The effect of selected immunostimulants on hemocytes of the false black widow Steatoda grossa (Theridiidae) spiders under chronic exposition to cadmium.

The aim of this study was to analyze whether, and to what extent, long-term exposure to cadmium, administered in sublethal concentrations by the oral route, caused changes in the immune potential of hemocytes in adult female Steatoda grossa spiders. We used artificial and natural immunostimulants, namely phorbol 12-myristate 13-acetate (PMA) and bacterial cell suspension based on Gram-positive (G+, Staphylococcus aureus) and Gram-negative (G-, Pseudomonas fluorescens) bacteria, to compare the status of hemocytes in nonstimulated individuals and those subjected to immunostimulation. After cadmium exposure, the percentage of small nongranular hemocytes in response to G+ cell suspension and PMA mitogen was decreased. Furthermore, in the cadmium-intoxicated spiders the percentage of plasmatocytes after immunostimulation remained lower compared to the complementary control group. Exposure to cadmium also induced several degenerative changes, including typical apoptotic and necrotic changes, in the analyzed types of cells. Immunostimulation by PMA mitogen and G+ bacterial suspension resulted in an increase in the number of cisterns in the rough endoplasmic reticulum of granulocytes, in both the control group and cadmium-treated individuals. These changes were accompanied with a low level of metallothioneins in hemolymph. Chronic cadmium exposure may significantly weaken the immune defense system of spiders during infections.

Tyrosine hydroxylase immunolabeling reveals the distribution of catecholaminergic neurons in the central nervous systems of the spiders Hogna lenta (Araneae: Lycosidae) and Phidippus regius (Araneae: Salticidae).

With over 48,000 species currently described, spiders (Arthropoda: Chelicerata: Araneae) comprise one of the most diverse groups of animals on our planet, and exhibit an equally wide array of fascinating behaviors. Studies of central nervous systems (CNSs) in spiders, however, are relatively sparse, and no reports have yet characterized catecholaminergic (dopamine [DA]- or norepinephrine-synthesizing) neurons in any spider species. Because these neuromodulators are especially important for sensory and motor processing across animal taxa, we embarked on a study to identify catecholaminergic neurons in the CNS of the wolf spider Hogna lenta (Lycosidae) and the jumping spider Phidippus regius (Salticidae). These neurons were most effectively labeled with an antiserum raised against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. We found extensive catecholamine-rich neuronal fibers in the first- and second-order optic neuropils of the supraesophageal mass (brain), as well as in the arcuate body, a region of the brain thought to receive visual input and which may be involved in higher order sensorimotor integration. This structure likely shares evolutionary origins with the DA-enriched central complex of the Mandibulata. In the subesophageal mass, we detected an extensive filigree of TH-immunoreactive (TH-ir) arborizations in the appendage neuromeres, as well as three prominent plurisegmental fiber tracts. A vast abundance of TH-ir somata were located in the opisthosomal neuromeres, the largest of which appeared to project to the brain and decorate the appendage neuromeres. Our study underscores the important roles that the catecholamines likely play in modulating spider vision, higher order sensorimotor processing, and motor patterning.

Ultrastructure of spider thread anchorages.

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.

Photoreceptor projection from a four-tiered retina to four distinct regions of the first optic ganglion in a jumping spider.

Jumping spiders have four pairs of eyes (ocelli) of which only the principal eyes (PEs) are used to detect features of objects. Photoreceptors in the retina of the PEs form four layers (PL1-4) and terminate in the first optic ganglion (FOG). Here, we focus on Hasarius adansoni because it has unique depth vision besides color vision and its FOG appears to contribute to the initial processing of these visual modalities. We first investigated the neuroanatomical organization of the FOG. The three-dimensional structure of the FOG revealed by synapsin immunostaining is horseshoe-shaped and consists of four terminal zones (TZ1-4). Then, we traced single photoreceptors through serial sections and found that green-sensitive receptors of PL1 and 2 terminate in TZ1 and 2, respectively, by keeping retinotopic organization. In contrast to TZ1 and 2, TZ3 receives terminals of ultraviolet-sensitive receptors from lateral regions of both PL3 and 4, while photoreceptors of the medial region of PL3 and 4 terminate in TZ4. We further studied details of photoreceptor terminals and the branching pattern of interneurons in the FOG in Golgi stained preparations. Photoreceptors have long lateral processes in each terminal zone. Some photoreceptors terminating in TZ3 have branches innervating TZ1, indicating that TZ1 receives different spectral information. A type of interneuron connects TZ1 and 2, while others have branches within a single terminal zone or in the entire FOG. These results suggest that TZ1 and 2 contribute to color, shape, and depth vision, while TZ3 and 4 have specific roles for UV vision.

