Coupling between sensory neurons in the olfactory epithelium

Coupling of olfactory sensory neurons (OSNs) in the olfactory epithelium of Necturus maculosus was demonstrated by dye-transfer with Lucifer yellow CH; however, the incidence of dye-transfer was low. Immunocytochemistry and Western blot analysis indicated that connexin 43, a gap junction channel subunit, was widely expressed by cells in the olfactory epithelium. Electrical coupling by presumptive gap junctions was assessed using electrophysiological recordings, heptanol block, tracer-uptake through hemi-junctions, and tracer-injection into tissue whole-mounts. Coupling, which involved pairs of OSNs only, was detected in approximately 3-10% of the OSN population; there was no evidence that OSNs were coupled into extended neural syncitia. These results suggest that coupling of OSNs by gap junctions is unlikely to have a general role in olfactory responses by mature (odor responsive) OSNs. Instead, the incidence of inter-neuronal coupling was small, similar to the fraction of immature OSNs, suggesting a possible role of gap junctions in the continual turnover and development of OSNs or possibly their senescence.     

Whole-cell recordings and photolysis of caged compounds in olfactory sensory neurons isolated from the mouse

Gene manipulation and molecular biological techniques for the study of olfaction are well developed in mice, while electrophysiological properties of mouse olfactory sensory neurons have been less extensively investigated. We used the whole-cell voltage-clamp technique in mouse isolated olfactory sensory neurons to investigate both voltage-gated and transduction currents. Voltage-gated currents were composed of transient inward currents followed by outward currents with transient and sustained components. Of the tested olfactory sensory neurons, 12% responded to the odorant cineole with an inward current. Caged compounds were introduced into the cytoplasm through the patch pipette and flash photolysis of caged cyclic nucleotides activated an inward current in 94% of the cells. When the flash was localized at the cilia, the response latency, rising time and duration were shorter than when the flash illuminated the soma. The amplitude of the photolysis response was dependent on light intensity and the relation was fitted by the Hill equation, with a Hill coefficient of 3.2. These results demonstrate that it is possible to obtain recordings in the whole-cell configuration from olfactory sensory neurons isolated from the mouse and that voltage-gated currents and transduction properties are largely similar to those of amphibians.     

Mitotic and meiotic chromosomes of the American lobster Homarus americanus (Nephropidae, Decapoda)

The chromosomes of H. americanus have been characterised by C-banding, fluorochrome banding and restriction endonuclease banding. Thanks to these techniques, it has been possible to identify mitotic and meiotic figures clearly and to study the distribution and structure of heterochromatic regions. Moreover, we have identified small supernumerary chromosomes, variable in number and often asynaptic in first meiotic metaphase.     

Copper transport by lobster (Homarus americanus) hepatopancreatic lysosomes

Lysosomes are known centers for sequestration of calcium and a variety of heavy metals in many invertebrate tissues, and as a result of this compartmentalization these organelles perform important detoxification roles in the animals involved. The present investigation uses a centrifugation method to isolate and purify hepatopancreatic lysosomes from the American lobster, Homarus americanus. Purified lysosomal preparations were used to characterize membrane transport mechanisms in these organelles for transferring and sequestering cytoplasmic copper following its absorption across the plasma membrane from dietary constituents. The copper-specific fluorescent dye, Phen Green, was employed to quantify transmembrane fluxes of this metal as has been recently used to investigate copper movements across hepatopancreatic mitochondrial and plasma membranes. Results indicated the presence of a vanadate-sensitive, calcium-stimulated, copper ATPase in the membranes of these organelles that displayed high affinity carrier-mediated transport kinetics and may significantly contribute to organismic copper homeostasis. Together with a putative bafilomycin-sensitive V-ATPase in the membrane of the same organelles, importing hydrogen ions into the organellar interior, this copper ATPase may function as part of a physiological mechanism for precipitate formation between metallic cations and anions. These ionic precipitate complexes may then act as a sink for excess metals and thereby reduce the circulating concentrations of these elements.     

