Oestradiol and diet modulate energy homeostasis and hypothalamic neurogenesis in the adult female mouse.

Leptin and oestradiol have overlapping functions in energy homeostasis and fertility, and receptors for these hormones are localised in the same hypothalamic regions. Although, historically, it was assumed that mammalian adult neurogenesis was confined to the olfactory bulbs and the hippocampus, recent research has found new neurones in the male rodent hypothalamus. Furthermore, some of these new neurones are leptin-sensitive and affected by diet. In the present study, we tested the hypothesis that diet and hormonal status modulate hypothalamic neurogenesis in the adult female mouse. Adult mice were ovariectomised and implanted with capsules containing oestradiol (E2 ) or oil. Within each group, mice were fed a high-fat diet (HFD) or maintained on standard chow (STND). All animals were administered i.c.v. 5-bromo-2'-deoxyuridine (BrdU) for 9 days and sacrificed 34 days later after an injection of leptin to induce phosphorylation of signal transducer of activation and transcription 3 (pSTAT3). Brain tissue was immunohistochemically labelled for BrdU (newly born cells), Hu (neuronal marker) and pSTAT3 (leptin sensitive). Although mice on a HFD became obese, oestradiol protected against obesity. There was a strong interaction between diet and hormone on new cells (BrdU+) in the arcuate, ventromedial hypothalamus and dorsomedial hypothalamus. HFD increased the number of new cells, whereas E2 inhibited this effect. Conversely, E2 increased the number of new cells in mice on a STND diet in all hypothalamic regions studied. Although the total number of new leptin-sensitive neurones (BrdU-Hu-pSTAT3) found in the hypothalamus was low, HFD increased these new cells in the arcuate, whereas E2 attenuated this induction. These results suggest that adult neurogenesis in the hypothalamic neurogenic niche is modulated by diet and hormonal status and is related to energy homeostasis in female mice.

An identified serotonergic neuron regulates adult neurogenesis in the crustacean brain.

New neurons are born and integrated into functional circuits in the brains of many adult organisms. In virtually all of these systems, serotonin is a potent regulator of neuronal proliferation. Specific neural pathways underlying these serotonergic influences have not, however, been identified and manipulated. The goal of this study was to test whether adult neurogenesis in the crustacean brain is influenced by electrical activity in the serotonergic dorsal giant neurons (DGNs) innervating the primary olfactory processing areas, the olfactory lobes, and higher order centers, the accessory lobes. Adult-born neurons occur in two interneuronal cell clusters that are part of the olfactory pathway. This study demonstrates that neurogenesis also continues in these areas in a dissected, perfused brain preparation, although the rate of neuronal production is lower than in brains from intact same-sized animals. Inclusion of 10(-9) M serotonin in the perfusate delivered to the dissected brain preparation restores the rate of neurogenesis to in vivo levels. Although subthreshold stimulation of the DGN does not significantly alter the rate of neurogenesis, electrical activation of a single DGN results in significant increases in neurogenesis in Cluster 10 on the same side of the brain, when compared with levels on the contralateral, unstimulated side. Measurements of serotonin levels in the perfusate using high-performance liquid chromatography established that serotonin levels are elevated about 10-fold during DGN stimulation, confirming that serotonin is released during DGN activity. This is the first identified neural pathway through which adult neurogenesis has been directly manipulated.

Hormonal and synaptic influences of serotonin on adult neurogenesis.