Nonlinearization: naturalistic stimulation and nonlinear dynamic behavior in a spider mechanoreceptor.

In a previous study, we used linear frequency response analysis to show that naturalistic stimulation of spider primary mechanosensory neurons produced different response dynamics than the commonly used Gaussian random noise. We isolated this difference to the production of action potentials from receptor potential and suggested that the different distribution of frequency components in the naturalistic signal increased the nonlinearity of action potential encoding. Here, we tested the relative contributions of first- and second-order processes to the action potential signal by measuring linear and quadratic coherence functions. Naturalistic stimulation shifted the linear coherence toward lower frequencies, while quadratic coherence was always higher than linear coherence and increased with naturalistic stimulation. In an initial attempt to separate the order of time-dependent and nonlinear processes, we fitted quadratic frequency response functions by two block-structured models consisting of a power-law filter and a static second-order nonlinearity in alternate cascade orders. The same cascade models were then fitted to the original time domain data by conventional numerical analysis algorithms, using a polynomial function as the static nonlinearity. Quadratic models with a linear filter followed by a static nonlinearity were favored over the reverse order, but with weak significance. Polynomial nonlinear functions indicated that rectification is a major nonlinearity. A complete quantitative description of sensory encoding in these primary mechanoreceptors remains elusive but clearly requires quadratic and higher nonlinear operations on the input signal to explain the sensitivity of dynamic behavior to different input signal patterns.

Changes in the midgut cells in the European cave spider, Meta menardi, during starvation in spring and autumn.

During the growth period, in surface habitats, spiders catch enough prey to feed normally. In contrast, in the cave entrance zone, prey may be relatively scarce. Meta menardi inhabits this cave section, resulting in temporary starvation. We studied structural changes in the midgut epithelial cells of M. menardi during a short-term and a medium-term controlled starvation to mimic the occasional starvation in caves, during spring and autumn. Digestive cells, secretory cells and adipocytes were examined before the experimental starvation, in the middle and at the end of starvation. We used light microscopy, transmission electron microscopy and specific histochemical methods for the detection of lipids, polysaccharides and proteins. Detection of lysosomes, autolysosomes and apoptosis was also carried out. The general structures of the cells did not change during the experimental starvation in either season, while some specific differences in the ultrastructure were observed. In both sexes, in both seasons, the amounts of lipids, glycogen and proteins decreased during starvation. Larger amounts of lipids were found in autumn, while there were no significant differences in the amounts of glycogen and proteins. In both sexes, in both seasons, autophagy and apoptosis intensified with starvation in progress, but more intensively in females. Thus, autumn individuals, in contrast to spring ones, compile energy-supplying stores to confront the subsequent winter deficiency of prey in caves, while the cellular ultrastructures undergo the same starvation-dependant changes at any time during the growth period.

A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum.

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

The complete mitochondrial genome of stretch spider Tetragnatha maxillosa (Araneae: Tetragnathidae).

The stretch spider Tetragnatha maxillosa (Araneae: Tetragnathidae) is found all over the world. In the present study, we investigated the complete mitochondrial genome of T. maxillosa and the mitogenome is a circular molecule of 14 414 bp in length, consists of 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a control region. The A + T content of the overall base composition of H-strand is 74.5% (T: 40.4%; C: 9.6%; A: 34.1%; G: 15.9%). COI gene begins with TTT as start codon, COII and COIII genes begin with TTG as start codon, ATP8, Cyt b, ND2, and ND4L genes begin with ATT as start codon, and other six protein-coding genes start with ATA. ATP6, ATP8, COI, COIII, ND1, ND3, and ND6 genes are terminated with TAA as stop codon, Cyt b, ND2, ND4, ND4L, and ND5 end with T, and COII ends with TAG. In addition, the phylogenetic relationships from neighbor-joining analyses based on the 13 concatenated PCGs indicated (Tetragnatha (Nephila (Argiope (Araneus, Neoscona)))).

First evidence of neurons in the male copulatory organ of a spider (Arachnida, Araneae).

