Characterisation of multiple immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis with different fixatives and antisera adsorbed with the homologous and the heterologous antigens

In the central nervous system of the pond snail Lymnaea stagnalis a large number of elements (cells and fibers) can be identified with antisera (a-FM) to the molluscan cardioactive tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2). Of these elements some are also reactive to antivasotocin (a-VT) and/or anti-gastrin (a-Gas). These observations suggest that the a-FM positive elements belong to more than one type. Previous results had already indicated that the immunoreactivity of many a-FM positive cells is influenced by the type of fixation. Taking into account the effects of three fixatives on the reactivity of the cells, and their staining characteristics with the two other antisera used, 8 a-FM positive types could be distinguished. Homologous and heterologous adsorptions were carried out to test the specificity of a-FM, a-VT and a-Gas. After homologous adsorptions no staining was obtained. After heterologous adsorptions only part of the multiple staining cells were identified. This indicates that in a-FM, a-VT and a-Gas in addition to (more) selective IgG molecules, less specific IgG molecules occur that can bind to other peptides than those used to raise the antisera (cross-reaction). The (more) selective IgG molecules in a-FM bind to 6 of the a-FM positive types, suggesting that in L. stagnalis a family of FMRFamide-like substances occurs. This conclusion is sustained by results obtained with a-FM adsorbed with fragments of FMRFamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells

We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.     

Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc,Helix aspersa,and in several other invertebrates

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 regionspecific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.     

Immunoreactivities of gastrin (G-) cells. I. dilution-dependent staining of G-cells by antisera and non-immune sera

Results of immunocytochemical studies reported by several laboratories suggest that gastrin (G-) cells of the stomach show immunoreactivities for various pituitary hormones (ACTH, met-enkephalin, beta-endorphin and growth hormone) in addition to gastrin. By reinvestigating the immunocytochemistry of G-cells we found that these cells exhibited reactivities towards a variety of antisera against enteric, pancreatic and hypophyseal hormones. Gastrin cells can also be "immunostained" by antisera towards proteins unrelated to any peptide hormones (e.g. alpha-fetoprotein antiserum) and by nonimmune sera. Thus the specificity of immunocytochemical findings in G-cells seems to be uncertain. According to our findings the polyvalent immunoreactivities of G-cells may be caused by a distinct binding capacity for IgG molecules. This binding of IgG to G-cells seems to be mediated by the Fab fragments of the IgG molecules which may behave like a basic dye and therefore "immunostain" anionic components within G-cells. Thus the significance of the immunocytochemical proof of peptide hormones within G-cells is limited unless extended specificity controls have been performed. The results of specificity controls performed in this study (adsorption controls, use of ascending dilutions of the primary and secondary antisera, comparison of crude antisera and affinity chromatographically purified antibodies) suggest that corticotropin-lipotropin related peptides are not contained in G-cells.     

Regulation of Drosophila FMRFamide neuropeptide gene expression

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell‐ or tissue‐specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide‐related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C‐terminal structure but different N‐terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide‐immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 347–358, 1999     

Localization of thyrotropin (TSH) in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of bovine and rat TSH¹, selective immunocytochemical staining was localized in a specific cell population in the pars distalis of the dog pituitary gland. These TSH cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. With the performic acidalcian blue (pH 0.2) -PAS-orange G procedure these cells stained blue-purple, demonstrating FSH/LH cells (blue or turquoise), ACTH/MSH cells (redpurple) and PRL cells (orange-red). The TSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunocytochemical double staining. In the pars distalis of adult male dogs the TSH cells were mostly shown to be smaller in size and less numerous than in bitches in the anestrous phase of the sexual cycle. Moreover, cytological alterations in the immunoreactive thyrotrophs in the pituitary of male and female dogs generally paralleled the spontaneous changes in thyroid function associated with thyroid atrophy and/or pituitary insufficiency, and thyroid hyperplasia or goiter. In conclusion, because of their specificity and high potency, the antisera to the -subunits of bovine and rat TSH represent an effective tool for the selective immunocytochemical localization of TSH in the dog pituitary. This allows the study of the morphology and function of TSH cells under different physiological, pathological and experimental conditions.Abbreviations for Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - TRH TSH Releasing Hormone - CG Chorionic Gonadotropin The authors are grateful to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.     