New neurons are incorporated into the adult brains of a variety of organisms, from humans and higher vertebrates, to non-vertebrates such as crustaceans. In virtually all of these systems serotonergic pathways appear to provide important regulatory influences over the machinery producing the new neurons. We have developed an in vitro preparation where adult neurogenesis can be maintained under highly controlled conditions, and are using this to test the influence of hormones on the production of neurons in the crustacean (Homarus americanus) brain. Serotonin levels have been manipulated in this in vitro preparation, and the resulting effects on the rate of neurogenesis have been documented. In addition we have compared in vitro influences of serotonin with results acquired from in vivo exposure of whole animals to serotonin. These experiments suggest that there are multiple mechanisms and pathways by which serotonin may regulate neurogenesis in the crustacean brain: (1) serotonin is effective in regulating neurogenesis at levels as low as 10(-10)M, suggesting that circulating serotonin may have hormonal influences on neuronal precursor cells residing in a vascular niche or the proliferation zones; (2) contrasting effects of serotonin on neurogenesis (up- vs. down-regulation) at high concentrations (10(-4)M), dependent upon whether eyestalk tissue is present or absent, indicate that serotonin elicits the release of substances from the sinus glands that are capable of suppressing neurogenesis; (3) previously demonstrated (Beltz, B.S., Benton, J.L., Sullivan, J.M., 2001. Transient uptake of serotonin by newborn olfactory projection neurons. Proc. Natl. Acad. Sci. USA 98, 12730-12735) serotonergic fibers from the dorsal giant neuron project directly into the proliferation zone in Cluster 10, suggest synaptic or local influences on neurogenesis in the proliferation zones where the final cell divisions and neuronal differentiation occur. Serotonin therefore regulates neurogenesis by multiple pathways, and the specific mode of influence is concentration-dependent.

Nitric oxide in the crustacean brain: regulation of neurogenesis and morphogenesis in the developing olfactory pathway.

Nitric oxide (NO) plays major roles during development and in adult organisms. We examined the temporal and spatial patterns of nitric oxide synthase (NOS) appearance in the embryonic lobster brain to localize sources of NO activity; potential NO targets were identified by defining the distribution of NO-induced cGMP. Staining patterns are compared with NOS and cyclic 3,5 guanosine monophosphate (cGMP) distribution in adult lobster brains. Manipulation of NO levels influences olfactory glomerular formation and stabilization, as well as levels of neurogenesis among the olfactory projection neurons. In the first 2 days following ablation of the lateral antennular flagella in juvenile lobsters, a wave of increased NOS immunoreactivity and a reduction in neurogenesis occur. These studies implicate nitric oxide as a developmental architect and also support a role for this molecule in the neural response to injury in the olfactory pathway.

Regulation of serotonin levels by multiple light-entrainable endogenous rhythms.

This study examined whether serotonin levels in the brain of the American lobster, Homarus americanus, are under circadian control. Using high-performance liquid chromatography and semi-quantitative immunocytochemical methods, we measured serotonin levels in the brains of lobsters at six time points during a 24-h period. Lobsters were maintained for 2 weeks on a 12 h:12 h light:dark cycle followed by 3 days of constant darkness. Under these conditions, brain serotonin levels varied rhythmically, with a peak before subjective dusk and a trough before subjective dawn. This persistent circadian rhythm in constant darkness indicates that serotonin levels are controlled by an endogenous clock. Animals exposed to a shifted light cycle for >10 days, followed by 3 days in constant darkness, demonstrate that this rhythm is light entrainable. Separate analyses of two pairs of large deutocerebral neuropils, the accessory and olfactory lobes, show that serotonin levels in these functionally distinct areas also exhibit circadian rhythms but that these rhythms are out of phase with one another. The olfactory and accessory lobe rhythms are also endogenous and light entrainable, suggesting the presence of multiple clock mechanisms regulating serotonin levels in different brain regions.

Neural pathways connecting the deutocerebrum and lateral protocerebrum in the brains of decapod crustaceans.

