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.

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.

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.

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).

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.

Glomerular organization in developing olfactory and accessory lobes of American lobsters: stabilization of numbers and increase in size after metamorphosis.

Olfactory glomeruli are columnar and radially arranged at the periphery of the primary chemosensory areas, the olfactory lobes (OLs), in the American lobster Homarus americanus. The number of olfactory glomeruli reaches nearly 100/lobe in midembryonic life, increases rapidly during larval life, and stabilizes at about 200 in juvenile and adult lobsters. The accessory lobes (ALs), higher order integration areas, are composed of cortical columns and of spherical glomeruli. Two populations of spherical glomeruli are defined, the cortical glomeruli located at the bases of the columns, and the medullary glomeruli scattered throughout the ALs. Both cortical columns and spherical glomeruli are seen for the first time in the second larval stage. There are about 1000 cortical columns and 1700 glomeruli/AL in the postlarva and these numbers remain constant during the life of the lobster. In both OLs and ALs, it is the size of the interglomerular spaces and of the glomeruli themselves that increases. Therefore, the data suggest that in both OLs and ALs the glomeruli were already generated when the lobster metamorphoses (stage III to IV) and switches from a planktonic to a benthic existence, and that the new sensory neurons that are formed at each molt in the antennulae grow into existing olfactory glomeruli. Stability of the glomerular population in the primary olfactory centers, once the full complement of glomeruli is acquired, has also been reported in insects, fish, and mammals.

Dopamine in the lobster Homarus gammarus: II. Dopamine-immunoreactive neurons and development of the nervous system.

Dopamine-immunoreactive neurons were revealed in lobster embryos, larvae, and postlarvae, and staining patterns were compared to neuronal labeling in the juvenile lobster nervous system (Cournil et al. [1994] J. Comp. Neurol. 344:455-469). Dopamine immunoreactivity is first detected by midembryonic life in 35-40 neuronal somata located anteriorly in brain and subesophageal ganglion. When the lobsters assume a benthic life during the first postlarval stage, an average of 58 cell bodies are labeled. The acquisition of dopamine in lobster neurons is a protracted event spanning embryonic, larval, and postlarval life and finally reaching the full complement of roughly 100 neurons in juvenile stages. Some of the dopaminergic neurons previously identified in the mature nervous system, such as the paired Br cells, L cells, and mandibular cells, are labeled in embryos and persist throughout development. In contrast, other neurons stain transiently for dopamine during the developmental period, but, by the adult stage, these neurons are no longer immunoreactive. Such transiently labeled neurons project to the foregut, the thoracic dorsal muscles, the neurohormonal pericardial plexus, and the pericardial pouches. It is proposed that these neurons are alive and functioning in adult lobster but that dopamine levels have been abolished, providing that neurotransmitter status is a dynamic, changing process.

Development of the olfactory and accessory lobes in the American lobster: an allometric analysis and its implications for the deutocerebral structure of decapods.

The allometric changes characterizing the growth of the deutocerebrum (midbrain) of the American lobster (Homarus americanus) are studied using computerized three-dimensional reconstructions of serial brain sections. During the embryogenesis of the midbrain, the paired accessory lobes (higher order processing areas) appear later than the paired olfactory lobes (primary olfactory centers), but the former grow faster from their emergence until metamorphosis. The accessory lobes, as they enlarge, shift progressively from a medial to a posterior position in the lateral deutocerebrum. In early juvenile stages the accessory lobes are one of the largest neuropils of the brain. However, these lobes stop growing in adult animals, whereas the brain and olfactory lobes continue to enlarge, albeit at a slow rate. The overall shape of the brain and the relative proportions and locations of the deutocerebral neuropils and associated cell clusters of various lobster ontogenetic stages are similar to those of selected adult decapods. In addition, the relation between deutocerebral organization and brain size seem parallel during lobster development and across crustacean species. Measurements of the brains of 13 species of decapods (illustrated in Sandeman et al. [1993] J. Exp. Zool. 265:112, plus Homarus) indicate the following trends: Small brains possess olfactory lobes but no accessory lobes, larger brains possess accessory lobes that are medial and small relative to the olfactory lobes, and the largest brains contain relatively voluminous posterior accessory lobes. These observations indicate that some differences in the organization of the deutocerebrum are related to absolute brain size in crustaceans and suggest that ontogenetic scaling of proportions may apply to the deutocerebral neuropils of decapods. Peramorphosis and paedomorphosis in the evolution of the decapod brain are considered.

Dopamine in the lobster Homarus gammarus. I. Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the nervous system of the juvenile.

As a catecholamine, dopamine belongs to a class of molecules that have multiple transmitter and hormonal functions in vertebrate and invertebrate nervous systems. However, in the lobster, where many central neurons have been identified and the peripheral innervation pattern is well known, the distribution of dopamine-containing neurons has not been examined in detail. Therefore, immunocytochemical methods were used to identify neurons likely to contain dopamine and tyrosine hydroxylase in the central nervous system of the juvenile lobster Homarus gammarus. Approximately 100 neuronal somata stain for the catecholamine and/or its synthetic enzyme in the brain and ventral nerve cord. The systems of neurons labeled with dopamine and tyrosine hydroxylase antibodies have the following characteristics: 1) the two systems are nearly identical; 2) every segmental ganglion contains at least one pair of labeled neurons; 3) the positions and numbers of cell bodies labeled with each antiserum are similar in the various segmental ganglia; 4) six labeled neurons are anatomically identified; two interneurons from the brain project within the ventral cord to reach the last abdominal ganglion, two neurons from the commissural ganglia are presumably neurosecretory neurons, and two anterior unpaired medial abdominal neurons project to the hindgut muscles; and 5) no cell bodies are labeled in the stomatogastric ganglion, but fibers and terminals in the neuropil are stained. The remarkably small numbers of labeled neurons and the presence of very large labeled somata with far-reaching projections are distinctive features consistent with other modulatory aminergic systems in both vertebrates and invertebrates.