An historical overview of the Republic of Ireland's lobster (Homarus gammarus Linnaeus) fishery,with reference to European and North American (Homarus americanus Milne Edwards) lobster landings

Historical developments of the Republic of Ireland's Homarus gammarus fishery are reviewed and compared in the context of landings in Europe and the North American H. americanus fishery. H. gammarus fishing has provided essential income for over a century to many Irish coastal communities. There have been significant fluctuations (greater than 460%) in recorded landings over this time and the relative importance of H. gammarus fishing has changed with the establishment of ancillary crustacean fisheries. In 1994, H. gammarus landings reached an all-time high of 714 tonnes, with a landed value of IR £6.3 million; in 1999 landings were 597 tonnes valued at IR £6.15 million. Current Republic of Ireland lobster fishery regulations are: (a) minimum carapace length of 85 mm, (b) ban on landing lobsters that have been `V-notched' or have a mutilated tail fan, and (c) prohibition of capturing lobsters by SCUBA diving. Chronological details on recent legislation, regulations and landings relating to Ireland's lobster fishery sector are reviewed. In the last 40 years landings of H. gammarus in the Republic of Ireland exhibit signs of an overexploited stock characterised by a reduction in catch per unit effort and there has been a general decline of total landings for H. gammarus throughout Europe. This contrasts with North America, where many regulations govern the H. americanus fishery and landings appear to be in a healthier state.     

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.This work was supported by funds from the University of Virginia Research and Development Committee.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Sustained primary culture of lobster (Panulirus argus) olfactory receptor neurons

Appropriate conditions were developed for primary sustained culture of olfactory neurons of the spiny lobster Panulirus argus. Neurons were cultured in a modified Liebowitz L15 media supplemented with Panulirus salts, basic minimal essential (BME) vitamins, L-glutamine, low dextrose, and either fetal calf serum (FCS) or lobster haemolymph. Neurite outgrowth and cell viability was strongly affected by choice of adherent substratum, presence of serum, and length of animal captivity. Neither nerve growth factor 7s (NGF-7s), HEPES, nor preconditioned media from the target organ, the olfactory lobe, had any gross effect on either longevity or neurite outgrowth. Five morphologically distinct neuronal cell types (8-16 mum soma diameter) could be defined based on their number and type of processes. All of these cells were electrically excitable (N = 50), and many (56%) produced either inward or outward currents in response to stimulation with single odors. The proportion of cells responding to odors increased (80%) when 10 cells were sequentially presented with a series of 3-5 odors. The finding that cultured cells maintain responsiveness to odors yet are morphologically more compact than their counterparts in situ, argues for the prospect of using these dissociated cultured olfactory receptor neurons to study signal transduction in olfaction.     

Analysis of the functional maturation of olfactory neurons in chicks before and after birth

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.     

Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium

During development, primary olfactory axons typically grow to their topographically correct target zone without extensive remodelling. Similarly, in adults, new axons arising from the normal turnover of sensory neurons essentially project to their target without error. In the present study we have examined axon targeting in the olfactory pathway following extensive chemical ablation of the olfactory neuroepithelium in the P2-tau:LacZ line of mice. These mice express LacZ in the P2 subpopulation of primary olfactory neurons whose axons target topographically fixed glomeruli on the medial and lateral surfaces of the olfactory bulb. Intraperitoneal injections of dichlobenil selectively destroyed the sensory neuroepithelium of the nasal cavity without direct physical insult to the olfactory neuron pathway. Primary olfactory neurons regenerated and LacZ staining revealed the trajectory of the P2 axons. Rather than project solely to their topographically appropriate glomeruli, the regenerating P2 axons now terminated in numerous inappropriate glomeruli which were widely dispersed over the olfactory bulb. While these errors in targeting were refined over time, there was still considerable mis-targeting after four months of regeneration.     

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley-Liss, Inc.     

