Sequence analysis, homology modeling, tissue expression, and potential functions of seven putative acetylcholinesterases in the spider Cupiennius salei.

Acetylcholine esterases (AChEs) are essential enzymes in cholinergic synapses, terminating neurotransmission by hydrolysing acetylcholine. While membrane bound AChEs at synaptic clefts efficiently perform this task, soluble AChEs are less stable and effective, but function over broader areas. In vertebrates, a single gene produces alternatively spliced forms of AChE, whereas invertebrates often have multiple genes, producing both enzyme types. Despite their significance as pesticide targets, the physiological roles of invertebrate AChEs remain unclear. Here, we characterized seven putative AChEs in the wandering spider, Cupiennius salei, a model species for neurophysiological studies. Sequence analyses and homology modeling predicted CsAChE7 as the sole stable, membrane-bound enzyme functioning at synaptic clefts, while the others are likely soluble enzymes. In situ hybridization of sections from the spider's nervous system revealed CsAChE7 transcripts co-localizing with choline acetyltransferase in cells that also exhibited AChE activity. CsAChE7 transcripts were also found in rapidly adapting mechanosensory neurons, suggesting a role in precise and transient activation of postsynaptic cells, contrasting with slowly adapting, also cholinergic, neurons expressing only soluble AChEs, which allow prolonged activation of postsynaptic cells. These findings suggest that cholinergic transmission is influenced not only by postsynaptic receptors but also by the enzymatic properties regulating acetylcholine clearance. We also show that acetylcholine is a crucial neurotransmitter in the spider's visual system and sensory and motor pathways, but absent in excitatory motor neurons at neuromuscular junctions, consistent with other arthropods. Our findings on sequence structures may have implications for the development of neurological drugs and pesticides.

Opsin knockdown specifically slows phototransduction in broadband and UV-sensitive photoreceptors in Periplaneta americana.

Photoreceptors with different spectral sensitivities serve different physiological and behavioral roles. We hypothesized that such functional evolutionary optimization could also include differences in phototransduction dynamics. We recorded elementary responses to light, quantum bumps (QBs), of broadband green-sensitive and ultraviolet (UV)-sensitive photoreceptors in the cockroach, Periplaneta americana, compound eyes using intracellular recordings. In addition to control photoreceptors, we used photoreceptors from cockroaches whose green opsin 1 (GO1) or UV opsin expression was suppressed by RNA interference. In the control broadband and UV-sensitive photoreceptors average input resistances were similar, but the membrane capacitance, a proxy for membrane area, was smaller in the broadband photoreceptors. QBs recorded in the broadband photoreceptors had comparatively short latencies, high amplitudes and short durations. Absolute sensitivities of both opsin knockdown photoreceptors were significantly lower than in wild type, and, unexpectedly, their latency was significantly longer while the amplitudes were not changed. Morphologic examination of GO1 knockdown photoreceptors did not find significant differences in rhabdom size compared to wild type. Our results differ from previous findings in Drosophila melanogaster rhodopsin mutants characterized by progressive rhabdomere degeneration, where QB amplitudes were larger but phototransduction latency was not changed compared to wild type.

Mechanotransduction channel Piezo is widely expressed in the spider, Cupiennius salei, mechanosensory neurons and central nervous system.

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

Suppression of Gq and PLC gene expression has a small effect on quantum bumps in vivo in Periplaneta americana.

Visual signal transmission by Drosophila melanogaster photoreceptors is mediated by a Gq protein that activates a phospholipase C (PLC). Mutations and deficiencies in expression of either of these proteins cause severe defects in phototransduction. Here we investigated whether these proteins are also involved in the cockroach, Periplaneta americana, phototransduction by silencing Gq α-subunit (Gqα) and phosphoinositide-specific phospholipase C (PLC) by RNA interference and observing responses to single photons (quantum bumps, QB). We found (1) non-specific decreases in membrane resistance, membrane capacitance and absolute sensitivity in the photoreceptors of both Gqα and PLC knockdowns, and (2) small changes in QB statistics. Despite significant decreases in expressions of Gq and PLC mRNA, the changes in QB properties were surprisingly modest, with mean latencies increasing by ~ 10%, and without significant decrease in their amplitudes. To better understand our results, we used a mathematical model of the phototransduction cascade. By modifying the Gq and PLC abundances, and diffusion rates for Gq, we found that QB latencies and amplitudes deteriorated noticeably only after large decreases in the protein levels, especially when Gq diffusion was slow. Also, reduction in Gq but not PLC lowered quantum efficiency. These results suggest that expression of the proteins may be redundant.

