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.

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.

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.

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.

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.

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.

Dynamic characterization of Drosophila antennal olfactory neurons indicates multiple opponent signaling pathways in odor discrimination.

Opponent signaling refers to processes in which antagonistic signals are produced by different, but closely related, stimuli. It allows enhanced discrimination and more accurate behavioral responses. We explored opponent signaling in the Drosophila melanogaster olfactory system by measuring frequency response functions between odorant concentrations and primary olfactory neuron responses. Random fluctuations in concentration of two aliphatic and two aromatic fruit odorants were used to modulate action potentials from basiconic antennal sensilla. We separated action potentials by two-dimensional cluster analysis using amplitude and cross-correlation with a median action-potential template. Frequency response functions were fitted with either bandpass or second-order low-pass functions and then divided into two polarity groups, excitatory and inhibitory, by fitting the frequency response functions. Cluster analysis gave two, three, or four action potential clusters for each sensillum recording. Sensilla were then grouped by the patterns of response polarities of the individual neurons into four sensillum types, one with four neurons and three with two neurons. All four odorant compounds produced a mixture of excitatory, inhibitory, and null responses in different neurons. Statistical analysis of frequency response parameters for individual odorants gave only weak correlation between dynamics and some neuron types, even when comparing the dynamics of excitatory and inhibitory responses to the same odorant. However, response dynamics were significantly different between aliphatic and aromatic compounds, and between the two aliphatic compounds. Each odorant caused opposing excitatory and inhibitory signals to be sent to the antennal lobe along at least two pairs of axonal pathways.

Activation of GABAA receptors modulates all stages of mechanoreception in spider mechanosensory neurons.

GABA(A) receptors mediate mainly inhibitory effects, but there are also many examples of excitatory effects in both mammalian and invertebrate preparations. Here, we aimed to create a complete, quantitative picture of GABA(A)-mediated excitation in a mechanosensory neuron where this phenomenon has been well established. We used muscimol to activate GABA(A) receptors in spider VS-3 neurons and measured the dynamic behavior independently and separately at each of three stages of mechanoreception (receptor current, receptor potential, and action potentials) before and during modulation. We calculated frequency response functions between each stage, estimated information as signal entropy, and estimated information capacity from coherence. Since coherence is sensitive to both noise and nonlinearity, we measured signal-to-noise separately at each stage by averaging responses to repeated mechanical inputs. Muscimol depolarized VS-3 neurons and, after brief inhibition, increased their firing rates. During this excitation, we found significant changes at each stage. Receptor current was attenuated but became more selective to high frequencies. Membrane impedance and time constant fell, favoring higher frequency transmission from receptor current to receptor potential. Action potential firing increased and had higher total entropy. Information capacity from signal-to-noise was always much higher than from coherence, confirming that intracellular noise does not limit signal transmission in these neurons. We conclude that GABA(A) receptor activation shifts each stage of mechanotransduction to higher frequency sensitivity, while the elevated firing rate increases the amount of information that can be encoded. These results show that a single neurotransmitter can finely modulate a sensory neuron's sensitivity and ability to transmit information.

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.

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.

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.

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.

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.

Regional distribution of calcium elevation during sensory transduction in spider mechanoreceptor neurons.

Spider mechanosensory VS-3 neurons receive peripheral efferent synaptic modulation, with regional variations in the types of efferent synapses and transmitter receptors. VS-3 somata possess a voltage-activated calcium current, but the levels and time courses of calcium changes in other regions are unknown. The roles of calcium in these neurons are not completely understood, but could include modulation of both mechanosensitivity and response dynamics. Here, we measured calcium concentration rises caused by single, mechanically induced action potentials in VS-3 sensory dendrites, somata and axons, using Oregon Green BAPTA-1 fluorescence. Calcium concentration rose by approximately 1 nM following each action potential. Time courses of calcium rise and fall were similar in the three regions but the rise in amplitude was about 50% higher in the sensory dendrite than in the soma. Antibody to the Ca(V)3.1(alpha(1g)) isotype of T-type calcium channel labeled all three neuronal regions. Some Ca(V)3.1 labeling colocalized with synapsin labeling, suggesting that calcium channels play some part in efferent modulation. We conclude that mechanically stimulated action potentials start near sensory dendrite tips and pass rapidly through the neurons to the axons, activating low voltage activated calcium channels in all three regions and causing calcium concentration to rise rapidly in each region. These results suggest important roles for calcium in several stages of mechanosensation.

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+.

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.

Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons.

In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70nM and rose to a maximum of about 400nM at firing rates above 20 action potentials per second.