The olfactory and accessory lobes of eureptantian decapod crustaceans are bilateral brain neuropil regions located within the deutocerebrum. Although the olfactory lobe seems to receive only primary olfactory inputs, the accessory lobe receives higher-order multimodal (including olfactory) inputs. The output pathways from both the olfactory and accessory lobes are provided by the axons of a large population of projection neurons, whose somata lie adjacent to the lobes. The axons of these neurons form a large tract that projects bilaterally to the medulla terminalis and hemiellipsoid body in the lateral protocerebrum. To gain insights into the ways in which olfactory information is processed on leaving the deutocerebrum, we examined the neuroanatomy of the projection neuron pathways of three species of eureptantian decapod crustaceans: the freshwater crayfish, Procambarus clarkii and Orconectes rusticus, and the clawed lobster, Homarus americanus. Projection neurons were labeled by focal injections of the lipophilic tracers DiI and DiA into the olfactory and accessory lobes. In all three species, projection neurons innervating the accessory lobe were found to exclusively innervate the neuropils of the hemiellipsoid body. In contrast, projection neurons innervating the olfactory lobes primarily target neuropil regions of the medulla terminalis. The results of this study indicate, therefore, that the projection neuron pathways from the olfactory and accessory lobes project to separate, largely nonoverlapping regions of the lateral protocerebrum. The implications of these findings for our understanding of the processing of olfactory information in the brains of decapod crustaceans are discussed.

Development and connectivity of olfactory pathways in the brain of the lobster Homarus americanus.

The main output pathways from the olfactory lobes (primary olfactory centers) and accessory lobes (higher-order integrative areas) of decapod crustaceans terminate within both of the main neuropil regions of the lateral protocerebrum: the medulla terminalis and the hemiellipsoid body. The present study examines the morphogenesis of the lateral protocerebral neuropils of the lobster, Homarus americanus, and the development of their neuronal connections with the paired olfactory and accessory lobes. The medulla terminalis was found to emerge during the initial stages of embryogenesis and to be the target neuropil of the output pathway from the olfactory lobe. In contrast, the hemiellipsoid body is first apparent during mid-embryonic development and is innervated by the output pathway from the accessory lobe. The dye injections used to elucidate these pathways also resulted in the labeling of a previously undescribed pathway linking the olfactory lobe and the ventral nerve cord. To increase our understanding of the morphology of the olfactory pathways in H. americanus we also examined the connectivity of the lateral protocerebral neuropils of embryonic lobsters. These studies identified several interneuronal populations that may be involved in the higher-order processing of olfactory inputs. In addition, we examined the neuroanatomy of ascending pathways from the antenna II and lateral antenna I neuropils (neuropils involved in the processing of chemosensory and tactile inputs). These studies showed that the ascending pathways from these neuropils innervate the same regions of the medulla terminalis and that these regions are different from those innervated by the olfactory lobe output pathway.

Transient uptake of serotonin by newborn olfactory projection neurons.

A life-long turnover of sensory and interneuronal populations has been documented in the olfactory pathways of both vertebrates and invertebrates, creating a situation where the axons of new afferent and interneuronal populations must insert into a highly specialized glomerular neuropil. A dense serotonergic innervation of the primary olfactory processing areas where these neurons synapse also is a consistent feature across species. Prior studies in lobsters have shown that serotonin promotes the branching of olfactory projection neurons. This paper presents evidence that serotonin also regulates the proliferation and survival of projection neurons in lobsters, and that the serotonergic effects are associated with a transient uptake of serotonin into newborn neurons.

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters.

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.

Agonistic behavior in naïve juvenile lobsters depleted of serotonin 5,7-dihydroxytryptamine.

We have been exploring the role of serotonin in fighting behavior in lobsters using a specific model of agonistic behavior, the establishment of hierarchical relationships between pairs of socially naive juvenile lobsters. We selected this model because the behavior is easily evoked, readily quantifiable, and the effects of experience are eleminated by using socially naive animals. In these studies we injected a specific neurotoxin, 5,7-dihydroxytryptamine, into juvenile lobsters over a 4-week period and then measured the effects on fighting behavior. This treatment reduces the levels of serotonin in the nervous system and immunocytochemical studies show a dramatic reduction in neuropil staining for the amine. Control animals received vehicle injection alone. All injected animals were paired against larger or smaller non-injected opponents, and three successive 30-min fights were carried out and statistically analyzed. The results were surprising: As with elevations of serotonin, reduced levels of serotonin increased the amount of time animals engaged in fighting behavior. No significant effects were seen on who initiated encounters, who retreated first, or who the eventual winner would be. Thus, in this model, elevation or reduction of serotonergic function increases the tendency of animals to engage in agonistic encounters.