RNA interference supports a role for Nanchung-Inactive in mechanotransduction by the cockroach, Periplaneta americana, tactile spine.

Proteins encoded by nanchung, inactive, nompC and piezo genes have been shown to play crucial roles in the initial detection of mechanical force by various insect auditory neurons, nociceptors and touch receptors. Most of this previous research has been performed on the larval and adult fruit fly, Drosophila melanogaster. We identified and assembled all four homologous genes in transcriptomes from the cockroach, Periplaneta americana. Injection of long double-stranded RNA (dsRNA) into the adult cockroach abdomen successfully reduced the expression of each gene, as measured by quantitative PCR (RT-qPCR). A simple electrophysiological assay was used to record action potential firing in afferent nerves of cockroach femoral tactile spines in response to a standardized mechanical step displacement. Responses of nanchung knockdown animals were significantly reduced compared to matched sham-injected animals at 14 and 21 days after injection, and inactive knockdowns similarly at 21 days. In contrast, responses of nompC and piezo knockdowns were unchanged. Our results support a model in which Nanchung and Inactive proteins combine to form a part of the mechanotransduction mechanism in the cockroach tactile spine.

Changes in electrophysiological properties of photoreceptors in Periplaneta americana associated with the loss of screening pigment.

Absence of screening pigment in insect compound eyes has been linked to visual dysfunction. We investigated how its loss in a white-eyed mutant (W-E) alters the photoreceptor electrophysiological properties, opsin gene expression, and the behavior of the cockroach, Periplaneta americana. Whole-cell patch-clamp recordings of green-sensitive photoreceptors in W-E cockroaches gave reduced membrane capacitance, absolute sensitivity to light, and light-induced currents. Decreased low-pass filtering increased voltage-bump amplitudes in W-E photoreceptors. Intracellular recordings showed that angular sensitivity of W-E photoreceptors had two distinct components: a large narrow component with the same acceptance angle as wild type, plus a relatively small wide component. Information processing was evaluated using Gaussian white-noise modulated light stimulation. In bright light, W-E photoreceptors demonstrated higher signal gain and signal power than wild-type photoreceptors. Expression levels of the primary UV- and green-sensitive opsins were lower and the secondary green-sensitive opsin significantly higher in W-E than in wild-type retinae. In behavioral experiments, W-E cockroaches were significantly less active in dim green light, consistent with the relatively low light sensitivity of their photoreceptors. Overall, these differences can be related to the loss of screening pigment function and to a compensatory decrease in the rhabdomere size in W-E retinae.

Electrical interactions between photoreceptors in the compound eye of Periplaneta americana.

The compound eye of Periplaneta americana contains two spectral classes of photoreceptors: narrow-band UV-sensitive and broad-band green-sensitive. In intracellular recordings, stimulation of green-sensitive photoreceptors with flashes of relatively bright UV/violet light produced anomalous delayed depolarization after the end of the normal light response, whereas stimulation of UV-sensitive photoreceptors with green light elicited biphasic responses characterized by initial transient hyperpolarization followed by prolonged delayed depolarization. To explore the basis for these findings, we used RNA interference to selectively suppress expression of the genes encoding green opsin (GO1), UV opsin (UVO) or both. The hyperpolarizing component in UV-sensitive photoreceptors was eliminated and the delayed depolarization was reduced after GO1 knockdown, suggesting that the hyperpolarization represents fast inhibitory interactions between green- and UV-sensitive photoreceptors. Green-sensitive photoreceptor responses of GO1 knockdowns to flashes of UV/violet were almost exclusively biphasic, whereas residual responses to green had normal kinetics. Knockdown of UVO reduced the responses of UV-sensitive photoreceptors but had minor effects on delayed depolarization in green-sensitive photoreceptors. Angular sensitivity analysis indicated that delayed depolarization of green-sensitive photoreceptors by violet light originates from excitation of (an)other photoreceptor(s) in the same ommatidium. The angle at which the maximal delayed depolarization was observed in green-sensitive photoreceptors stimulated with violet light did not match the angle of the maximal transient depolarization. In contrast, no significant mismatch was observed for delayed depolarization elicited by green light. These results suggest that the cellular sources of the normal transient and additional delayed depolarization by violet light are separate and distinct.