A critical examination of the occurrence of FMRFamide immunoreactivity in the brain of guinea pig and rat

Summary Several reports (cf. Weber et al. (1981) Science 214: 1248–1251) have described the extensive occurrence, in rat brain, of material immunologically related to the molluscan neuropeptide FMRFamide. We have reexamined these data in guinea pig and rat, using six different antisera to FMRFamide. Immunoreactive perikarya and fibres were found to be distributed throughout the rodent brain (Table 1). This distribution was roughly similar to that found by Weber and coworkers. However, solid-phase absorption of the antisera with bovine pancreatic polypeptide, which shares an arginine and an amidated aromatic amino acid (RYamide) with FMRFamide, showed that staining in most regions could be due to crossreactivity with bovine pancreatic polypeptide-like (or neuropeptide Y-like) material. Double-labelling experiments with antisera to FMRFamide and bovine pancreatic polypeptide led to the same conclusion. The only structures where no apparent crossreactivity occurred were perikarya and fibres in the nucleus dorsomedialis, ventromedialis, periventricularis and paraventricularis hypothalami and fibres in the area lateralis hypothalami, nucleus parabrachialis, substantia grisea centralis mesencephali, various parts of the formatio reticularis, and spinal cord. Hence only these structures might contain material which is more related to the molluscan retrapeptide.Used one-letter abbreviations of amino acids D aspartic acid - F phenylalanine - G glycine - H histidine - L leucine - M methionine - P proline - R arginine - T threonine - V valine - W tryptophan - Y tyrosine     

Nonspecific immunocytochemical reactions with certain neurohormonal peptides and basic peptide sequences

Immunocytochemical staining experiments on filter paper or nitrocellulose models reveal that many, but not all, neurohormonal peptides, as well as poly-L-lysine, strongly bind a number of labeled reporter molecules, including colloidal gold- or peroxidase-labeled IgG, protein A, streptavidin, and albumin. Peptides displaying this type of (nonspecific) binding are basic; they include ACTH, VIP, opioid peptides, and poly-L-lysine. Pre-absorption of labeled probes with excess ACTH[1-24] or poly-L-lysine abolishes or greatly reduces binding not only to the homologous but also to the heterologous peptides tested. A number of cell types previously reported to display nonspecific immunoglobulin binding contain one or several of the basic neurohormonal peptides shown to display nonspecific absorption of labeled IgG, protein A, streptavidin, and albumin. This nonspecific absorption is reversed neither by high salt nor high pH conditions, nor by a number of detergents and blocking proteins. One dynorphin antiserum also displays nonspecific binding to the peptides as well as to pancreatic glucagon cells, and this nonspecific staining can be blocked by basic peptide pre-absorption (whether homologous or heterologous). These results suggest a need for caution when immunocytochemical studies of a number of basic polypeptides are interpreted, and also suggest the inclusion of novel control procedures in immunocytochemistry.     

FMRFamide- and neurotensin-immunoreactive elements in the intestine of some polyclad and triclad flatworms (Turbellaria)

By means of immunohistochemistry with antisera to tetrapeptide FMRFamide and regulatory peptides neurotensin and calcitonin intestines of marine turbellarians Notoplana atomata, N. humilis (Polycladida) and Procerodes littoralis (Tricladida) were investigated. In all flatworms polymorphous cells and processes reacting with antibodies to FMRFamide and neurotensin but not with calcitonin were revealed. These cell elements are localized both in the epithelium and beneath it. FMRFamide-immunoreactive cells and processes of investigated turbellarians and neurotensin-immunoreactive elements in P. littoralis obviously belong to the nervous system, while intraepithelial neurotensin-immunoreactive cells of polyclads share some morphological features with endocrine-like cells.     