Serotonin depletion in vivo inhibits the branching of olfactory projection neurons in the lobster deutocerebrum.

Serotonin depletion during embryogenesis has been shown previously to retard the growth of the olfactory and accessory lobes of the lobster deutocerebrum (Benton et al., 1997). The present study was undertaken to determine whether morphological changes in the interneurons innervating these lobes contribute to this growth retardation. We examined the effects of in vivo serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) on the morphology of the olfactory projection neurons, one of two major classes of interneurons that innervate both lobes. Intracellular dye fills of olfactory projection neurons in normal embryos showed that each neuron extensively innervates either the olfactory or accessory lobe before projecting to neuropil regions in the protocerebrum. In embryos injected with 5,7-DHT, however, the deutocerebral arbors of 13.5% of the olfactory projection neurons examined were either markedly reduced compared with normal neurons or absent. Affected neurons also exhibited a number of additional aberrant morphological features suggesting that these neurons represent cells that were affected during their initial morphogenesis. Olfactory projection neurons with aberrant morphologies were also encountered, although less frequently (7.5% of the neurons examined), in control (sham-injected) embryos indicating that the sham injections can affect the development of the brain. This observation provides insights into the nature of effects seen in control embryos in previous experiments (Benton et al., 1997). The results of the present study indicate that in vivo serotonin depletion inhibits the branching of olfactory projection neurons and suggest, therefore, that one of the functions of serotonin during normal development is to promote the ingrowth of these neurons into the deutocerebral neuropils.

An unusual case of a mutant lobster embryo with double brain and double ventral nerve cord.

We report the rare finding of a Siamese twin embryo of the American lobster Homarus americanus. Immunohistochemical labeling of this mutant with an antibody directed against Drosophila synaptic proteins revealed that the embryo had a structurally normal visual system with two compound eyes and eyestalk Anlagen but twin brains and twin ventral nerve cords. We have analyzed the patterns of connectivity of the components of the nervous system and have concluded that the wiring pattern in this nervous system provides a logical and elegant way of connecting the parts of the twin system in this unusual mutation.

Crustacean hyperglycemic hormone in the lobster nervous system: localization and release from cells in the subesophageal ganglion and thoracic second roots.

Crustacean hyperglycemic hormones (CHHs) are neuropeptides involved in the regulation of hemolymph glucose. The primary source of CHHs has been identified as the neurosecretory neurons of the eyestalk X-organ and its associated neurohemal organ, the sinus gland. We have identified another source of CHH-like peptides in the nervous system. With the use of immunocytochemistry, cells in the second roots of the thoracic ganglia have been observed to stain positively for CHH-reactive material. We also identified a pair of cells in the subesophageal ganglion that contain large amounts of CHH-reactive material. Depolarization of these cells with elevated potassium mediates a calcium-dependent release of CHH-like material from the ganglion as quantified with an enzyme-linked immunosorbent assay (ELISA).

A new look at embryonic development of the visual system in decapod crustaceans: neuropil formation, neurogenesis, and apoptotic cell death.

In recent years, comparing the structure and development of the central nervous system in crustaceans has provided new insights into the phylogenetic relationships of arthropods. Furthermore, the structural evolution of the compound eyes and optic ganglia of adult arthropods has been discussed, but it was not possible to compare the ontogeny of arthropod visual systems, owing to the lack of data on species other than insects. In the present report, we studied the development of the crustacean visual system by examining neurogenesis, neuropil formation, and apoptotic cell death in embryos of the American lobster, Homarus americanus, the spider crab, Hyas araneus, and the caridean shrimp, Palaemonetes argentinus, and compare these processes with those found in insects. Our results on the patterns of stem cell proliferation provide evidence that in decapod crustaceans and hemimetabolous insects, there exist considerable similarities in the mechanisms by which accretion of the compound eyes and growth of the optic lobes is achieved, suggesting an evolutionary conservation of these mechanisms.