Phenotypic plasticity in Periplaneta americana photoreceptors.

Plasticity is a crucial aspect of neuronal physiology essential for proper development and continuous functional optimization of neurons and neural circuits. Despite extensive studies of different visual systems, little is known about plasticity in mature microvillar photoreceptors. Here we investigate changes in electrophysiological properties and gene expression in photoreceptors of the adult cockroach, Periplaneta americana, after exposure to constant light (CL) or constant dark (CD) for several months. After CL, we observed a decrease in mean whole-cell capacitance, a proxy for cell membrane area, from 362 ± 160 to 157 ± 58 pF, and a decrease in absolute sensitivity. However, after CD, we observed an increase in capacitance to 561 ± 155 pF and an increase in absolute sensitivity. Small changes in the expression of light-sensitive channels and signaling molecules were detected in CD retinas, together with a substantial increase in the expression of the primary green-sensitive opsin (GO1). Accordingly, light-induced currents became larger in CD photoreceptors. Even though normal levels of GO1 expression were retained in CL photoreceptors, light-induced currents became much smaller, suggesting that factors other than opsin are involved. Latency of phototransduction also decreased significantly in CL photoreceptors. Sustained voltage-activated K+ conductance was not significantly different between the experimental groups. The reduced capacitance of CL photoreceptors expanded their bandwidth, increasing the light-driven voltage signal at high frequencies. However, voltage noise was also amplified, probably because of unaltered expression of TRPL channels. Consequently, information transfer rates were lower in CL than in control or CD photoreceptors. These changes in whole-cell capacitance and electrophysiological parameters suggest that structural modifications can occur in the photoreceptors to adapt their function to altered environmental conditions. The opposing patterns of modifications in CL and CD photoreceptors differ profoundly from previous findings in Drosophila melanogaster photoreceptors.

Multiple Biogenic Amine Receptor Types Modulate Spider, Cupiennius salei, Mechanosensory Neurons.

The biogenic amines octopamine (OA), tyramine (TA), dopamine (DA), serotonin (5-HT), and histamine (HA) affect diverse physiological and behavioral processes in invertebrates, but recent findings indicate that an additional adrenergic system exists in at least some invertebrates. Transcriptome analysis has made it possible to identify biogenic amine receptor genes in a wide variety of species whose genomes have not yet been sequenced. This approach provides new sequences for research into the evolutionary history of biogenic amine receptors and allows them to be studied in experimentally accessible animal models. The Central American Wandering spider, Cupiennius salei, is an experimental model for neurophysiological, developmental and behavioral research. We identified ten different biogenic amine receptors in C. salei transcriptomes. Phylogenetic analysis indicated that, in addition to the typical receptors for OA, TA, DA, and 5-HT in protostome invertebrates, spiders also have α1- and α2-adrenergic receptors, but lack TAR2 receptors and one invertebrate specific DA receptor type. In situ hybridization revealed four types of biogenic amine receptors expressed in C. salei mechanosensory neurons. We used intracellular electrophysiological experiments and pharmacological tools to determine how each receptor type contributes to modulation of these neurons. We show that arachnids have similar groups of biogenic amine receptors to other protostome invertebrates, but they lack two clades. We also clarify that arachnids and many other invertebrates have both α1- and α2-adrenergic, likely OA receptors. Our results indicate that in addition to an OAß-receptor that regulates rapid and large changes in sensitivity via a Gs-protein activating a cAMP mediated pathway, the C. salei mechanosensory neurons have a constitutively active TAR1 and/or α2-adrenergic receptor type that adjusts the baseline sensitivity to a level appropriate for the behavioral state of the animal by a Gq-protein that mobilizes Ca2+.

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.

Distinct roles of light-activated channels TRP and TRPL in photoreceptors of Periplaneta americana.

