Encoding of plant odour information in insects: peripheral and central mechanisms

Insects are suitable model organisms for studying mechanisms underlying olfactory coding and olfactory learning, by their unique adaptation to host plants in which the chemical senses are essential. Recent molecular biological studies have shown that a large number of genes in insects and other organisms are coding for olfactory receptor proteins. In general, one receptor type seems to be expressed in each neurone. The functional characterisations of olfactory receptor neurones have been extensive in certain insect species, demonstrating a fine-tuning of single neurones to biologically relevant odourants; both insect and plant produced volatiles. Stained neurones of the same functional type have been shown to project in one and the same glomerular unit in the primary olfactory centre, the antennal lobe. This corresponds to molecular biological studies, showing projections in one glomerulus by neurones expressing the same receptor type. Comparison of these findings with physiological and morphological characterisations of antennal lobe neurones has indicated correspondence between input and output of the glomerular units. Examples are presented from studies of heliothine moths. From the antennal lobe, the olfactory information is further conveyed to the mushroom bodies, particularly important for learning, and the lateral protocerebrum, a premotoric area. The three brain areas are regions of synaptic plasticity important in learning of odours, which is well studied in the honeybee but also in species of moths.     

NADPH-diaphorase activity in the nodose ganglion of normal and vagotomized guinea-pigs

The activity and distribution of reduced nico-tinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) in the nodose ganglion of normal and vagotomized guinea-pigs were examined by light and electron microscopy. Light microscopy confirmed a remarkable increase in the number of NADPH-diaphorase-reactive neurons in the nodose ganglion following unilateral cervical vagotomy. The increase was present at 5 days but became more prominent at 10 days and was sustained until at least 30 days after vagotomy when compared with the non-lesioned side. The NADPH-diaphorase reaction product was associated with the membrane of the rough endoplasmic reticulum, Golgi apparatus, mitochondria and nucleus of the nodose neurons. In animals killed 5 days post-operation, there was no noticeable degeneration in the nodose neurons. However, at 10 days, the mitochondria in some neurons appeared swollen and vacuolated with disrupted cristae. These changes were accentuated in some nodose neurons 20 and 30 days after vagotomy but there was no evidence of cell death. All the degenerating neurons exhibited NADPH-diaphorase activity. The increase in NADPH-diaphorase activity in the neuronal somata after vagotomy suggests that the enzyme is involved in either the retrograde degeneration or the recovery of the lesioned neurons. Received: 15 June 1995 / Accepted: 15 February 1996     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.     

Detection of NADPH-diaphorase activity in Paramecium primaurelia

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.     

Electron microscopic studies of the lung of the frog

Summary Scanning and transmission electron microscopy were used to study the inner architecture of the frog lung. In some specimens the alveolar surface mucus layer was removed to permit the examination of underlying features. The inner surface of the frog's lung is covered by a layer of microvilli belonging to only one type of epithelial cells. The boundaries of these epithelial cells are demarcated by small ridges. Different degrees of lung expansion cause variations of the surface topography. The morphology of certain surface features is examined in detail. Several methods of drying the specimens are compared.The author wishes to thank Dr. I. E. Richter, Institut für allgemeine und experimentelle Pathologie der Bundeswehr, Mainz, for the opportunity to do these investigations and for helpful discussions.     

The sorting behaviour of olfactory and vomeronasal axons during regeneration

Summary In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.     

Electron microscopic examination of uncultured soil-dwelling bacteria

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.     

Ultrastructural localization of NADPH-diaphorase and colocalization of nitric oxide synthase in endothelial cells of the rabbit aorta

This is the first report on the ultrastructural pattern of distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in endothelial cells, using the rabbit aorta, and its colocalization with the neuronal isoform (type I) of nitric oxide synthase. About 30% of the endothelial cells showed a positive reaction for NADPH-d compared to about 6% for nitric oxide synthase immunoreactivity. Simultaneous double histochemical-immunocytochemical labelling procedures indicate that all of the cells displaying nitric oxide synthase-positive reactivity also contained NADPH-d; the remainder of NADPH-d-positive endothelial cells were negative for this isoform of nitric oxide synthase. Nitric oxide synthase-immunogold labelling was mostly associated with free ribosomes, while NADPH-d activity was distributed largely in patches in the cytoplasm and in association with the cell membrane.     

Mushroom body input connections form independently of sensory activity in Drosophila melanogaster

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Electron microscopic study of Burkholderia cepacia biofilms

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilm) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated.     

