Evolution of identified arthropod neurons: the serotonergic system in relation to engrailed-expressing cells in the embryonic ventral nerve cord of the american lobster homarus americanus milne edwards, 1873 (malacostraca,pleocyemata, homarida)

One of the long-standing questions in zoology is that on the phylogenetic relationships within the Arthropoda. Comparative studies on structure and development of the nervous system can contribute important arguments to this discussion. In the present report, the arrangement of serotonin- and engrailed-expressing cells was examined in the embryonic ventral nerve cord of the American lobster Homarus americanus Milne Edwards, 1873 (Malacostraca, Pleocyemata, Homarida), and the spatial relationship of these two cell classes was explored by a double-labelling approach. The goal of this study was to determine whether the lobster serotonergic neurons are homologous to similar cells present in representatives of the Hexapoda and other Arthropoda. The results indicate that, in fact, these neurons in the lobster ventral nerve cord have corresponding counterparts in many other mandibulate taxa. Based on the finding of these homologies, the arrangement of serotonergic neurons in a model trunk ganglion of the mandibulate ground pattern was reconstructed as comprising an anterior and a posterior pair of serotonergic neurons per hemiganglion, each cell with both an ipsilateral and a contralateral neurite. Starting from this ground pattern, the evolutionary diversification of this class of neurons within the Mandibulata is discussed.     

Morphology and monoaminergic modulation of Crustacean Hyperglycemic Hormone-like immunoreactive neurons in the lobster nervous system

Neuronal somata located near branch points in the second thoracic nerve roots of the lobster are immunoreactive for Crustacean Hyperglycemic Hormone (CHH)-like peptides, a family of putative stress hormones. We have employed intracellular dye injection, immunostaining, and confocal imaging to observe the anatomy of these root neurons, which are morphologically diverse and dye coupled. Some root neurons contribute to neurosecretory structures at the points of exit of the root from the nerve cord. Other CNS-projecting root neurons send projections into the T5-A1 interganglionic connectives. Neurosecretory elements of the serotonin (5HT) and octopamine (OCT) systems, implicated in postural control and aggression, terminate densely in the vicinity of the second thoracic root neurons. We have confirmed by double immunostaining for 5HT and CHH-like peptides that the endings of the 5HT neurons are in close apposition to root neurons in the superficial regions of the root. We have also extended previous studies documenting electrophysiological responses of the root neurons to 5HT or OCT. Bath-applied 5HT and OCT inhibit the spontaneous bursting activity of root neurons at concentrations higher than 100 nM. The root neurons desensitize to the persistent presence of high concentrations of 5HT, but not OCT, in the bath. Nanomolar concentrations of OCT, but not 5HT have an excitatory effect on the spontaneous bursting activity of root neurons. This region of the lobster nervous system is of continuing interest, as identified neurons of three neuromodulatory systems implicated in stress and aggression converge and interact at the level of identified neurons.     

Biochemistry and ultrastructure of serotonergic nerve endings in the lobster: serotonin and octopamine are contained in different nerve endings

In this article we report that the distribution of serotonin in the lobster nervous system parallels the distribution of octopamine and that the same tissues that contain endogenous serotonin can synthesize it from tryptophan. Octopamine and serotonin are highly concentrated in a neurosecretory region of the second thoracic roots in association with a group of neurosecretory cells. The roots possess separate high-affinity uptake systems for both serotonin and tryptophan. Radioactive serotonin, accumulated in tissues during incubations with either tritiated serotonin or tritiated tryptophan, can be released, in a calcium-dependent manner, by depolarization with potassium. A detailed morphological examination of the second thoracic roots shows four distinct categories of nerve endings in the vicinity of the neurosecretory cells. Octopamine is synthesized in one of these types of endings and serotonin in another. The high-affinity uptake systems for serotonin and tryptophan are found only in association with the endings that make serotonin. These endings and all the biochemical parameters of serotonin metabolism in the roots are selectively destroyed by previous injection of animals with the neurotoxin 5,7-dihydroxytryptamine.     

The innervation of the closer muscle of the mesothoracic spiracle of the locust.