From embryo to adult: persistent neurogenesis and apoptotic cell death shape the lobster deutocerebrum.

Neuronal plasticity and synaptic remodeling play important roles during the development of the invertebrate nervous system. In addition, structural neuroplasticity as a result of long-term environmental changes, behavioral modifications, age, and experience have been demonstrated in the brains of sexually mature insects. In adult vertebrates, persistent neurogenesis is found in the granule cell layer of the mammalian hippocampus and the subventricular zone, as well as in the telencephalon of songbirds, indicating that persistent neurogenesis, which is presumably related to plasticity and learning, may be an integral part of the normal biology of the mature brain. In decapod crustaceans, persistent neurogenesis among olfactory projection neurons is a common principle that shapes the adult brain, indicating a remarkable degree of life-long structural plasticity. The present study closes a gap in our knowledge of this phenomenon by describing the continuous cell proliferation and gradual displacement of proliferation domains in the central olfactory pathway of the American lobster Homarus americanus from early embryonic through larval and juvenile stages into adult life. Neurogenesis in the deutocerebrum was examined by the in vivo incorporation of bromodeoxyuridine, and development and structural maturation of the deutocerebral neuropils were studied using immunohistochemistry against Drosophila synapsin. The role of apoptotic cell death in shaping the developing deutocerebrum was studied using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling method, combined with immunolabeling using an antiphospho histone H3 mitosis marker. Our results indicate that, in juvenile and adult lobsters, birth and death of olfactory interneurons occur in parallel, suggesting a turnover of these cells. When the persistent neurogenesis and concurrent death of interneurons in the central olfactory pathway of the crustacean brain are taken into account with the life-long turnover of olfactory receptor cells in crustacean antennules, a new, highly dynamic picture of olfaction in crustaceans emerges.

Distribution and functional anatomy of amine-containing neurons in decapod crustaceans.

One of the lessons learned from studying the nervous systems of phylogenetically distant species is that many features are conserved. Indeed, aminergic neurons in invertebrate and vertebrate systems share a multitude of common characteristics. In this review, the varied roles of serotonin, octopamine, dopamine, and histamine in decapod crustaceans are considered, and the distributions of the amine-containing cells are described. The anatomy of these systems reinforces the idea that amine neurons are involved in widespread modulation and coordination within the nervous system. Many aminergic neurons have long projections, linking multiple regions with a common input, and therefore are anatomically perfected as "gain setters." The developmental patterns of appearance of each amine in the crustacean nervous system are described and compared. The developmental picture suggests that transmitter acquisition is distinctive for each amine, and that the pace of acquisition may be co-regulated with target maturation. The distinctive roles that transmitters play during specific developmental periods may, ultimately, provide important clues to their functional contributions in the mature organism.

Neurogenesis in the thoracic neuromeres of two crustaceans with different types of metamorphic development