Electrophysiological studies in Drosophila melanogaster and Periplaneta americana have found that the receptor current in their microvillar photoreceptors is generated by two light-activated cationic channels, TRP (transient receptor potential) and TRPL (TRP-like), each having distinct properties. However, the relative contribution of the two channel types to sensory information coding by photoreceptors remains unclear. We recently showed that, in contrast to the diurnal Drosophila in which TRP is the principal phototransduction channel, photoreceptors of the nocturnal P. americana strongly depend on TRPL. Here, we perform a functional analysis, using patch-clamp and intracellular recordings, of P. americana photoreceptors after RNA interference to knock down TRP (TRPkd) and TRPL (TRPLkd). Several functional properties were changed in both knockdown phenotypes: cell membrane capacitance was reduced 1.7-fold, light sensitivity was greatly reduced, and amplitudes of sustained light-induced currents and voltage responses decreased more than twofold over the entire range of light intensities. The information rate (IR) was tested using a Gaussian white-noise modulated light stimulus and was lower in TRPkd photoreceptors (28 ± 21 bits/s) than in controls (52 ± 13 bits/s) because of high levels of bump noise. In contrast, although signal amplitudes were smaller than in controls, the mean IR of TRPLkd photoreceptors was unchanged at 54 ± 29 bits/s1 because of proportionally lower noise. We conclude that TRPL channels provide high-gain/high-noise transduction, suitable for vision in dim light, whereas transduction by TRP channels is relatively low-gain/low-noise and allows better information transfer in bright light.

EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision.

KEY POINTS: The principles underlying the evolutionary selection of ion channels for expression in sensory neurons are unclear. Photoreceptor depolarization in the diurnal Drosophila melanogaster is predominantly provided by light-activated transient receptor potential (TRP) channels, whereas repolarization is mediated by sustained voltage-gated K+ channels of the Shab family. In the present study, we show that phototransduction in the nocturnal cockroach Periplaneta americana is predominantly mediated by TRP-like channels, whereas membrane repolarization is based on EAG channels. Although bright light stimulates Shab channels in Drosophila, further restricting depolarization and improving membrane bandwidth, it strongly suppresses EAG conductance in Periplaneta. This light-dependent inhibition (LDI) is caused by calcium and is abolished by chelating intracellular calcium or suppressing eag gene expression. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth. This makes EAG unsuitable for light response conditioning during the day and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects. ABSTRACT: The principles underlying evolutionary selection of ion channels for expression in sensory neurons are unclear. Among species possessing microvillar photoreceptors, the major ionic conductances have only been identified in Drosophila melanogaster. In Drosophila, depolarization is provided by light-activated transient receptor potential (TRP) channels with a minor contribution from TRP-like (TRPL) channels, whereas repolarization is mediated by sustained voltage-gated K+ (Kv) channels of the Shab family. Bright light stimulates Shab channels, further restricting depolarization and improving membrane bandwidth. In the present study, data obtained using a combination of electrophysiological, pharmacological and molecular knockdown techniques strongly suggest that in photoreceptors of the nocturnal cockroach Periplaneta americana the major excitatory channel is TRPL, whereas the predominant delayed rectifier is EAG, a ubiquitous but enigmatic Kv channel. By contrast to the diurnal Drosophila, bright light strongly suppresses EAG conductance in Periplaneta. This light-dependent inhibition (LDI) is caused by calcium entering the cytosol and is amplified following inhibition of calcium extrusion, and it can also be abolished by chelating intracellular calcium or suppressing eag gene expression by RNA interference. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth, impairing information transfer. LDI is also observed in the nocturnal cricket Gryllus integer, whereas, in the diurnal water strider Gerris lacustris, the delayed rectifier is up-regulated by light. Although LDI is not expected to reduce delayed rectifier current in the normal illumination environment of nocturnal cockroaches and crickets, it makes EAG unsuitable for light response conditioning during the day, and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects.

Expression of Cys-loop receptor subunits and acetylcholine binding protein in the mechanosensory neurons, glial cells, and muscle tissue of the spider Cupiennius salei.

The central and peripheral nervous system transcriptomes of the spider Cupiennius salei have 15 Cys-loop receptor subunits and an acetylcholine-binding protein (AChBP). Twelve subunits are predicted to form anion channels gated by γ-aminobutyric acid (GABA), glutamate, histamine, or changes in pH, and three are putative ACh-gated cation channels. Spiders have a variety of mechanosensilla and proprioceptive organs that are innervated by efferents in their peripherally located parts, and efferents also innervate muscle fibers. We investigated Cys-loop gene expression in muscle tissue by qPCR and localized this expression in mechanosensilla via in situ hybridization. The cuticular mechanosensory neurons had only CsGABArdl and CspHCl2 subunits, whereas the muscle tissue expressed a wider variety of subunits, especially CsGABAgrd, CsGABAA ß, CsGluCl1 and CspHCl, but very low levels of the CsGABArdl or CsnACh subunits. An nACh non-α subunit was expressed in a group of unidentified cells in the hypodermis and at low level in the muscle tissue, but the physiological function of this subunit is unknown. The CsnAChα subunit was not expressed in sensory neurons and was expressed at extremely low level in the muscle tissue. None of the probes gave signals in proprioceptive joint receptors, suggesting that efferent innervation to this sense organ employs other receptor types. CsAChBP and a glia-specific homeodomain CsREPO were both expressed in glial cells that surround sensory neurons and also in muscle tissue, probably around the nerve endings of the neuromuscular junction. These locations have large numbers of synapses, suggesting that AChBP may have a function in modulating synaptic transmission. J. Comp. Neurol. 525:1139-1154, 2017. © 2016 Wiley Periodicals, Inc.