The closer muscle of the mesothoracic spiracle of the locust, Schistocerca gregaria is innervated by two excitatory motoneurones and also by processes of a peripherally located neurosecretory cell. Within the muscle, ultrastructural studies show the presence of two types of excitatory nerve terminal which differ in the content of dense cored vesicles and in their distribution. The ventral segment of the muscle is innervated predominantly by terminals with small clear vesicles and only an occasional dense-cored vesicle. The central part of the muscle is innervated predominantly by terminals with small clear vesicles and larger numbers of dense-cored vesicles. The dorsal segment of the muscle is innervated exclusively by a neurosecretory type innervation. The small neurohaemal organ of the median nerve close to the spiracle muscle is immunoreactive to an antibody raised against bovine pancreatic polypeptide but no immunoreactive processes enter the muscle itself. The muscle possesses specific octopaminergic receptors that increase cyclic AMP levels and the possibility that the neurosecretory input to the muscle is provided by either a central or peripheral octopamine containing neurone is discussed.     

Octopamine Uptake and Metabolism in the Insect Nervous System

Several insect tissues were examined for their ability to take up octopamine in the presence and absence of sodium ions. The cockroach Malpighian tubules, ovary, and ventral nerve cord showed the highest level of sodium-dependent uptake. The adult firefly lantern exhibited substantial sodium-independent uptake. Some of these tissues were also examined for their ability to metabolize octopamine by N-acetylation. Measurable N-acetyltransferase activity was present in the cockroach ventral nerve cord, tobacco hornworm CNS, and firefly light organ. N-Acetylation is proposed to be the major metabolic pathway for octopamine in the cockroach (Periplaneta americana) nervous system. Several classes of compounds, including octopamine receptor agonists, tricyclic antidepressants, amphetamines, chloroethylbenzylamines, and some experimental insecticides, were tested for their ability to inhibit octopamine uptake and metabolism. The sodium-insensitive component of uptake was not inhibited by most compounds tested, but the sodium-sensitive component was strongly inhibited by xylamine, N-ethyl-N-chloroethyl-o-bromobenzylamine, and their aziridinium ions (60-100%). These compounds also effectively inhibited N-acetyl-transferase (IC50 values at or below 1 microM). Other good inhibitors of N-acetyltransferase included desipramine, synephrine, and an experimental insecticide, CGA 132427. Formamidine pesticides had limited effect on both processes, and neither action seems likely to be involved in their octopaminergic actions in vivo. Cocaine was unique in stimulating N-acetyltransferase activity. When inhibition of sodium-sensitive uptake is compared with inhibition of N-acetyltransferase in the cockroach ventral nerve cord, two groups of inhibitors are discernible. Type 1 compounds inhibit uptake without an effect on N-acetyltransferase, whereas type 2 compounds inhibit both processes. These results suggest a functional linkage between the uptake and acetylation of octopamine.     

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.     

Octopaminergic modulation of the femoral chordotonal organ in the stick insect

The modulatory action of DL-octopamine on the multicellular femoral chordotonal organ (fCO) of the stick insect Cuniculina impigra was examined using extracellular recordings from the fCO nerve and intracellular recordings from single sensory neurons. To determine the octopaminergic effect on position, velocity and/or acceleration sensitivity of mechanoreceptors direct mechanical stimulations with defined parameters were applied to the fCO apodeme. The spontaneous activity in the fCO nerve was enhanced in a dose-dependent manner by octopamine (threshold at 5 × 10^?7 M). This was based on enhanced activity of position sensitive neurons as the fCO activity for all position stimuli was shifted to higher values. Intracellular recordings of single sensory cells showed that velocity-sensitivity of single sensory cells was not altered by octopamine. Similarly, the response of fCO afferents to ramp-and-hold stimuli revealed that acceleration sensitivity was unaffected by octopamine. The observed alterations in the fCO activity indicate that responses to static stimuli are enhanced while responses to motion stimuli are not affected by octopamine. These findings suggest that the octopaminergic modulation of the fCO may affect the animals' posture and those leg movements that rely on position information.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

Serotonin-containing Cells in the Nervous System and Other Tissues During Ontogeny of a Lancelet,Branchiostoma floridae

