Lyriform Slit Sense Organ Research Articles

Random Stimulation of Spider Mechanosensory Neurons Reveals Long-Lasting Excitation by GABA and Muscimol

gamma-Aminobutyric acid type A (GABA(A)) receptor activation inhibits many primary afferent neurons by depolarization and increased membrane conductance. Deterministic (step and sinusoidal) functions are commonly used as stimuli to test such inhibition. We found that when the VS-3 mechanosensory neurons innervating the spider lyriform slit-sense organ were stimulated by randomly varying white-noise mechanical or electrical signals, their responses to GABA(A) receptor agonists were more complex than the inhibition observed during deterministic stimulation. Instead, there was rapid excitation, then brief inhibition, followed by long-lasting excitation. During the final excitatory phase, VS-3 neuron sensitivity to high-frequency signals increased selectively and their linear information capacity also increased. Using experimental and simulation approaches we found that the excitatory effect could also be achieved by depolarizing the neurons without GABA application and that excitation could override the inhibitory effect produced by increased membrane conductance (shunting). When the VS-3 neurons were exposed to bumetanide, an antagonist of the Cl(-) transporter NKCC1, the GABA-induced depolarization decreased without any change in firing rate, suggesting that the effects of GABA can be maintained for a long time without additional Cl(-) influx. Our results show that the VS-3 neuron's response to GABA depends profoundly on the type of signals the neuron is conveying while the transmitter binds to its receptors.

Intracellular recording from a spider vibration receptor

The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

Mechanotransduction in spider slit sensilla

Mechanoreception is a vital constituent of several sensory modalities and a wide range of internal regulatory processes, but fundamental mechanisms for neural detection of mechanical stimuli have been difficult to characterize because of the morphological properties of most mechanoreceptors and the nature of the stimulus itself. An invertebrate preparation, the VS-3 lyriform slit sense organ of the spider, Cupiennius salei, has proved useful because it possesses large mechanosensory neurons, whose cell bodies are close to the sites of sensory transduction, and accessible to intracellular recording during mechanotransduction. This has made it possible to observe and experiment with all the major stages of mechanosensation. Here, we describe several important findings from this preparation, including the estimated number, conductance and ionic selectivity of the ion channels responsible for mechanotransduction, the major voltage-activated ion channels responsible for action potential encoding and control of the dynamic properties of the neurons, the location of action potential initiation following mechanical stimulation, and the efferent control of mechanoreception. While many details of mechanosensation remain to be discovered, the VS-3 system continues to offer important opportunities to advance our understanding of this crucial physiological process.

From stress and strain to spikes: mechanotransduction in spider slit sensilla.

This review focuses on the structure and function of a single mechanoreceptor organ in the cuticle of spiders. Knowledge emerging from the study of this organ promises to yield general principles that can be applied to mechanosensation in a wide range of animal systems. The lyriform slit sense organ on the antero-lateral leg patella of the spider Cupiennius salei is unusual in possessing large sensory neurons, whose cell bodies are close to the sites of sensory transduction, and accessible to intracellular recording during mechanotransduction. This situation, combined with recent technical developments, has made it possible to observe and experiment with all the major stages of mechanosensation. Important findings include the approximate size, number and ionic selectivity of the ion channels responsible for mechanotransduction, the types of voltage-activated ion channels responsible for action potential encoding, and the mechanisms controlling the dynamic properties of transduction and encoding. Most recently, a complex efferent system for peripheral modulation of mechanosensation has been discovered and partially characterized. Much remains to be learned about mechanosensation, but the lyriform slit sense organ system continues to offer important opportunities to advance our understanding of this crucial sense.

Structural correlates of mechanosensory transduction and adaptation in identified neurons of spider slit sensilla.