The mode of embryonic and larval development and the ethology of metamorphosis in the spider crab and the American lobster are very different, and we took advantage of this to compare neuronal development in the two species. The goals of this study were to discover whether the differences in the maturation of the neuromuscular system in the pereopods and the metamorphic changes of motor behavior between the two species are reflected at the level of the developing nervous system ('neurometamorphosis'). Furthermore, we wanted to broaden our understanding of the mechanisms that govern neuronal development in arthropods. Proliferation of neuronal stem cells in thoracic neuromeres 4-8 of the lobster Homarus americanus and the crab Hyas araneus was monitored over the course of embryonic and larval development using the in vivo incorporation of bromodeoxyuridine (BrdU). Neuropil structure was visualized using an antibody against Drosophila synapsin. While proliferation of neuronal precursors has ceased when embryogenesis is 80 % complete (E80%) in the lobster thoracic neuromeres, proliferation of neuroblasts in the crab persists throughout embryonic development and into larval life. The divergent temporal patterns of neurogenesis in the two crustacean species can be correlated with differences in larval life style and in the degree of maturation of the thoracic legs during metamorphic development. Several unusual aspects of neurogenesis reported here distinguish these crustaceans from other arthropods. Lobsters apparently lack a postembryonic period of proliferation in the thoracic neuromeres despite the metamorphic remodeling that takes place in the larval stages. In contrast, an increase in mitotic activity towards the end of embryonic development is found in crabs, and neuroblast proliferation persists throughout the process of hatching into the larval stages. In both E20% lobster embryos and mid-embryonic crabs, expression of engrailed was found in a corresponding set of neurons and putative glial cells at the posterior neuromere border, suggesting that these cells have acquired similar specific identities and might, therefore, be homologous. None of the BrdU-labeled neuroblasts (typically 6-8 per hemineuromere over a long period of embryogenesis) was positive for engrailed at this and subsequent stages. Our findings are discussed in relation to the spatial and temporal patterns of neurogenesis in insects.

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster, Homarus americanus.

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A approximately 90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas.

Amines and peptides in the brain of the American lobster: immunocytochemical localization patterns and implications for brain function.

The distributions of serotonin- (5HT-), substance P- (SP-), small cardioactive peptideb- (SCPb-), and histamine- (HA-) like immunoreactivities were examined in the adult lobster supraesophageal ganglion. Vibratome sections were labeled using avidin-biotin-peroxidase immunocytochemical methods. The localization patterns for each substance were assessed in 21 regions within the median protocerebrum, deutocerebrum, and tritocerebrum. Each immunoreactivity has a unique distribution within the brain; however, most regions are immunoreactive for more than one neurotransmitter. Of particular interest are SP-immunoreactive protocerebral neurons that contact olfactory projection neurons and appear homologous to those found in other crustaceans. Regional differences in immunolabeling within the deutocerebral olfactory and accessory lobes suggest that specific areas within individual olfactory lobe glomeruli serve distinct functions in olfactory processing, and that subpopulations of accessory lobe glomeruli are innervated by different groups of neurons. This detailed comparison of the labeling patterns also has allowed us to define the anatomical connectivity between several cell body clusters, fiber tracts, and neuropil areas in the lobster brain.

Developmental expression of the octopamine phenotype in lobsters, Homarus americanus.

We have used immunocytochemical methods to examine the sequence of appearance of octopamine-immunoreactive neurons during development, and to try to correlate that appearance with the emergence of behavioral or physiological capabilities. The first octopamine neurons express their transmitter phenotype at approximately 43% of embryonic development. The last cells show immunostaining at the 3rd larval stage. In the wild, therefore, immunoreactivity in cells appears over a 9-12 month period. In contrast, serotonin-immunoreactive neurons stain early in embryonic development and the last serotonin-immunoreactive cells appear at about the same time the first octopamine-immunoreactive neurons show staining. The pattern of appearance of octopamine-immunoreactive cells is cell type-specific. A pair of brain cells and the descending interneurons stain first. Additional brain cell staining is seen throughout embryonic development. The ascending interneurons appear next, and a general anterior-posterior gradient typifies their emergence over a relatively short portion of embryonic life (E 48-62%). The neurosecretory cell staining appears last, is segment-specific, begins at about 62% development, and continues to the 3rd larval stage. The emergence of immunostaining for amine neurotransmitters within groups of identified neurons at precise times in development may specify possible functional units. With at least one group of cells, this possibility seems plausible: the three pairs of claw octopamine neurosecretory cells show immunostaining as a unit.