Abstract Serotonin-containing cells are described by immunohistochemistry throughout lancelet ontogeny. Such cells are first detected in the 2-day larva: these are (1) enterochromaffin cells in the inner epithelium of the gut and (2) anterior serotonergic neurons at the rostral end of the nerve cord. In the 6-day larva, relatively low levels of serotonin appear in ventro-lateral perikarya and cell processes of intraspinal serotonergic neurons scattered along the nerve cord. In the 18-day (early metamorphic) larva, antero-lateral serotonergic neurons are detected near the rostral end of the nerve cord as two small, bilateral clusters of perikarya with axons that descend the nerve cord; at later developmental stages, these axons extend almost to the posterior end of the body. In the 21-day (mid-metamorphic) larva, serotonin can no longer be detected in the anterior serotonergic neurons, but serotonin-containing cells are found subjacent to the inner epithelium of the digestive caecum and in the peribranchial epithelium covering the primary gill bars. In the discussion, we suggest that the anterior serotonergic neurons may play a role in larval photoreception and that the antero-lateral serotonergic neurons may be homologous to vertebrate hindbrain neurons with axons descending the spinal cord to modulate undulation (if this homology is valid, the anterior limit of the lancelet hindbrain would be roughly 100 μm behind the rostral tip of the nerve cord).     

Distribution of tachykinin-related neuropeptide in the developing central nervous system of the moth Spodoptera litura

Neuropeptides with similarities to vertebrate tachykinins, designated tachykinin-related peptides (TRPs), have been identified in several insect species. In this investigation we have utilized an antiserum raised to one of the locust TRPs, locustatachykinin-I (LomTK-I), to determine the distribution pattern of LomTK-like immunoreactive (LTKLI) neurons in the developing nervous system of the moth Spodoptera litura. A number of LTKLI neurons could be followed from the larval to the adult nervous system: a set of median neurosecretory cells (MNCs) in the brain, a pair of brain descending neurons and a few sets on neurons in the ventral nerve cord. The distribution of LTKLI neurons in the adult brain is very similar to that seen in other insect species with prominent arborizations in the central body, antennal lobes, mushroom body calyces, optic lobe neuropils and other distinct neuropil areas in the protocerebrum and tritocerebrum. A new finding is the presence of LTKLI neurosecretory cells with axon terminals in the anterior aorta and corpora cardiaca, suggesting for the first time a neurohormonal role of tachykinin-related peptide(s) in insects. During postembryonic development the number of LTKLI neurons in the ventral nerve cord decreases somewhat, whereas the number increases in the brain. Thus the functional roles of TRPs may change to some extent during development.     

DISTRIBUTION OF AMINO ACIDS AND OF EXO- AND ENDOPEPTIDASES ALONG VERTEBRATE AND INVERTEBRATE NERVES

Abstract— The proximo-distal gradients for representative peptidases, peptidylpeptide hydrolases, and amino acids were measured in segments of peripheral nerve from invertebrates and vertebrates and in the lobster brain and ventral cord.
Crustacean nerve was characterized by a large pool of free amino acids totaling 100–200 μmoles/g wet wt. In lobster nerve, the principal free amino acid was aspartic acid which comprised 55 per cent of the free pool, whereas in the rat sciatic nerve it comprised only 5 per cent. The principal free amino acid in rat sciatic nerve was taurine (32 per cent of the pool) and in lobster brain glycine comprised 30 per cent of the pool. No consistent patterns emerged for the gradients along the nerves for amino acids and hydrolytic enzymes. In the leg nerve of the lobster, concentrations of aspartic acid and arginine were higher in the proximal region, and concentrations of proline and alanine were higher in the distal region. Concentrations of most amino acids were higher in the proximal regions of crab nerve, of lobster brain and ventral cord, and of rat sciatic nerve.
Rat sciatic nerve exhibited a pronounced proximo-distal increase in activity of aminopeptidase (Leu-Gly-Gly). In lobster leg nerve, activity of neutral proteinase was higher in the proximal segment, whereas activity of acid proteinase was higher in the distal segment. The best examples of proximo-distal gradients were found in lobster brain and ventral cord; activities of endopeptidases, arylamidases (Leu- and Arg-βNA), and aminopeptidase were higher in the supra-esophageal ganglia or cephalothorax segments than in the distal regions.     

