Histochemical evidence for catecholamines as neurotransmitters in the statocyst of Octopus vulgaris

Summary Formaldehyde-induced fluorescence (Falck-Hillarp technique) provided histochemical evidence for the presence of catecholamines in the sensory epithelia (macula and crista) of the Octopus statocyst. A specific bright green fluorescence occurred in the neuronal plexus beneath the receptor cell layers of the epithelia and in the appropriate nerves. The histochemical findings are discussed with reference to the well-known neuronal and synaptic organization of the epithelia and to relevant results in cephalopods as well as in other molluscs. All data support the hypothesis that in the receptor systems of the Octopus statocyst catecholamines (probably dopamine and/or noradrenaline) act as neurotransmitters in the efferent fibre system.     

Efferent activity in theOctopus statocyst nerves

Summary Single unit electrophysiological recordings were obtained from efferent fibres in the statocyst nerves ofOctopus vulgaris. A preparation comprising the CNS and a single statocyst was employed. 42% of the efferents displayed a level of resting activity; transient changes in this activity occurred at irregular intervals.The responses of the efferent units were examined during sinusoidal oscillations of the statocyst at stimulus frequencies between 0.01–1 Hz, and amplitudes up to 35°. 84% of the units showed activity synchronised with the imposed oscillations; the time taken to establish this response varied for different units (Fig. 1).The lowest stimulus frequency at which a unit could be entrained varied for different units, with those units with a resting level of activity having the lowest thresholds. The peak firing frequency of the efferents was found to increase with increasing stimulus frequency or amplitude (Fig. 3). However, the change in firing frequency was much smaller than that reported for the statocyst afferents to similar stimuli.The efferent units of the posterior crista nerve were found to respond to clockwise or anticlockwise rotations (Fig. 4), with the individual units having unipolar responses. The phase response of the units changed little with increasing stimulus amplitude but an increase in phase lag occurred with an increase in the stimulus frequency (Fig. 5). The form of this relationship (Fig. 6) was similar to that reported for the statocyst crista afferents.The principal source of the input to the efferents in these experiments was shown to be afferents from the contralateral statocyst. These results are discussed and compared with data from the vertebrate semicircular canal system.     

Non-inhibition of Acetylcholinesterase by cyclosal Nucleotides

Abstract

An acetylcholinesterase (AChE) assay based on the Rappaport method was established to investigate the behaviour of several cycloSal nucleotides against AChE from electrophorus electricus and human sources (purified enzymes). AChE is a physiologically essential enzyme as it catalyzes the hydrolysis of the neurotransmitter acetylcholine. No inhibition was observed in any of the cases.     

Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17°C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 × 10^–4 M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.     

Immunocytochemical applications in neuroanatomy

Summary In the present paper we review immunocytochemical methods for anterograde tracing with the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L), combined PHA-L tracing — neurotransmitter immunocytochemistry, and the immunocytochemical localization of receptor proteins. These methods will be mainly illustrated by examples from tracing- and neurotransmitter studies on the cholinergic basal forebrain system. The morphology of PHA-L labeled neurons strongly resembles that of Golgi impregnated neurons. The complete axonal trajectories and patterns of presynaptic endings of PHA-L labeled neurons are visualized, both for light- and electron microscopic application.PHA-L-tracing can very well be combined with second immunocytochemical labeling procedures. In this way, traced pathways can be studied in their relation to chemically identified fiber systems or target neurons. Application of immunocytochemistry for the localization of the muscarinic acetylcholine receptor, albeit in its early stages, holds great promise for the near future.     

The origin ofSenecio vulgaris (Asteraceae)

Senecio vulgaris L. (2n = 40) is suggested to be of autotetraploid origin fromS. vernalis Waldst. & Kit. (2n = 20). This conclusion is based on results obtained from experimental hybridisations and cytological observations, and the consideration of morphological affinities, patterns of geographical distribution and hybrid formation under natural conditions. The morphological differences between the two species are related to a difference in the breeding system. WhilstS. vernalis is self-incompatible,S. vulgaris is self-compatible and strongly self-pollinating. Equally, other self-pollinating taxa traditionally associated withS. vulgaris are shown to have evolved independently from outbreeding relatives. Within a narrow frame of relationship, annual weeds have evolved in different cytotaxonomic circumstances.     

