Sensory transduction in an insect mechanoreceptor: extended bandwidth measurements and sensitivity to simulus strenght

The dynamic properties of sensory transduction in an insect mechanoreceptor, the femoral tactile spine of the cockroach, Periplaneta americana, have been studied by measurement of the frequency response function between randomly varying movement of the tactile spine and afferent action potentials from the sensory neuron which innervates it. The frequency response function of the mechanoreceptor has been characterized over a frequency range which is more than ten times larger than has previously been used for this preparation. Also the effects of varying the amplitude of the stimulating signal have been studied by the use of a range of input signal strengths from about 0.5 to 10 m R.M.S. displacement. The measured frequency response functions can all be well fitted by a theoretical relationship which is a fractional exponent of complex frequency, provided that the time delay caused by conduction of the action potentials from the sensory dendrite to the recording electrodes is taken into account. Under small signal conditions the exponent of complex frequency is close to 0.5 but with larger displacements its value decreases to about half this value. The overall sensitivity of the receptor, as measured by the gain of the frequency response function at a natural frequency of 1 radian/s, is not significantly altered by changes in the input movement amplitude, so that the receptor behaves linearly in this respect. However, the mean rate of action potential occurrence is not linearly related to input movement amplitude. These results are discussed in terms of current theories of sensory transduction and the possible role of tubular bodies in the dynamic behaviour of insect cuticular mechanoreceptors.     

Sensory Regeneration in Arthropods: Implications of Homoeosis and of Ectopic Sensilla

SYNOPSIS. The seemingly antithetic attributes of rigorous connectivityon one hand and vigorous regeneration on the other, are combinedin the arthropod nervous system This apparent paradox is largelyresolved by the comparison of normal postembryonic developmentwith regeneration, which is also restricted to immature stagesIt is also becoming apparent that growth and interaction betweenneurons is more flexible than had been assumed. Normal sensory regeneration in situ is highly specific in restoringlost function The crucial event in regeneration as in embryonicdevelopment, is the establishment of first contacts betweenperiphery and center Thereafter regeneration follows an acceleratedrecapitulation of normal postembryonic development. Data from ectopic grafts, homoeotic mutants and homoeotic regeneratesaddress four components of sensory development and regenerationa) Positional information in the epidermis determines receptortype and central projection, b) Passage from periphery to ganglionis non specific Ectopic neurons reach mismatched ganglia c)Within neuropile the specific projection is a product of interactionbetween intrinsic programs of the neuron and pathways expressedas specific surface markers d) Fine tuning of synaptic relationshipscan occur in response to changed milieu The current elucidationof the genetic basis of metameric segment determination, andthe identification of specific gene products as markers of pathwaysopen the way to the understanding of neural specificity in developmentand regeneration at the molecular level.     

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.     

Specificity of the collagenase from the insect Hypoderma lineatum

Specificity of the collagenase from the larvae Hypoderma lineatum, a serine protease related to trypsin, has been investigated by using native collagen and non-collagenous substrates. At 25 degrees C and neutral pH the degradation of collagen by the larval enzyme in solution results in a 52% loss of specific viscosity, without loss of helicity. Electron microscopy of segment-long-spacing crystallites of the digest shows the occurrence of one cleavage region between bands 41 and 44 whereas Edman degradation indicates several cleavage loci in this region. Hypoderma collagenase differs from proteinases I and II from the crab Uca pugilator, which catalyse cleavages in multiple regions of the collagen molecule, and also from vertebrate collagenases, which cleave collagen only between residues 775 and 776. Apart of specific action on collagen, Hypoderma collagenase degrades the oxidized chain B of insulin; the major cleavage occurs at the Leu15-Tyr16 bond followed by two minor cleavages at the Arg22-Gly23 and Lys29-Ala30 bonds. The larval enzyme has no action on synthetic peptide substrates of trypsin or chymotrypsin.     