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus

Summary Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP_B-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP_B- and anti-MIP_C-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.     

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Immunoreactivities of gastrin (G-) cells

Summary Results of immunocytochemical studies reported by several laboratories suggest that gastrin (G-) cells of the stomach show immunoreactivities for various pituitary hormones (ACTH, met-enkephalin, -endorphin and growth hormone) in addition to gastrin. By reinvestigating the immunocytochemistry of G-cells we found that these cells exhibited reactivities towards a variety of antisera against enteric, pancreatic and hypophyseal hormones. Gastrin cells can also be immunostained by antisera towards proteins unrelated to any peptide hormones (e.g. -fetoprotein antiserum) and by nonimmune sera. Thus the specificity of immunocytochemical findings in G-cells seems to be uncertain. According to our findings the polyvalent immunoreactivities of G-cells may be caused by a distinct binding capacity for IgG molecules. This binding of IgG to G-cells seems to be mediated by the Fab fragments of the IgG molecules which may behave like a basic dye and therefore immunostain anionic components within G-cells. Thus the significance of the immunocytochemical proof of peptide hormones within G-cells is limited unless extended specificity controls have been performed. The results of specificity controls performed in this study (adsorption controls, use of ascending dilutions of the primary and secondary antisera, comparison of crude antisera and affinity chromatographically purified antibodies) suggest that corticotropin-lipotropin related peptides are not contained in G-cells.Supported by a grant of the Deutsche Forschungsgemeinschaft, SFB 87-G2     

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreactic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust

Summary Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified doral unpaired median (DUM)heart1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co²⁺/Ni²⁺-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina

Summary Immunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide.Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRFamide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y.Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRFamide-like tissue antigen by pancreatic polypeptide antibodies.Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.     

Neuropeptide localization in varicosities of Aplysia sensory neurons is regulated by target and neuromodulators evoking long-term synaptic plasticity

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. We examined with immunocytochemical methods whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT that evoke long-term facilitation or FMRFamide that evoke long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7 compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoked changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites. © 1996 John Wiley & Sons, Inc.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

The immunogenicity of the liposome-associated outer membrane proteins (OMPs) of <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The outer membrane proteins (OMPs) are the most immunogenic and attractive of the Moraxella catarrhalis vaccine antigens that may induce the protective immune response. The aim of this study was to determine the effectiveness of two types of OMP-associated phosphatidylcholine (PC) liposomal formulations (OMPs-PC, PC-OMPs) and of Zwittergent-based proteomicelles (OMPs-Z) in potentiating an anti-OMP systemic immune response in mice. The immunogenicities of the above preparations were evaluated by assessing serum anti-OMP IgG and IgA reactivity in the post-immunized mouse antisera using ELISA and Western blotting. Additionally, the cross-reactivity of the most effective anti-OMP response was determined using heterologous sera from both humans and mice. Both the proteoliposomes and the proteomicelles showed high immunogenic properties and did not elicit any distinct quantitative differences in the antibody titer or qualitative differences in the pattern of the mouse antisera. The post-immunized mouse antisera predominantly recognized a ∼60-kDa OMP of M. catarrhalis. That protein was also found to be a highly cross-reactive antigen interacting with a panel of pooled mouse antisera produced by immunization either with whole cells or the purified OMPs of heterologous M. catarrhalis strains. Furthermore, normal sera collected from healthy children were found to be preferentially reactive with the 60-kDa OMP. The serum-specific IgG, IgA and IgM were respectively detected via immunoblotting in 90%, 85% and 30% of heterologous human sera. This similar immunogenic effectiveness of both OMP-associated liposomal formulations could contribute to the practical use of such formulations in the future in human vaccination. Moreover, the highly cross-reactive 60-kDa OMP seems to be an important antigenic marker of M. catarrhalis, and, as it is responsible for the induction of an antibody-mediated and long-lasting immune response, studying it may partially aid us in understanding the relatively low degree of pathogenicity of the bacterium in immunocompetent individuals.