Efferent projections from the lateral septal nucleus to the anterior hypothalamus in the rat: A study combining Phaseolus vulgaris-leucoagglutinin tracing with vasopressin immunocytochemistry

Summary The tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral septum of the rat at different rostrocaudal locations to study the efferent septal projections to the anterior hypothalamus. For spatial correlation of these septofugal elements with the vasopressinergic system a dual immunocytochemical technique was used (i) to demonstrate nerve fibers and their corresponding bouton-like structures labeled with the tracer, and (ii) to identify vasopressin in the same section. The hypothalamic paraventricular and supraoptic nuclei, the accessory hypothalamic magnocellular system, and the suprachiasmatic nucleus are recipients of PHA-L-labeled fibers from all parts of the lateral septum. Close appositions between (i) these axons and their varicosities, and (ii) vasopressin-immunoreactive perikarya and their processes, putatively indicating functional interrelationships, were observed in all these nuclear areas, especially in their neuropil formations.Abbreviations F fornix - OC optic chiasm - OT optic tract - PVN paraventricular nucleus - SCN suprachiasmatic nucleus - SON supraoptic nucleus - III third ventricle     

Secondary hair cells and afferent neurones of the squid statocyst receive both inhibitory and excitatory efferent inputs

Summary Intracellular recordings were obtained from the hair cells and afferent neurones of the angular acceleration receptor system of the statocyst of the squid,Alloteuthis subulata. Electrical stimulation of the efferent fibres in the crista nerve (minor) evoked responses in all of the secondary hair cells recorded from (n=211). 48% of the secondary air cells responded with a small depolarization, 15% with a hyperpolarization, and 37% with a depolarization followed by a hyperpolarization. The depolarizations and hyperpolarizations had mean stimulus to response delays of 6.7 ms and 24 ms, and reversal potentials of about –1 mV and –64 mV, respectively. Both types of potential increased in amplitude, up to a point, when the stimulus shock was increased and facilitation and/or summation effects could be obtained by applying multiple shocks. These data, together with the fact that both responses could be blocked by bath application of cobalt or cadmium, indicate that the secondary hair cells receive both inhibitory and excitatory efferent inputs and that these are probably mediated via chemical synapses. No efferent responses were seen in the primary hair cells but both depolarizing and hyperpolarizing efferent responses were obtained from the afferent neurones.     

Two-Component Signal Transduction Systems of Desulfovibrio vulgaris: Structural and Phylogenetic Analysis and Deduction of Putative Cognate Pairs

A large number of two-component signal transduction systems (TCSTS) including 59 putative sensory histidine kinases (HK) and 55 response regulators (RR) were identified from the Desulfovibrio vulgaris genome. In this study, the structural and phylogenetic analyses of all putative TCSTSs in D. vulgaris were performed. The results showed that D. vulgaris contained 21 hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Despite the low sequence similarity that restricted the resolution of the phylogenetic analyses, most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in Escherichia coli and Bacillus subtilis. An attempt was made in this investigation to identify the possible cognate HK-RR pairs not linked on the chromosome in D. vulgaris based on similar expression patterns in response to various environmental and genetic changes. Expression levels of all HK and RR genes were measured using whole-genome microarrays. Five groups of HK-RR genes not linked on the chromosome were identified as possible cognate pairs in D. vulgaris. The results provided a preliminary list of possible cognate HK-RR pairs and constitute a basis for further exploration of interaction and physiological function of TCSTSs in D. vulgaris. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Gail Simmons]     

Susceptibility of dry bean (Phaseolus vulgaris L.) to Agrobacterium infection: Transformation of cotyledonary and hypocotyl tissues

A germinating-seed assay was developed to determine the susceptibility of dry bean (Phaseolus vulgaris L.) to infection by Agrobacterium tumefaciens. Seedlings infected one to three days after germination were more susceptible to A. tumefaciens infection than seedlings germinated for five to seven days and the galls that formed on the one to three day seedlings were significantly larger. Nineteen genotypes of dry bean were screened with this assay and all were equally susceptible to nopaline, octopine and agropine biotypes of A. tumefaciens. In addition, cotyledonary nodes and hypocotyls of P. vulgaris were inoculated with disarmed strain A. tumefaciens strain C58Z707 and the avirulent A. rhizogenes strain A4RS (pRiB278b), respectively. Both strains contain the binary plasmid pGA482 which has the neomycin phosphotransferase II (NPT II) gene nested between T-DNA borders. From these infected tissues, callus and root tissues, respectively capable of growing in the presence of kanamycin were obtained. These tissues displayed NPT II activity and integrated copies of the NPT II gene were detected from putative transformed root cultures by genomic blot hybridization.     

Localization of choline acetyltransferase-expresing neurons in the larval vissual system of Drosophila melanogaster

Choline acetyltransferease (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5 flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.This work was supported by a grant from the National Institute of Neurological Disorders and Stroke.     

