Non-varicose substance P immunoreactive nerve fibres enter or leave the ganglia in all nerves associated with them, traversing the ganglia in larger or smaller bundles. Networks, mainly single-stranded, of varicose substance P immunoreactive nerve fibres also permeate the ganglia, forming a loose meshwork among the neurons. Similar networks are present in the lumbar paravertebral ganglia. In all these ganglia, neuronal somata do not in general show substance P immunoreactivity. The various nerves connected with the inferior mesenteric ganglion have been cut, in single categories and in various combinations, and the ganglion examined, after intervals of up to six days. Cutting the colonic or hypogastric nerves, which connect the ganglion with the hindgut and pelvic organs, leads to accumulation of substance P immunoreactive material in their ganglionic stumps, extending retrogradely to intraganglionic non-varicose fibres traceable through into the intermesenteric and lumbar splanchnic nerves. There is some local depletion of intraganglionic varicose networks. Cutting the intermesenteric nerve, which connects the coeliac-superior mesenteric ganglion complex with the ganglion, leads to accumulation of substance P immunoreactive material in its cranial stump and depletion of its distal stump; a minimal depletion is detectable in the inferior mesenteric ganglion itself. Cutting the lumbar splanchnic nerves, which connect the ganglion with the upper lumbar spinal cord and dorsal root ganglia, leads to accumulation of substance P immunoreactive material in their proximal stumps and total depletion of their distal, ganglionic stumps; in the ganglion there is subtotal loss of non-varicose substance P immunoreactive fibres and of varicose nerve networks, and the few surviving non-varicose fibres are traceable across the ganglion from the intermesenteric nerve to the colonic and hypogastric nerves. Cutting the intermesenteric and lumbar splanchnic nerves virtually abolishes substance P immunoreactive elements from the ganglion within three days postoperatively. It is concluded that these arise centrally to the ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensory receptors on the hindgut of the cockroach, Leucophaea maderae Fabricius (Dictyoptera: Blaberidae)

In the cockroach, Leucophaea maderae, the hindgut is innervated by the proctodeal nerves, branches of nerves XI of the terminal abdominal ganglion.Phasic afferent activity was recorded from the 11th-proctodeal nerve in response to distention of the hindgut by saline injection. With methylene blue staining, eight multipolar sensory receptors of type II (Dethier, 1963, The Physiology of Insect Senses. Methuen, London) were identified on the ventral and lateral surfaces of the rectum, four on each side. The afferent activity in the 11th-proctodeal nerve most likely originated in these receptors.     

Distribution of SchistoFLRFamide-like immunoreactivity in the adult ventral nervous system of the locust,Schistocerca gregaria

SchistoFLRFamide (PDVDHVFLRF-NH₂) is one of the major endogenous neuropeptides of the FMRF-amide family found in the nervous system of the locust,Schistocerca gregaria. To gain insights into the potential physiological roles of this neuropeptide we have examined the distribution of SchistoFLRFamide-like immunoreactivity in the ventral nervous system of adult locusts by use of a newly developed N-terminally specific antibody. SchistoFLRFamide-like immunoreactivity in the ventral nerve cord is found in a subgroup of the neurones that are immunoreactive to an antiserum raised against bovine pancreatic polypeptide (BPP). In the suboesophageal ganglion three groups of cells stain, including one pair of large posterior ventral cells. These cells are the same size, in the same location in the ganglion and have the same branching pattern as a pair of BPP immunoreactive cells known to innervate the heart and retrocerebral glandular complex of the locust. In the thoracic and abdominal ganglia two and three sets of cells, respectively, stain with both the SchistoFLRFamide and BPP antisera. In the abdominal ganglia the immunoreactive cells project via the median nerves to the intensely immunoreactive neurohaemal organs.     

The result of evolutionary limb loss on the complement of motor neurons in a crayfish limb nerve revealed by serial homology

Many macruran decapod crustaceans show sexual dimorphism of abdominal appendages adapted for use as secondary reproductive organs. Not only does the Australian crayfish, Cherax destructor, show no external, abdominal dimorphism, but both males and females have lost the pleopods of the first abdominal segment entirely. The first nerves of the abdominal ganglia of crayfish and lobsters carry the axons of the pleopod motor neurons. We used intracellular cobalt infusion into the first nerves of the first and second abdominal ganglia to reveal the motor neuron complement of these ganglia in males and females. The first nerves of the second abdominal ganglia of both males and females have approximately 37 motor neurons associated with them. The homologous nerves in the first abdominal segment, where there are no pleopods, have only 8 or 9 motor neurons associated with them. The evolutionary implications of this difference are discussed.