Iron uptake by hepatopancreas brush border membrane vesicles (BBMV) of the lobster (Homarus americanus)

The uptake of (55)Fe(2+) and solubilized (55)Fe(3+) into brush border membrane vesicles prepared from the hepatopancreas of the Atlantic lobster (Homarus americanus) was investigated. Non-specific surface binding of (55)Fe(2+) at equilibrium to the vesicular surface approximated 57% of total (55)Fe(2+) uptake. (55)Fe(2+) uptake showed temperature sensitivity and was trans-stimulated by a Ca(2+) gradient (at 5mM) directed out. Equilibrated (59)Fe(2+) exchanged for both Cd(2+) and cold Fe(2+). The data obtained in this study are suggestive that at least a portion of ferrous iron absorption may occur by a divalent exchanger mechanism.     

A naturalistic analysis of autobiographical memories triggered by olfactory visual and auditory stimuli

The emotional and content qualities of autobiographical memories evoked by three memory cue items (campfire, fresh-cut grass, popcorn) presented in olfactory, visual and auditory form were examined using a new repeated measures paradigm. Results revealed that memories recalled by odors were significantly more emotional and evocative than those recalled by the same cue presented visually or auditorily. However, there were no differences in the content features (vividness, specificity) of memories as a function of cue-form. These findings support previous research in both laboratory and naturalistic settings and is the first comparative sensory memory study to include auditory variants of memory cues. The present data contribute to a growing body of evidence indicating that there is a privileged relationship between olfaction and emotion during recollection. Various subject factors such as age, sex and region of residence were also examined and some were found to affect the quality of memories in interaction with the specific memory cue items, indicating that prior experience is a primary influence in autobiographical memory. Questions for future investigation regarding how odor-evoked memories may be different from other memory experiences are suggested.     

The complete mitochondrial genome of the American lobster, Homarus americanus (Crustacea, Decapoda)

Although relatively a large number of the complete mitochondrial genome sequences have been determined from various decapod species (29 mtDNA sequences reported so far), the information for the infraorder Astacidea (including lobsters, crayfishes, and their relatives) is very limited and represented by only one complete sequence from the Australian freshwater crayfish species Cherax destructor. In this study, we determined the complete mitochondrial DNA sequence of Homarus americanus, the first representative of the family Nephropidae to be fully characterized. Comparison of the gene arrangement reveals that H. americanus mtDNA is identical to those of other pancrustacean species but differs from the other astacidean species (C. destructor). Based on these data, it can be assumed that an idiosyncratic gene order discovered in C. destructor mtDNA may be secondarily acquired from the ancestral lineage of the Astacidea.     

Effects of starvation on moult cycle and hepatopancreas of Stage I lobster(Homarus americanus) larvae

Effects of feeding and starvation on the moult cycle and on the ultrastructure of hepatopancreas cells were studied in Stage I lobster larvae (Homarus americanus Milne-Edwards). The relative significance of yolk and first food was quite different in larvae originating from two females. This difference was evident also in the amounts of stored lipid in the R-cells of the larval hepatopancreas. Most larvae from one hatch were, in principle, able to develop exclusively with yolk reserves (without food) to the second instar. The larvae from the second hatch showed lecithotrophic development only to the transition between late intermoult and early premoult (Stages C/D₀ of Drachs's moult cycle) of the first larval instar. When initial starvation in this group lasted for 3 days or more, the point of no return (PNR) was exceeded. After the PNR, consumption of food was still possible, but development ceased in the transition C/D₀ or in late premoult (D_3–4). It is suggested that these stages of the moult cycle are critical points were cessation of development and increased mortality are particularly likely in early larval lobsters under nutritional stress. Examination of hepatopancreas R-cells suggested that the PNR is caused by an irreversible loss of the ability to restore lipid reserves depleted during initial starvation. Initial periods of starvation ending before the PNR prolonged mainly Stage D₀ of the same instar (I). During this delay, structural changes in the R-cells caused by the preceding period of starvation were reversed: reduced lipid inclusions, swollen mitochondria, an increased number of residual bodies indicating autolysis, and a reduction of the microvillous processes. Continually starved larvae which showed lecithotrophic development throughout the first instar and were then re-fed after moulting successfully, had later a prolonged intermoult (Stage C) period in the second instar. This shows that, despite occasional lecithotrophy, food is an important factor in early larval development of the lobster.