Sensory inputs stimulate progenitor cell proliferation in an adult insect brain

Although most brain neurons are produced during embryonic and early postnatal development, recent studies clearly demonstrated in a wide range of species from invertebrates to humans that new neurons are added to specific brain structures throughout adult life. Hormones, neurotransmitters, and growth factors as well as environmental conditions modulate this neurogenesis. In this study, we address the role of sensory inputs in the regulation of adult neural progenitor cell proliferation in an insect model. In some insect species, adult neurogenesis occurs in the mushroom bodies, the main sensory integrative centers of the brain, receiving multimodal information and often considered as the analog of the vertebrate hippocampus. We recently showed that rearing adult crickets in enriched sensory and social conditions enhanced neuroblast proliferation in the mushroom bodies. Here, by manipulating hormonal levels and affecting olfactory and/or visual inputs, we show that environmental regulation of neurogenesis is in direct response to olfactory and visual stimuli rather than being mediated via hormonal control. Experiments of unilateral sensory deprivation reveal that neuroblast proliferation can be inhibited in one brain hemisphere only. These results, obtained in a relatively simple brain, emphasize the role of sensory inputs on stem cell division.     

Location and dynamic properties of the spike generator in an insect mechanosensory neuron

1.The cereal bristle hairs of the cockroach, Periplaneta americana, are each innervated by one mechanosensory cell and 1–5 chemosensory cells. In transepithelial recordings, chemo- and mechanosensory spikes could be discriminated from each other by their relative amplitude. 2. When current steps were applied via the sensory hair, trains of impulses were triggered whatever the polarity of the current. 3. All responses adapted to the current, but the time course of adaptation was fitted by a power law for outward currents and an exponential law for inward currents. 4. During application of outward currents, the spikes showed a negative initial phase on which a small positive component was superimposed; strong polarizations produced purely negative spikes. More classical spikes with a positive initial phase were induced by inward currents. 5. The present work supports the hypothesis of a direct excitability of the apical dendrite in cereal bristle mechanoreceptors and confirms previous results suggesting that spikes are normally triggered within that region during mechanical stimulations. It is also established, for the first time, that adaptation to currents may be different in the apical dendrite and in more basal regions of the same mechanosensory neuron.Abbreviation RP receptor potential - TEV transepithelial voltage - TTX tetrodotoxin     

Defining Specificity Determinants of cGMP Mediated Gustatory Sensory Transduction in Caenorhabditis elegans

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.     

Sensory control of leg movement in the stick insect Carausius morosus

Hind legs with crossed receptor-apodemes of the femoral chordotonal organ when making a step during walking often do not release the ground after reaching the extreme posterior position. After putting a clamp on the trochanter (stimulation of the campaniform sensilla) the leg is no longer protracted during walking. However, during searching-movements the same leg is moved very far forwards. The anatomical situation of the campaniform sensilla on the trochanter and the sensory innervation of the trochanter is described. After removal of the hair-rows and continuously stimulating the hair-plate at the thorax-coxa-joint the extreme anterior and posterior positions of the leg in walking are displaced in the posterior direction. Front and middle legs operated in this way sometimes do not release the ground at the end of retraction. In searching-movements the same leg is moved in a normal way. If only one side of a decerebrated animal goes over a step, then on the other side a compensatory effect is observed. The main source of this compensatory information appears to be the BF1-hair-plates. If the animal has to drag a weight the extreme anterior and posterior positions of the middle and hind legs are displaced in the anterior direction. Crossing the receptor-apodeme of the femoral chordotonal organ, when it causes the leg to remain in the protraction phase, displaces the extreme posterior position of the ipsilateral leg in front of the operated one in the posterior direction. Influences of different sources on the extreme posterior position can superimpose. A model is presented which combines both a central programme and peripheral sensory influence. The word programme used here means that it does not only determine the motor output but also determines the reactions to particular afferences. The fact that the reaction to a stimulus depends on the internal state of the CNS is also represented by the model.Supported by Deutsche Forschungsgemeinschaft     

Sensory organs in a cavernicolous insect after cryofixation without cryoprotectants: a comparative ultrastructural study

Three different sensory organs (one mechanoreceptor, one chemoreceptor and one chemo-mechanoreceptor) of the antennae of the cavernicolous Coleoptera Speonomus hydrophilus and S. zophosinus have been compared by means of electron microscopy after chemical fixation or cryofixation without cryoprotectant; when cryofixation succeeds, cells and organelles have a different appearance from the chemically fixed ones: they have rounded and smooth outlines and are more electron-dense; some very fine details, such as tubulinc sub-units of microtubules, are perceptible after cryofixation only. However, chemical fixation gives much more reproducible results than cryofixation; moreover, when good cryofixation is achieved preservation of cells and organelles is satisfactory only for the peripheral region; large ice-crystals more or less destroying the central zone, except the nerves. In short, chemical fixation and cryofixation appear as complementary techniques: chemical fixation must be used as a routine fixation and cryofixation when specially good cell and molecular preservation must be achieved for peripheral tissues only.     