Patterns of neurogenesis in the midbrain of embryonic lobsters differ from proliferation in the insect and the crustacean ventral nerve cord

Neurogenesis persists throughout life in the olfactory pathway of many decapod crustaceans. However, the relationships between precursor cells and the temporal characteristics of mitotic events in these midbrain regions have not been examined. We have conducted studies aimed at characterizing the sequence of proliferative events that leads to the production of new deutocerebral projection neurons in embryos of the American lobster, Homarus americanus. In vivo bromodeoxyuridine (BrdU) labeling patterns show that three distinct cell types are involved in neurogenesis in this region. Quantitative and temporal analyses suggest that the clearing time for BrdU is 2-3 days in lobster embryos, and that the sequence of proliferative events in the midbrain is significantly different from the stereotypical pattern for the generation of neurons in the ventral nerve cord ganglia of insects and crustaceans. The unusual pattern of proliferation in the crustacean midbrain may be related to the persistence of neurogenesis throughout life in these regions.     

Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana)

Serotonin-like immunoreactivity was mapped in the central nervous system (CNS) of the cockroach, Periplaneta americana. Immunoreactive staining occurred in every ganglion of the CNS. The largest numbers of immunoreactive somata were detected in the optic lobes and the brain, and lowest numbers in the first and second thoracic ganglia. Dense stained fibers, ramifications, and varicosities were found in all ganglia, and numerous axon like processes occurred in all interganglionic connectives. Immunoreactive processes were not, however, detected in most of the peripherally projecting nerve roots. Processes were found only in roots of the suboesophageal ganglion and the tritocerebral lobes of the brain. A comparison of the map for serotonin immunoreactivity with one generated for the pentapeptide transmitter proctolin suggests that the two systems overlap only in the suboesophageal ganglion and the tritocerebrum. The amine and peptide may co-occur in neurons in these regions. The serotonin immunoreactive system appeared significantly different from the octopaminergic system of the ventral nerve cord. Seventy-two potentially identifiable immunoreactive cells were located in the cockroach CNS. Some of these may be suitable for physiological study of the functional role of serotonin.     

Localization of octopaminergic neurones in insects

This paper reviews data on the localization of octopaminergic neurones revealed by immunocytochemistry in insects, primarily the locusts Schistocerca gregaria and Locusta migratoria, cricket Gryllus bimaculatus, and cockroach Periplaneta americana. Supporting evidence for their octopaminergic nature is mentioned where available. In orthopteran ventral ganglia, the major classes of octopamine-like immunoreactive (-LI) neurones include: (1) efferent dorsal and ventral unpaired median (DUM, VUM) neurones; (2) several intersegmentally projecting DUM interneurones in the suboesophageal ganglion; other DUM interneurones are probably GABAergic; (3) a pair of anterior median cells in the prothoracic ganglion; (4) a single pair of ventral cells in most thoracic and some other ganglia; these appear to be plurisegmentally projecting interneurones. Eight categories of octopamine-LI neurones occur in the orthopteran brain. The basic projections of three types are described here: one class project to the optic lobes to form wide field projections. Another type descends to cross into the tritocerebral commissure and may invade the contralateral brain hemisphere. A further class is the median neurosecretory cells with axons in the nervi corpori cardiaci I. Available data for the honey bee Apis mellifera and moth Manduca sexta indicate that the octopamine-LI cell types found in orthopterans also occur in holometabolous insects. Immunocytochemical evidence suggests that some octopaminergic DUM cells contain an FMRFamide-related peptide and the amino acid taurine as putative cotransmitters.     

Serotonin-immunoreactive neurons in the ventral nerve cord of Crustacea: a character to study aspects of arthropod phylogeny

The number of serotonin-expressing neurons in the nervous system of Euarthropoda is small and their neurites have a characteristic branching pattern. They can be identified individually, which provides a character well suited for phylogenetic analyses. In order to gain data that may be useful in the ongoing discussion on insect–crustacean relationships, we documented the pattern of serotonin immunoreactive neurons in the ventral nerve cord of four crustacean species: the phyllocarid malacostracan Nebalia bipes Fabricius, 1780 (Phyllocarida, Leptostraca) and the entomostracans Artemia salina Linnaeus, 1758 (Branchiopoda, Anostraca, Sarsostraca), Triops cancriformis Bosc, 1801 (Branchiopoda, Phyllopoda, Calmanostraca, Notostraca), and Leptestheria dahalacensis Rüppell, 1837 (Branchiopoda, Phyllopoda, Diplostraca, Conchostraca, Spinicaudata). In the entomostracan taxa investigated, the pattern of serotonergic cells in the thoracic hemiganglia comprises an anterior and a posterior bilateral pair of neurons with ipsi- and/or contralateral neurites. Comparing these data to existing information on serotonin-immunoreactivity in the ventral nerve cord of other malacostracan and entomostracan groups enabled us to determine several features of these thoracic neurons being part of the ground pattern of these taxa. Our data demonstrate that studying individually identifiable neurons in Arthropoda can be used to analyse the phylogeny of this taxon.     