Transcriptome Analysis of the Central and Peripheral Nervous Systems of the Spider Cupiennius salei Reveals Multiple Putative Cys-Loop Ligand Gated Ion Channel Subunits and an Acetylcholine Binding Protein.

Invertebrates possess a diverse collection of pentameric Cys-loop ligand gated ion channel (LGIC) receptors whose molecular structures, evolution and relationships to mammalian counterparts have been intensely investigated in several clinically and agriculturally important species. These receptors are targets for a variety of control agents that may also harm beneficial species. However, little is known about Cys-loop receptors in spiders, which are important natural predators of insects. We assembled de novo transcriptomes from the central and peripheral nervous systems of the Central American wandering spider Cupiennius salei, a model species for neurophysiological, behavioral and developmental studies. We found 15 Cys-loop receptor subunits that are expected to form anion or cation permeable channels, plus a putative acetylcholine binding protein (AChBP) that has only previously been reported in molluscs and one annelid. We used phylogenetic and sequence analysis to compare the spider subunits to homologous receptors in other species and predicted the 3D structures of each protein using the I-Tasser server. The quality of homology models improved with increasing sequence identity to the available high-resolution templates. We found that C. salei has orthologous γ-aminobutyric acid (GABA), GluCl, pHCl, HisCl and nAChα LGIC subunits to other arthropods, but some subgroups are specific to arachnids, or only to spiders. C. salei sequences were phylogenetically closest to gene fragments from the social spider, Stegodyphus mimosarum, indicating high conservation within the Araneomorphae suborder of spiders. C. salei sequences had similar ligand binding and transmembrane regions to other invertebrate and vertebrate LGICs. They also had motifs associated with high sensitivity to insecticides and antiparasitic agents such as fipronil, dieldrin and ivermectin. Development of truly selective control agents for pest species will require information about the molecular structure and pharmacology of Cys-loop receptors in beneficial species.

Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels.

Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100-1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596-708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction.

Co-localization of Gamma-Aminobutyric Acid and Glutamate in Neurons of the Spider Central Nervous System.

Spider sensory neurons with cell bodies close to various sensory organs are innervated by putative efferent axons from the central nervous system (CNS). Light and electronmicroscopic imaging of immunolabeled neurons has demonstrated that neurotransmitters present at peripheral synapses include γ-aminobutyric acid (GABA), glutamate and octopamine. Moreover, electrophysiological studies show that these neurotransmitters modulate the sensitivity of peripheral sensory neurons. Here, we undertook immunocytochemical investigations to characterize GABA and glutamate-immunoreactive neurons in three-dimensional reconstructions of the spider CNS. We document that both neurotransmitters are abundant in morphologically distinct neurons throughout the CNS. Labeling for the vesicular transporters, VGAT for GABA and VGLUT for glutamate, showed corresponding patterns, supporting the specificity of antibody binding. Whereas some neurons displayed strong immunolabeling, others were only weakly labeled. Double labeling showed that a subpopulation of weakly labeled neurons present in all ganglia expresses both GABA and glutamate. Double labeled, strongly and weakly labeled GABA and glutamate immunoreactive axons were also observed in the periphery along muscle fibers and peripheral sensory neurons. Electron microscopic investigations showed presynaptic profiles of various diameters with mixed vesicle populations innervating muscle tissue as well as sensory neurons. Our findings provide evidence that: (1) sensory neurons and muscle fibers are innervated by morphologically distinct, centrally located GABA- and glutamate immunoreactive neurons; (2) a subpopulation of these neurons may co-release both neurotransmitters; and (3) sensory neurons and muscles are innervated by all of these neurochemically and morphologically distinct types of neurons. The biochemical diversity of presynaptic innervation may contribute to how spiders filter natural stimuli and coordinate appropriate response patterns.