Spider males have evolved a remarkable way of transferring sperm by using a modified part of their pedipalps, the so-called palpal organ. The palpal organ is ontogenetically derived from tarsal claws; however, no nerves, sensory organs or muscles have been detected in the palpal bulb so far, suggesting that the spider male copulatory organ is numb and sensorily blind. Here, we document the presence of neurons and a nerve inside the male palpal organ of a spider for the first time. Several neurons that are located in the embolus are attached to the surrounding cuticle where stresses and strains lead to a deformation (stretching) of the palpal cuticle on a local scale, suggesting a putative proprioreceptive function. Consequently, the male copulatory organ of this species is not just a numb structure but likely able to directly perceive sensory input during sperm transfer. In addition, we identified two glands in the palpal organ, one of which is located in the embolus (embolus gland). The embolus gland appears to be directly innervated, which could allow for rapid modulation of secretory activity. Thus, we hypothesize that the transferred seminal fluid can be modulated to influence female processes.

Evolutionary morphology of the male reproductive system, spermatozoa and seminal fluid of spiders (Araneae, Arachnida)--current knowledge and future directions.

The male reproductive system and spermatozoa of spiders are known for their high structural diversity. Spider spermatozoa are flagellate and males transfer them to females in a coiled and encapsulated state using their modified pedipalps. Here, we provide a detailed overview of the present state of knowledge of the primary male reproductive system, sperm morphology and the structural diversity of seminal fluids with a focus on functional and evolutionary implications. Secondly, we conceptualized characters for the male genital system, spermiogenesis and spermatozoa for the first time based on published and new data. In total, we scored 40 characters for 129 species from 56 families representing all main spider clades. We obtained synapomorphies for several taxa including Opisthothelae, Araneomorphae, Dysderoidea, Scytodoidea, Telemidae, Linyphioidea, Mimetidae, Synotaxidae and the Divided Cribellum Clade. Furthermore, we recovered synspermia as a synapomorphy for ecribellate Haplogynae and thus propose Synspermiata as new name for this clade. We hope that these data will not only contribute to future phylogenetic studies but will also stimulate much needed evolutionary studies of reproductive systems in spiders.

Sperm dynamics in spiders (Araneae): ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772).

Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

Multiple origins of sex chromosome fusions correlated with chiasma localization in Habronattus jumping spiders (Araneae: Salticidae).

Entelegyne spiders rarely show fusions yielding neo-Y chromosomes, which M. J. D. White attributed to a constraint in spiders, namely their proximal chiasma localization acting to upset meiotic segregation in males with fusions. Of the 75 taxa of Habronattus and outgroups studied, 47 have X1 X2 0 sex chromosomes in males, 10 have X1 X2 Y, 15 have X1 X2 X3 Y, 2 have X0, and one has both X1 X2 0 and X1 X2 X3 Y. Chromosome numbers and behavior suggest neo-Ys formed by an autosome-X fusion to make X1 X2 Y, with a second fusion to an autosome to make X1 X2 X3 Y. Phylogeny shows at least 8-15 gains (or possibly some losses) of neo-Y (i.e., X-autosome fusions), a remarkable number for such a small clade. In contrast to the many X-autosome fusions, at most one autosome-autosome fusion is indicated. Origins of neo-Y are correlated significantly with distal localization of chiasmata, supporting White's hypothesis that evolution of neo-Y systems is facilitated by looser pairing (distal chiasmata) at meiosis. However, an alternative (or contributing) explanation for the correlation is that X-autosome fusions were selected to permit isolation of male-favored alleles to the neo-Y chromosome, aided by distal chiasmata limiting recombination. This intralocus sexual conflict hypothesis could explain both the many X-autosome fusions, and the stunning complexity of male Habronattus courtship displays.

Morphology and composition of the spider major ampullate gland and dragline silk.

Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

Calcium buffering and clearance in spider mechanosensory neurons.