We used isolated but functionally intact preparations of the lyriform slit-sense organ VS-3 from the leg of the spider, Cupiennius salei Keys, to examine the role of prominent fine-structural elements for mechanosensory transduction and adaptation. Slit sensilla act as strain sensors in the cuticular exoskeleton; each slit is innervated by two mechanosensitive neurons. Punctate mechanical deformation at four points along the dendrites demonstrated that mechanical excitability is confined to membrane sites at the extreme dendrite tips that are enclosed by cuticular slit structures. Depletion of microtubules in VS-3 neurons by prolonged mechanical stimulation and application of 5 mmol l(-1) colchicine did not disrupt the generation of a receptor potential. Hence, putative gating mechanisms of the mechanically activated membrane channels at the dendrite tips appear to be largely independent of microtubular structures. The discrete adaptation pattern in each of the two partner neurons, rapidly adapting versus slowly adapting, did not depend on the distinct mode of dendrite attachment to cuticular slit structures, and even persisted in isolated neurons after their dendrite tips and auxiliary structures were lost. We suggest that the two discrete adaptation patterns are based on intrinsic differences in the action potential encoding process rather than differences in stimulus transformation or mechanotransduction.

Peripheral Synapses at Identified Mechanosensory Neurons in Spiders: Three-Dimensional Reconstruction and GABA Immunocytochemistry

The mechanosensory organs of arachnids receive diverse peripheral inputs. Little is known about the origin, distribution, and function of these chemical synapses, which we examined in lyriform slit sense organ VS-3 of the spider Cupiennius salei. The cuticular slits of this organ are each associated with two large bipolar mechanosensory neurons with different adaptation rates. With intracellular recording, we have now been able to correlate directly the staining intensity of a neuron for acetylcholinesterase with its adaptation rate, thus allowing us simply to stain a neuron to identify its functional type. All rapidly adapting neurons stain more heavily than slowly adapting neurons. Immunostaining of whole-mount preparations reveals GABA-like immunoreactive fibers forming numerous varicosities at the surface of all sensory neurons in VS-3; peripheral GABA-like immunoreactive somata are lacking. Sectioning the leg nerve procures rapid degeneration of most fiber profiles, confirming that the fibers are efferent. Punctate synapsin-like immunoreactivity colocalizes to these varicosities, although some synapsin-like immunoreactive puncta are GABA-immunonegative. Fibers with similar immunoreactivities are also associated with trichobothria, tactile hairs, internal joint receptors, i.e. other types of spider mechanosensory organs. In organ VS-3, immunoreactivity is most dense across the initial axon segment. The exact distribution of peripheral synapses was reconstructed from a 10-microm-long electron micrograph series of the dendritic, somatic, and initial axon regions of acetylcholinesterase-stained VS-3 neurons. These reveal a pattern similar to that of the synapsin-like immunoreactivity. Two different types of synapse were distinguished on the basis of their presynaptic vesicle populations. Many peripheral synapses thus appear to derive from efferent GABA-like immunoreactive fibers and probably provide centrifugal inhibitory control of primary mechanosensory activities.

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei: synapsin-like immunoreactivity.

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5-6.8 micrometer long and located 0.5-6.6 micrometer apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results. Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.

Ionic selectivity of mechanically activated channels in spider mechanoreceptor neurons.

The lyriform slit-sense organ on the patella of the spider, Cupiennius salei, consists of seven or eight slits, with each slit innervated by a pair of mechanically sensitive neurons. Mechanotransduction is believed to occur at the tips of the dendrites, which are surrounded by a Na+-rich receptor lymph. We studied the ionic basis of sensory transduction in these neurons by voltage-clamp measurement of the receptor current, replacement of extracellular cations, and application of specific blocking agents. The relationship between mechanically activated current and membrane potential could be approximated by the Goldman-Hodgkin-Katz current equation, with an asymptotic inward conductance of approximately 4.6 nS, indicating that 50-230 channels of 20-80 pS each would suffice to produce the receptor current. Amiloride and gadolinium, which are known to block mechanically activated ion channels, also blocked the receptor current. Ionic replacement showed that the channels are not permeable to choline or Rb+, but are partly permeable to Li+. The receptor current was inward at all membrane potentials (-200 to +200 mV) and never reversed, indicating high selectivity for Na+ over K+. This situation contrasts strongly with insect mechanoreceptors, vertebrate hair cells, and mechanically activated ion channels in nonsensory cells, most of which are either unselective for monovalent cations or selective for K+.