Upstream open reading frames and Kozak regions of assembled transcriptome sequences from the spider Cupiennius salei. Selection or chance?

We assembled a new set of mRNA sequences from the leg hypodermis transcriptome of the wandering spider, Cupiennius salei. Each sequence was assembled to exhaustion in the 5' direction to detect all upstream open reading frames (uORFs) both in-frame and out-of-frame with the main open reading frame (mORF). We also counted nucleotide probabilities before and after the START codon of the mORF to establish the optimum Kozak consensus sequence. More than 80% of 5' sequences had uORFs before the mORF with a range of 1-16 uORFs. Kozak consensus strengths of uORFs were significantly weaker than mORFs. Random scrambling of 5' nucleotide positions did not give significantly different numbers, sizes, or Kozak consensus strengths of uORFs. Random simulations of 5' sequences using either equal or experimental distributions of nucleotides gave similar numbers of uORFs, with similar sizes and Kozak consensus strengths to experimental data. Abundance of mRNA for each gene was estimated by counting matching Illumina reads to assembled genes. Abundance was negatively correlated with numbers of uORFs, but not with 5' length. Our data are compatible with a random model of 5' mRNA sequence structure.

Carbon dioxide and fruit odor transduction in Drosophila olfactory neurons. What controls their dynamic properties?

We measured frequency response functions between odorants and action potentials in two types of neurons in Drosophila antennal basiconic sensilla. CO2 was used to stimulate ab1C neurons, and the fruit odor ethyl butyrate was used to stimulate ab3A neurons. We also measured frequency response functions for light-induced action potential responses from transgenic flies expressing H134R-channelrhodopsin-2 (ChR2) in the ab1C and ab3A neurons. Frequency response functions for all stimulation methods were well-fitted by a band-pass filter function with two time constants that determined the lower and upper frequency limits of the response. Low frequency time constants were the same in each type of neuron, independent of stimulus method, but varied between neuron types. High frequency time constants were significantly slower with ethyl butyrate stimulation than light or CO2 stimulation. In spite of these quantitative differences, there were strong similarities in the form and frequency ranges of all responses. Since light-activated ChR2 depolarizes neurons directly, rather than through a chemoreceptor mechanism, these data suggest that low frequency dynamic properties of Drosophila olfactory sensilla are dominated by neuron-specific ionic processes during action potential production. In contrast, high frequency dynamics are limited by processes associated with earlier steps in odor transduction, and CO2 is detected more rapidly than fruit odor.

GABA and glutamate receptors have different effects on excitability and are differentially regulated by calcium in spider mechanosensory neurons.

GABA and glutamate receptors belonging to the ligand-gated chloride-channel family are primary targets of insecticides and antiparasitics, so their molecular structure, pharmacology and biophysical properties have attracted significant attention. However, little is known about the physiological roles of these channels or how they regulate neuronal excitability and animal behavior. Mechanosensory neurons of VS-3 slit sensilla in the patella of the tropical wandering spider, Cupiennius salei, react to the GABA(A)-receptor agonists, GABA and muscimol, with depolarization and an increase in intracellular [Ca(2+)] and, during random noise stimulation, with a mixed inhibitory-excitatory response. We established that the GABA(A)-receptors in all VS-3 neurons are identical, but there are at least two types of glutamate receptors and some neurons do not respond to glutamate at all. Immunohistochemistry with antibodies against Drosophila inhibitory glutamate receptor (GluCls) α-subunit suggests that in addition to VS-3 neurons, these receptors may also be present in the efferent neurons surrounding the sensory neurons. Most VS-3 neurons were inhibited but not depolarized by glutamate during random stimulation, but some depolarized and had a similar excitatory-inhibitory response to glutamate as to muscimol. The membrane-permeable Ca(2+)-chelator BAPTA-AM abolished muscimol effects but potentiated glutamate effects, indicating that GABA and glutamate receptors are differentially modulated by Ca(2+), leading to diverse regulation of neuronal excitability. We hypothesize that this could be achieved by different Ca(2+)-triggered phosphorylation processes at each receptor type. These findings are important for understanding the significance of Ca(2+)-mediated regulation of transmitter receptor molecules and its role in controlling excitability.

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.