Spider VS-3 mechanoreceptor neurons have a low-voltage-activated Ca2+ current that raises intracellular calcium concentration [Ca2+] when they are depolarized by agonists of GABAA receptors or fire action potentials. The Ca2+ rise produces negative feedback by modulating the mechanoreceptor current and regulates Ca2+- and voltage-activated K+ currents. However, nothing is known about Ca2+ buffering in VS-3 neurons. Dynamic changes in VS-3 neuron intracellular [Ca2+] were measured using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (OG488) to understand Ca2+ buffering and clearance. Intracellular OG488 concentration increased slowly over more than 2 h as it diffused through a sharp intracellular microelectrode and spread through the cell. This slow increase was used to measure endogenous Ca2+ buffering and clearance by the added buffer technique, with OG488 acting as both added exogenous buffer and Ca2+ indicator. [Ca2+] was raised for brief periods by regular action potential firing, produced by pulsed electric current injection through the microelectrode. The resulting rise and fall of [Ca2+] were well fitted by the single compartment model of Ca2+ dynamics. With earlier ratiometric [Ca2+] estimates, these data gave an endogenous Ca2+ binding ratio of 684. Strong Ca2+ buffering may assist these neurons to deal with rapid changes in mechanical inputs.

Silk secretion from tarantula feet revisited: alleged spigots are probably chemoreceptors.

Controversial views have been expressed about whether tarantula feet can secrete fine silk threads that could prevent them from falling off smooth vertical surfaces. Two studies have claimed that 'ribbed hairs' on the tarsi of tarantulas produce silk. We examined these ribbed hairs in several tarantula species using light and scanning electron microscopy, and compared them with the silk-producing spigots on the abdominal spinnerets. We found that, morphologically, these ribbed hairs correspond very closely to known chemosensitive hairs in spiders; they have a distinct socket, a bent hair shaft with fine cuticular ridges, an eccentric double lumen within the hair shaft, and a blunt tip with a subterminal pore. Spigots on the spinnerets have a large bulbous base instead of a socket, a long shaft with a scaly surface and a central terminal pore. We never observed any silk threads coming out of these ribbed hairs under the electron microscope. By contrast, silk threads exiting the spigots on the spinnerets were common. Interestingly, ribbed hairs also occur on the spinnerets, often side by side with the silk-producing spigots. Our conclusion is that the ribbed hairs are chemoreceptors, not spigots. Observations of live tarantulas clinging inverted to glass coverslips confirmed that some substance is produced by the ribbed hairs, but it remains unclear whether this secretion is actually silk. In any case, the thousands of adhesive setae on the tarsi of legs and pedipalps almost certainly far outweigh any potential contribution from the sparsely distributed trails secreted by the ribbed hairs.

Cytogenetical characterization of six orb-weaver species and review of cytogenetical data for Araneidae

The family Araneidae is the third largest among spiders and the third most studied from a cytogenetical point of view. In spite of this, only 2% of all araneids have been karyotyped. The majority of araneids analyzed possess 2n ?=? 24 chromosomes in males; however, the study of additional species could reveal unusual karyotype characteristics. Thus, the aim of this work is to analyze chromosomally, for the first time, six species belonging to three araneid genera from Brazil. The specimens of Alpaida leucogramma (White 1841), Alpaida truncata (Keyserling 1865), Alpaida veniliae (Keyserling 1865), Parawixia kochi (Taczanowski 1873), Parawixia velutina (Taczanowski 1878) and Wagneriana sp. were collected in Parque Nacional de Ilha Grande and in the municipality of Rio Claro. The gonads were treated with colchicine and hypotonic solution before fixation with Carnoy I solution. The results were 2n¡Î? ?=? 24 (11II+X1X2) in A. leucogramma and P. velutina, and 2n¡Î? ?=? 22 (10II+X1X2) in A. truncata, A. veniliae, P. kochi and Wagneriana sp. When the chromosomal morphologies were established, we observed telocentric chromosomes in all specimens save one female specimen of P. velutina. The univalent sex chromosomes were easily recognized on diplotenes. The unpaired metacentric element found in one female specimen of P. velutina with 2n ?=? 25 probably arises by centric fusion/fission. Araneidae is a megadiverse family composed of ¡­3000 species distributed mainly in the tropics; thus the analysis of more species may provide new insights about orb-weaver chromosome evolution.

Chromosomal characteristics and karyotype evolution of Oxyopidae spiders (Araneae, Entelegynae).