Serotonin-containing neurons in lobsters: their role as gain-setters in postural control mechanisms.

1. The electrophysiological properties of two pairs of identified serotonin-containing neurons in the fifth thoracic (T5) and first abdominal (A1) ganglia of the lobster, Homarus americanus, were studied with the use of intracellular recording methods. Intracellular dye injection combined with immunocytochemistry verified the neurochemical status of the recorded neurons. 2. The serotonin-containing neurons usually are spontaneously active at 0.5-1.0 Hz and produce large, overshooting action potentials with a prominent after-hyperpolarization. The action potentials appear to be generated by a pacemaking mechanism endogenous to the cells. Extracellular recordings from thoracic connectives and from second thoracic roots show that action potentials from the cells in A1 and T5 are propagated rostrally along their axons and invade axon collaterals that innervate neurohemal organs in the second thoracic roots and the pericardial organs. These observations suggest that these serotonin-containing cells may function in part as important neurosecretory cells in the lobster. 3. Members of the pairs of serotonin-containing cells are not synaptically connected. They receive prominent inhibitory inputs in the form of inhibitory postsynaptic potentials (IPSPs), which exhibit discrete size classes and probably arise from several sources. Most IPSPs are temporally synchronized among the two pairs of serotonin-containing cells. 4. The serotonin-containing cells respond to stimulation of postural command fibers, with flexion command fibers exciting and extension command fibers inhibiting the cells, suggesting that these cells are a part of the postural flexion circuitry. 5. Intracellular activation or inhibition of the serotonin-containing cells has no effect on the spontaneous readout of postural motor programs recorded from motor nerve roots. Coactivation of the serotonin-containing cells and command fibers, or inhibition of the serotonin-containing cells while activating command fibers, however, shows that the cells act as "gain-setters," modulating the interaction between command inputs and motoneuron outputs. 6. About 24% of the motor neuron units analyzed are influenced by the serotonin-containing cells. There is a bias toward facilitation of the readout of flexion motor programs, particularly with stimulation of strong and moderate flexion command fibers. 7. The serotonin-containing cells in T5 and A1 ganglia are hypothesized to serve two functions, one tonic and the other phasic, in modulating behavioral output in lobsters. Tonic firing of the cells should result in a sustained release of serotonin from central and peripheral sets of nerve terminals, which, in turn, could influence peripheral and central targets of the amine.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.

SCPB-and FMRFamide-like immunoreactivities in lobster neurons: colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.

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.

FMRFamidelike peptides of Homarus americanus: distribution, immunocytochemical mapping, and ultrastructural localization in terminal varicosities.

The distribution of FMRFamidelike peptides was studied in the nervous system of the lobster Homarus americanus by using immunocytochemical and radioimmunological techniques. By radioimmunoassay FMRFamidelike immunoreactivity (FLI) was found in low levels (ca. 1 pmol/mg protein) throughout the ventral nerve cord and in much higher amounts (60-100 pmol/mg protein) in the neurosecretory pericardial organs. Immunocytochemical studies showed FLI in approximately 300-350 cell bodies, and in distinct neuropil regions, neuronal fiber tracts, and varicose endings. Specificity of the immunostaining was tested by preabsorbing the antiserum with FMRFamide, with peptides having similar carboxyl termini to FMRFamide (Met-enkephalin-Arg-Phe, Phe-Met-Arg-Tyr-amide), with several amidated peptides (alpha-melanocyte-stimulating hormone, substance P, oxytocin), and with proctolin, a peptide found widely distributed in the lobster nervous system. Of these substances, only FMRFamide blocked the staining. In addition to the pericardial organs, significant levels of FLI were found in neurosecretory regions associated with thoracic second roots and in the connective tissue sheath that surrounds the ventral nerve cord. In all three regions, immunocytochemical studies showed the FLI to be localized to fine fibers and associated terminal varicosities lying close to the surface of the tissue, with no obvious target in their immediate vicinity. When examined at the ultrastructural level, the immunoreactive varicosities of the thoracic second roots and of the ventral nerve cord sheaths were found a few microns from the surface of the tissue and contained electron-dense granules. In the immunoreactive nerve cord sheath endings, in addition to the large, dense granules, small, clear vesicles were found. The appearance and location of these terminals suggest a neurohormonal role for FMRFamidelike peptides in lobsters. The observation that low levels of FLI are found in the hemolymph supports this suggestion. In addition, the localization of FLI to particular neuronal somata, fiber tracts, and neuropil regions suggests possible functional roles for these peptides in (1) integration of visual and olfactory information, (2) function of the anterior and posterior gut, and (3) the control of exoskeletal muscles.