Electron microscopic examination of the extracellular polymeric substances in anaerobic granular biofilms

Scanning electron microscopy revealed that collapsed extracellular polymeric substances (EPS) surrounded bacteria present in granular sludge. Treatment of granular sludge with whole-cell antiserum and staining with polycationic ferritin demonstrated that bacteria were enveloped by extensive EPS. Antibody stabilization permitted a visualization of the EPS which more closely resembled its natural hydrated state. The EPS was seen to completely fill the intercellular spaces in the microcolonies. Both pure and mixed microcolonies were observed to be enclosed by EPS. The presence of these large amounts of EPS indicates that this extracellular layer is important in maintaining the structural integrity of granular sludge.     

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2–4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.     

Scanning electron microscopic study of the olfactory epithelium of four coldwater hillstream teleosts from Garhwal hills (India)

This contribution deals with the scanning electron microscopic surface structure of olfactory epithelium in four hillstream teleosts from the glacialfed river Alaknanda in Garhwal Himalaya (UP, India). The closely related species—Schizothorax plagiostomus, Schizothorax richardsonii (both bottom dweller, bottom feeder, herbiomnivorous) andSchizothoraichthys progastus (column dweller, column feeder, carniomnivorous) reveal the predominance of different types of olfactory receptor cell types separately in their respective olfactory epithelium while the distinctly related speciesCrossocheilus latius latius with similar nature as first two (i.e. bottom dweller, bottom feeder, herbiomnivorous) displays the presence of more microvillous cells in the olfactory epithelium. Possibly, the occurrence of particular receptor cells in a fish species is related to the ecological and feeding behaviours with distinct mechanism of olfaction.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10

We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis.     

Maxillary palp glomeruli and ipsilateral projections in the antennal lobe of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The antennal lobe was examined by Golgi-silver impregnation to differentiate the glomeruli depending on the source and types of inputs. Thirty-five of the 43 ‘identified’ olfactory glomeruli were Golgi-silver impregnated in the present study. Seven glomeruli compared to three, reported previously, were found to be targets of maxillary palp chemosensory neurons. These include glomeruli VA3, VC2, VM5, VA7m/VA7l of the ventral antennal lobe and DC2, DC3, DM5 of the dorsal antennal lobe. The number of glomeruli receiving the maxillary palp sensory projections tallies with the number ofDrosophila olfactory receptors (seven) reported to be expressed exclusively in the maxillary palp. Twenty-eight Golgi-impregnated glomeruli were found to receive input from the antennal nerve. The ratio of glomeruli serving the maxillary palp to those serving the antenna (∼1:5) matches with the ratio ofDrosophila olfactory receptors expressed in these two olfactory organs respectively. In addition to glomerulus V, glomeruli VP1-3, VL1, VL2a/2p and VC3m/3l were found to receive ipsilateral projections. Thus, additional ipsilateral glomeruli have been identified.     

Expansion and Accelerated Evolution of 9-Exon Odorant Receptors in Polistes Paper Wasps

Independent origins of sociality in bees and ants are associated with independent expansions of particular odorant receptor (OR) gene subfamilies. In ants, one clade within the OR gene family, the 9-exon subfamily, has dramatically expanded. These receptors detect cuticular hydrocarbons (CHCs), key social signaling molecules in insects. It is unclear to what extent 9-exon OR subfamily expansion is associated with the independent evolution of sociality across Hymenoptera, warranting studies of taxa with independently derived social behavior. Here, we describe OR gene family evolution in the northern paper wasp, Polistes fuscatus, and compare it to four additional paper wasp species spanning ∼40 million years of evolutionary divergence. We find 200 putatively functional OR genes in P. fuscatus, matching predictions from neuroanatomy, and more than half of these are in the 9-exon subfamily. Most OR gene expansions are tandemly arrayed at orthologous loci in Polistes genomes, and microsynteny analysis shows species-specific gain and loss of 9-exon ORs within tandem arrays. There is evidence of episodic positive diversifying selection shaping ORs in expanded subfamilies. Values of omega (d_N/d_S) are higher among 9-exon ORs compared to other OR subfamilies. Within the Polistes OR gene tree, branches in the 9-exon OR clade experience relaxed negative (relaxed purifying) selection relative to other branches in the tree. Patterns of OR evolution within Polistes are consistent with 9-exon OR function in CHC perception by combinatorial coding, with both natural selection and neutral drift contributing to interspecies differences in gene copy number and sequence.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.