Organization of statocysts in the Otoplanidae (Plathelminthes): an ultrastructural analysis with implications for the phylogeny of the Proseriata

Summary The statocyst of otoplanids is enveloped by a bipartite capsule which consists of two different extracellular matrices. This capsule encircles three different types of aciliary cells: several peripherally located flattened parietal cells, one central statolith forming cell (lithocyte) and two clusters of accessory cells. Intracapsular lumina exist which are different from extracapsular intercellular spaces. The accessory cells most probably represent those structures that are mainly involved in nervous conduction. These cells extend cytoplasmatic processes towards different peripheral regions of the statocyst where processes of outer nerve cells penetrate the capsule. The statocyst does not seem to represent a more evolved equilibrium receptor system but may function as a relatively simple aciliary sense organ suitable for positive geotactic behaviour. The otoplanid statocyst corresponds to statocysts in other lithophorous proseriates but not to statocysts in other taxa of the free-living Plathelminthes. The monophyly of a taxon Lithophora within the Proseriata is corroborated by this autapomorphic characteristic.Abbreviations ac accessory cell(s) - c capsule of the statocyst - ce cerebrum - ci cephalic intestine - co capsule opening - cp cell process(es) of accessory cell(s) and cell(s) containing filaments - ecm extracellular matrix - fc cell(s) containing filaments - ic intercellular spaces within the capsule - mc muscle cell(s) - n lobed nucleus of the lithocyte - nac nucleus (nuclei) of accessory cell(s) - nc nerve cell(s) - npc nucleus (nuclei) of parietal cell(s) - pc parietal cell(s) - s statolith - sc statolith cell (lithocyte)     

Effect of aromatic plant species on vesicular-arbuscular mycorrhizal establishment in Pistacia terebinthus

The inoculation of Pistacia terebinthus with vesicular-arbuscular mycorrhizal (VAM) fungi and the spread of the infection were studied using a mixed cropping system, under glasshouse conditions, with Salvia officinalis, Lavandula officinalis and Thymus vulgaris colonized by Glomus mosseae as an inoculation method. This method was compared with soil inoculum placed under the seed or distributed evenly in the soil. Indirect inoculation with all the aromatic plants tested significantly increased VAM root colonization of P. terebinthus compared with the use of soil inoculum, although the effect on plant growth was different for each one of the aromatic species used as inoculum source. Inoculation with L. officinalis and T. vulgaris were the best treatments resulting in high VAM colonization and growth enhancement of P. terebinthus.     

Putative neurotransmitters in the retinae of three urodele species (Triturus alpestris,Salamandra salamandra,Pleurodeles waltli)

Summary The immunocytochemical localization of several substances with putative neurotransmitter or modulator properties was investigated in the retinae of three urodele species. Gamma-aminobutyric acid-like immunoreactive labelling appeared in different types of amacrine and horizontal cells. In addition, labelled fibres in the optic nerve were detected. It was not possible to determine whether these fibres were ganglion-cell axons or part of an efferent projection. Endogenous serotonin was found in several populations of amacrine cells including stratified and diffuse types. Glucagon-like immunoreactivity appeared in one bistratified amacrine cell type, and neurotensin-like immunoreactivity was detected in a single monostratified amacrine cell type. Metenkephalin-like-immunoreactive labelling was rare but found in several sublaminae of the inner plexiform layer. Thus each peptide-like-immunoreactive cell type makes up a distinct and unique population of cells and probably has a special functional role in retinal processing. There are striking similarities in the peptide-like immunoreactive patterns of Triturus alpestris and Necturus maculosus whereas in Ambystomatidae the peptide-like-immunoreactive systems appear to be differently organized. This supports the hypothesis that Salamandridae and Proteidae are more closely related to each other than to the Ambystomatidae.Abbreviations GABA gamma-aminobutyric acid - GCL ganglion cell layer - Glu glucagon - HRP horseradish peroxidase - INL inner nuclear layer - IPL inner plexiform layer - IR immunoreactive or immunoreactivity - M-enk metenkephalin - Neu neurotensin - OFL optic fibre layer - ONL outer nuclear layer - OPL outer plexiform layer - Ser serotonin This work forms part of the doctoral thesis of Gaby Gläsener, Faculty of Biology, Technical University of Darmstadt, Federal Republic of Germany. Supported by a research grant from the Deutsche Forschungsgemeinschaft (Hi 306/1-1)     

Plant species effect on the spatial patterns of soil properties in the Mu-us desert ecosystem,Inner Mongolia,China

Although Artemisia ordosica Krasch. and Sabina vulgaris Ant. are the dominant shrub species in the Mu-us desert ecosystem, they differ in their botanical traits. We investigated the spatial patterns of soil properties using geostatistical analysis to examine the effect of plant species on these spatial patterns. Comparison among three microsite types (under A. ordosica, under S. vulgaris, and the opening between vegetation) showed that A. ordosica generally had less effect than S. vulgaris on local soil properties. The long life-span, prostrate life-form, and evergreen leaf-habit of S. vulgaris may lead to a higher accumulation of organic and fine materials under S. vulgaris. The range of spatial autocorrelation found in the mass of organic matter on the soil surface was smaller than that of the coverage of S. vulgaris (11.5 m) which corresponded to the canopy patch size of this species, and was longer than the canopy patch size of A. ordosica. The ranges of total C and N, and pH (11.7–15.6 m) were similar to that of S. vulgaris. The range of available P (106.3 m) was comparable to that of the coverage of A. ordosica (86.2 m) considered to be the scale of the distribution of this species. The ranges of silt+clay and exchangeable K, Ca, and Mg (31.0–46.7 m) were not related to plant presence, and were similar to that of topography (43.1 m).     