Generation of specific behaviors in a locust by local release into neuropil of the natural neuromodulator octopamine

The natural insect neuromodulator octopamine (OCT) was released iontophoretically into regions of neuropil in locust metathoracic ganglia. A narrowly-defined site was found on one side of the ganglion at which release caused a prolonged bout of repetitive flex-extend-flex movements of the tibia on the injected side, at a frequency of from 2-3.5 Hz. When a bout had terminated, repetition of the OCT release caused an extremely similar bout to occur, and again with further treatments, indefinitely. OCT iontophoresis at the equivalent site on the contralateral side caused the contralateral flexor to make stepping movements. Two sites were found, in each half of the ganglion, at which similar OCT release evoked a bout of flight motor activity at 10 Hz. The flight bout involved both sides synchronously and nearly equally, except for a slightly greater motor output on the injected side. Evoked bouts lasted from 20 sec to 25 min depending on the preparation and amount of OCT released. At a site in the 6th abdominal ganglion of mature female locusts OCT release suppressed ongoing rhythmic oviposition digging evoked by severing the ventral nerve cord. A number of previously undescribed DUM neurons was encountered and their dendritic patterns, which are distinctive, determined following dye injection. A hypothesis, termed the Orchestration Hypothesis is presented, which considers how modulator neurons such as locust octopaminergic neurons, might be involved in the generation of specific behaviors.     

A developmental study of serotonin-immunoreactive neurons in the larval central nervous system of the spider crabHyas araneus (Decapoda,Brachyura)

Larval development in crabs is characterized by a striking double metamorphosis in the course of which the animals change from a pelagic to a benthic life style. The larval central nervous system has to provide an adequate behavioural repertoire during this transition. Thus, processes of neuronal reorganization and refinement of the early larval nervous system could be expected to occur in the metamorphosing animal. In order to follow identified sets of neurons throughout metamorphosis, whole mount preparations of the brain and ventral nerve cord of laboratory reared spider crab larvae (Hyas araneus) were labelled with an antibody against the neurotransmitter serotonin. The system of serotonin-immunoreactive cell bodies, fibres and neuropils is well-developed in newly hatched larvae. Most immunoreative structures are located in the protocerebrum, with fewer in the suboesophaegeal ganglia, while the thoracic and abdominal ganglia initially comprise only a small number of serotonergic neurons and fibres. However, there are significant alterations in the staining pattern through larval development, some of which are correlated to metamorphic events. Accordingly, new serotonin-immunoreactive cells are added to the early larval set and the system of immunoreactive fibres is refined. These results are compared to the serotonergic innervation in other decapod crustaceans.     

Ventral nerve cord in Phoronopsis harmeri larvae

The nervous system organization is considered a phylogenetically important character among metazoans. The phylum Phoronida is included in a supraphyletic taxon known as Lophotrochozoa. Many lophotrochozoans possess a metameric ventral nerve cord as adults or larvae. Phoronids do not exhibit external metamery either as larvae or as adults. The current study describes the ventral nerve cord in the young larva of Phoronopsis harmeri. This structure is apparent both in the serotonergic and FMRF-amidergic nervous system in young larvae. The ventral nerve cord extends from the mouth to the tentacular ridge. Both serotonergic and FMRF-amidergic components consist of two ventrolateral nerves, each with several unipolar neurons. The ventrolateral nerves connect to each other by means of thin repetitive transversal nerves ("commissures"). The abundance of neurons and nerves in the epidermis of the oral field of actinotrocha larva likely reflects the importance of this area in collection of food particles. The ventral nerve cords of the actinotrocha and the metatrochophore differ in their positions with respect to ciliated bands: the cord is located between the preoral and postoral ciliated bands in the actinotrocha but between the postoral ciliated band and telotroch in the metatrochophore. The presence of the ventral nerve cord, which contains repetitive elements (neurons and "commissures"), in the early development of P. harmeri may recapitulate some stages of nervous system development during phoronid phylogeny. The larval nervous system does not contain nervous centers under the tentacular ridge that can correlate with the catastrophic metamorphosis and unique body plan of phoronids.     

Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.