Induction of anhydrobiosis in fat body tissue from an insect

The larva of the African chironomid Polypedilum vanderplanki can withstand complete desiccation. Our previous reports revealed that even when the larva is dehydrated without a brain, it accumulated a great amount of trehalose and successfully went into anhydrobiosis. In this paper we determined the viability after rehydration in tissues from the larvae followed by complete dehydration. Only fat-body tissues that were the main producer of trehalose could be preserved in a dry state at room temperature for an extended period of more than 18 months in a viable form. Thus we have confirmed that the central nervous system is not involved in the induction of anhydrobiosis, even in this complex multicellular organism.     

The Ganglionic Origins and Central Projections of Primary Sensory Neurons Innervating the Upper and Lower Lips in the Rat

Retrograde and transganglionic transport of horseradish peroxidase (HRP) was used to investigate the neurons innervating the upper and the lower lips and their central projections in the rat. Both the upper and the lower lips were observed to be innervated by a very large number of trigeminal sensory neurons, with their cell bodies located in the maxillary and the mandibular parts of the trigeminal ganglion, respectively. The central projections of neurons innervating the upper lip formed a long continuous column starting rostrally at midlevels of the trigeminal main sensory nucleus (5P) and extending caudally through the CI dorsal horn, with occasional fibers reaching the C3 segment. The heaviest projections appeared in the middle portions of 5P and nucleus interpolaris (51), as well as in the rostral part of nucleus caudalis (5C). A small but consistent projection to the solitary tract nucleus, originating from cells in the inferior vagal ganglion, was observed in the upper-lip experiments. The central projections from neurons innervating the lower lip also appeared as a long column located dorsally or dorsomedially to the projections from the upper lip. The most prominent projections from the lower lip were located in the caudal part of 5P, the middle part of 5I, and the caudal two-thirds of 5C. Sparse projections could be traced as far caudally as C4. At 5C and cervical levels, some labeling appeared contralaterally in the same location as on the ipsilateral side.     

Effect of Potassium on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L. Specificity and Membrane Location

Rates of ¹⁴C-photosynthate unloading from excised seed-coathalves of Phaseolus vulgaris L. plants were stimulated by externalKCI concentrations in excess of 10 mM with an optimal responseat 100–150 mM KCI. The cellular pattern of ¹⁴C-photosynthatemetabolism was not altered by KCI but the treatment preferentiallystimulated the release of sucrose from the seed-coats. Photosynthateunloading was insensitive to Cl^– and was stimulated bya range of membrane-permeable cations (Na⁺, Mg²⁺ and tetraphenylphosphoniumion) in addition to K⁺. The K⁺ ionophore, valinomycin, abolishedthe K⁺ stimulation of ¹⁴C-photosynthate unloading. A switchto a wash solution containing K⁺ elicited a rapid burst of ¹⁴C-photosynthateunloading; the rate constant for the final phase of ¹⁴C-efflux(probably across the tonoplast) was unaffected by K⁺. The KCItreatment did not change the passive permeability of eitherthe plasmalemma or tonoplast. While sucrose influx across theplasmalemma was insensitive to K⁺, sucrose transfer to the vacuolewas slowed. The results obtained support the postulate thatK⁺ (and other membrane permeable cations) preferentially stimulatesucrose efflux across the plasmalemma of the unloading cellsby serving to carry positive charge in the opposite direction. Phaseolus vulgaris, bean, photosynthate unloading, potassium stimulation, seed-coat     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Plant tannins and insect herbivores: an appraisal

Abstract. 1. The bioassays with tannins and insects, and the ecological studies on insects implicating tannins, are summarized and discussed.
2. Because of the great variation now shown in all aspects of the insect-tannin relationship, the difficulty of making generalizations is stressed.
3. The significance of plant tannins for insect herbivores is reconsidered in the light of recent work and little-known older work, which illustrate the very varied nature of its effects.     

Biophysical characterization of an insect lysozyme from Manduca sexta

Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active protein. Recombinant MS-lys presented a globular structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed that in solution MS-lys has a quasi-monodisperse size distribution, with a rod-like structure similar to nucleation clusters reported in egg lysozyme pre-crystallization stages. These results show that MS-lys is an excellent candidate for crystallization, folding and denaturation studies.