Isozyme evidence and the origin ofSenecio vulgaris (Compositae)

An electrophoretic survey of isozyme variation was conducted to test the hypothesis thatSenecio vulgaris L. (2n = 40) is of autotetraploid origin fromS. vernalis Waldst. & Kit. (2n = 20). It was established thatS. vulgaris exhibited fixed heterozygosity at three loci examined, showed disomic inheritance at all polymorphic loci, and contained a gene (Est-1) and an allele (Aat-3b) which were not present in the single population ofS. vernalis surveyed. From this it is concluded thatS. vulgaris is not of autotetraploid origin. Instead, the genetic evidence is in keeping with an allopolyploid origin ofS. vulgaris with the possibility thatS. vernalis acted as one of its two parents.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

水陆生境和氮沉降对香菇草(Hydrocotyle vulgaris)入侵湿地植物群落的影响

大气氮沉降对湿地外来植物入侵的影响已成为生态学研究的热点之一。选用梭鱼草(Pontederia cordata)、水菖蒲(Acorus calamus)、黄花鸢尾(Iris wilsonii)和粉绿狐尾藻(Myriophyllum aquaticum)模拟湿地植物群落,设置有无香菇草入侵、水生和陆生生境(水位分别为15 cm和0 cm)及有无氮沉降处理(15 g N m~(-2)a(-1)和0)交叉组成的8种处理组合,进行为期70d的温室控制实验,以分析水陆生境及氮沉降对香菇草入侵湿地植物群落的影响。结果表明:(1)水生生境下,香菇草的总生物量、叶生物量、叶片数和节点数与陆生生境相比显著降低;水生生境下氮沉降处理对香菇草各指标无显著影响,陆生生境下氮沉降处理的香菇草叶片数、节点数显著多于无氮沉降处理。(2)实验周期内氮沉降和香菇草入侵没有对群落的多样性指数及群落内4种湿地植物的生物量产生显著影响;水生生境下植物群落的总生物量及梭鱼草和粉绿狐尾藻的生物量与陆生生境相比显著提高。(3)水生生境下香菇草的相对优势度相比陆生生境下显著下降;氮沉降只在陆生生境下显著提高了香菇草的相对优势度。因此,香菇草向水生生境扩散和入侵的能力不强,其入侵在短时间内对湿地植物群落影响较小。研究结果可以为入侵植物生态学研究提供案例借鉴。     

Histamine is a major mechanosensory neurotransmitter candidate in Drosophila melanogaster

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.     

RFamide neuropeptide actions on molluscan proboscis smooth muscle: interactions with primary neurotransmitters

The potency (muscle force-generated) of a number of long-chain RFamide neuropeptides was examined in mechanical experiments with the radular-retractor and radular-sac muscles of gastropods Buccinum undatum and Neptunea antiqua. Many of the heptapeptides, octapeptides and the decapeptide LMS were found to induce greater contraction than FMRFamide in both smooth muscles and in both species. RFamide neuropeptides interacted with the neurotransmitter acetylcholine in an additive way and RFamide-induced contractions were inhibited by the neuromodulator serotonin. Pre-treatment with a calcium-free saline completely abolished acetylcholine-induced responses but only partially inhibited RFamide responses in the muscles, suggesting that acetylcholine acts to cause influx of extracellular calcium for contraction. In contrast, RFamide neuropeptides may mobilise intracellular calcium to maintain sustained tonic force in calcium-free conditions. This suggests that an additional involvement of a fast calcium channel may be present in the RFamide responses, since loss of the usual superimposed twitch activity is observed. Force regulation in these muscles appears to result from a complex interaction of RFamide neuropeptides with the primary transmitter acetylcholine and the neuromodulator serotonin.Abbreviations ACh acetylcholine - Ala alanine - Arg arginine - Asn asparagine - Asp aspartic acid - Cys cysteine - FLRFamide Phe-Leu-Arg-Phe-NH₂ - FMRFamide Phe-Met-Arg-Phe-NH₂ - Gln glutamine - Glu glutamic acid - Gly glycine - His histadine - Ile isoleucine - Leu leucine - LMS leucomyosuppressin - Met methionine - Nle norleucine - Phe phenylalanine - Pro proline - SCP_B (small cardioactive peptide B) Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-NH₂ - Ser serine - Val valine