We made a cytogenetic analysis of four species of Oxyopidae and compared it with the karyotype data of all species of this family. In Hamataliwa sp, the mitotic cells showed 2n♂ = 26+X(1)X(2) and telocentric chromosomes. The 2n♂ = 28, which has been described for only one oxyopid spider, is the highest diploid number reported for this family. Peucetia species exhibited distinct karyotype characteristics, i.e., 2n♂ = 20+X(1)X(2) in P. flava and 2n♂ = 20+X in P. rubrolineata, revealing interspecific chromosome variability within this genus. However, both Peucetia species exhibited telocentric chromosomes. The most unexpected karyotype was encountered in Oxyopes salticus, which presented 2n♂ = 10+X in most individuals and a predominance of biarmed chromosomes. Additionally, one male of the sample of O. salticus was heterozygous for a centric fusion that originated the first chromosomal pair and exhibited one supernumerary chromosome in some cells. Testicular nuclei of Hamataliwa sp and O. salticus revealed NORs on autosomal pairs, after silver impregnation. The majority of Oxyopidae spiders have their karyotype differentiated by both reduction in diploid number chromosome number and change of the sex chromosome system to X type; however, certain species retain the ancestral chromosome constitution 2n = 26+X1X2. The most remarkable karyotype differentiation occurred in O. salticus studied here, which showed the lowest diploid number ever observed in Oxyopidae and the second lowest registered for Entelegynae spiders.

Ca²(+) /calmodulin-dependent protein kinase II mediates the octopamine-induced increase in sensitivity in spider VS-3 mechanosensory neurons.

G-protein-coupled octopamine (OA) receptors mediate their effects by Ca²(+) signaling or adjusting intracellular cAMP levels. Depending on OA concentration and cell type, activation of OA receptors in excitable cells triggers excitatory or inhibitory effects, but the mechanisms by which Ca²(+) or cAMP mediates these effects are not well understood. We investigated signaling mechanisms that are potentially activated by OA, and OA effects on excitability and frequency sensitivity in mechanosensory neurons innervating the VS-3 slit sensilla on the patella of the spider Cupiennius salei. These neurons are directly innervated by octopaminergic efferents, and possess OA receptors that were immunoreactive to an antibody against an OA receptor highly expressed in mushroom bodies. OA application enhanced VS-3 neuron sensitivity, especially at high stimulation frequencies. This enhancement lasted for at least 1 h after OA application. Changes in sensitivity were also detected when the Ca²(+) ionophore ionomycin or the cAMP analog 8-Br-cAMP was applied. However, the cAMP pathway was unlikely to mediate the OA effect, as the protein kinase A inhibitor RP-cAMPS did not diminish this effect. In contrast, the OA-induced sensitivity enhancement was significantly reduced by KN-62, an inhibitor of Ca²(+) /calmodulin-dependent protein kinase II (CaMKII), and by the Ca²(+) chelator BAPTA-AM. OA depolarized the neurons by 3.8 mV from resting potential, well below the threshold for opening of voltage-activated Ca²(+) channels. OA also reduced the amplitudes of voltage-activated K(+) currents. We propose that OA receptors in VS-3 neurons activate inositol 1,4,5-trisphosphate, leading to Ca²(+) release from intracellular stores. The Ca²(+) surge switches on CaMKII, which modulates voltage-activated K(+) channels, resulting in persistent enhancement in excitability.

Feedback modulation of transduction by calcium in a spider mechanoreceptor.

Calcium ions play important roles in the adaptation of auditory hair cells, and there is evidence that they are involved in modifying the sensitivity and adaptation of a variety of vertebrate and invertebrate mechanoreceptors. However, there is little direct evidence concerning the concentration changes, signaling pathways or ultimate effects of these proposed modulatory mechanisms. We measured receptor potential, receptor current and action potentials intracellularly during mechanotransduction in a group of sensory neurons of the spider Cupiennius salei, which possesses low-voltage-activated calcium channels. Simultaneously, we elevated intracellular [Ca(2+) ] by UV light release from cage molecules, and observed increases in [Ca(2+) ] as changes in calcium-sensitive dye fluorescence. Increases of 10-15% in [Ca(2+) ] caused reductions of approximately 40% in receptor potential and approximately 20% in receptor current. Mechanically evoked action potential firing caused much larger increases in [Ca(2+) ], and the firing rate fell as [Ca(2+) ] rose during mechanical stimulation. Release of caged calcium just before mechanical stimulation significantly reduced peak firing. Dose-response measurements suggested that the binding of one or two intracellular calcium ions per channel reduces the probability of the mechanotransduction channel being open. Our data indicate that calcium regulates sensitivity in these mechanoreceptor neurons by negative feedback from action potentials onto transduction channels.