Acetylcholine and histamine are transmitter candidates in identifiable mechanosensitive neurons of the spider Cupiennius salei: an immunocytochemical study

Histochemical and indirect immunocytochemical techniques were used to search for neuroactive substances and transmitter candidates in identified sensory neurons of two types of cuticular mechanoreceptors in the spider Cupiennius salei Keys.: (1) in lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits each innervated by 2 bipolar neurons), and (2) in tactile hairs (each supplied by 3 bipolar sensory cells). All neurons are mechanosensitive. A polyclonal antibody against choline acetyltransferase (ChAT) strongly labeled all cell bodies and afferent fibers of both mechanoreceptor types. Western blot analysis using the same antibody against samples of spider sensory hypodermis and against samples from the central nervous system demonstrated a clear band at 65 kDa, corresponding to the molecular mass of ChAT in insects. Moreover, staining for acetylcholine esterase (AChE) revealed AChE activity in one neuron of each mechanoreceptor type. Incubation with a polyclonal antibody against histamine clearly labeled one neuron in each set of sensilla, whereas activity in the remaining one or two cells was near background. All mechanoreceptor preparations treated with a polyclonal antiserum against serotonin tested negative, whereas sections through the central nervous system of the same spiders were clearly labeled for serotonin. The presence of ChAT-like immunoreactivity and AChE implicates acetylcholine as a transmitter candidate in the two mechanoreceptive organs. We assume that histamine serves as a mechanosensory co-transmitter in the central nervous system and may also act at peripheral synapses that exist in these sensilla.

Intracellular characterization of identified sensory cells in a new spider mechanoreceptor preparation.

1. We have developed an isolated mechanoreceptor-organ preparation in which the intact sensory structures are available for mechanical stimulation and electrical recording. The anterior lyriform slit sense organ on the patella of the spider, Cupiennius salei Keys., consists of seven or eight cuticular slits, each innervated by a pair of large bipolar sensory neurons. The neurons are fusiform, and the largest somata are < or = 120 microns long. The innervation of the organ was characterized by light microscopy of neurons backfilled with neuronal tracers. Intracellular recording was used to measure the passive and active electrical properties of the neurons, in several cases followed by identification with Lucifer yellow injection. Both neurons of each pair from one slit responded with action potentials to depolarization by a step current injection. Approximately half of the sensory neurons adapted very rapidly and generated only one or two action potentials in response to a sustained depolarizing step, while a second group produced a burst of action potentials that adapted to silence in approximately 1 s or less. Recordings from identified neuron pairs indicated that each pair consists of one rapidly adapting and one bursting neuron. Measurements of cell membrane impedances and time constants produced estimates of neuronal size that agreed with the morphological measurements. This new preparation offers the possibility of characterizing the mechanisms underlying transduction and adaptation in primary mechanosensory neurons.

Morphology, sense organs, and regeneration of the forelegs (whips) of the whip spider Heterophrynus elaphus (Arachnida, Amblypygi).

In the whip spider Heterophrynus elaphus the first pair of legs is specialized to serve sensory functions. The morphology of these "whips" and the sensory organs of their tarsi and tibiae are described using scanning and transmission electron microscopy. The tarsus is normally subdivided into 74 segments and bears 7 types of sensory hairs: bristles, club sensilla, two types of porous sensilla, two types of rod sensilla, and leaflike hairs. In addition there are modified claws, 3 kinds of slit sense organs, a "pit organ," a "plate organ," and probably a joint receptor. The tibia is usually subdivided into 33 segments. In addition to bristles the tibia bears 7 trichobothria at constant locations and a lyriform slit sense organ. The functional and systematic implications of these findings are discussed.

Proprioceptors and sensory nerves in the legs of a spider, Cupiennius salei (Arachnida, Araneida)

Retrograde CoS-impregnation was used to trace and map the course of sensory nerves and the distribution and innervation of the various proprioceptor types in all leg segments of Cupiennius salei, a Ctenid spider. 1. Sensory nerve branches. In both the tibia and femur, axons of all proprioceptor types ascend in just two lateral nerves which do not merge with the main leg nerve until they reach the next proximal joint region. In the short segments — coxa, trochanter, patella, and tarsus — axons of the internal joint receptors often run separately from those of the other sensilla. Axons of the large lyriform slit sense organ at the dorsal metatarsus and of the trichobothria join with only a few hair axons and form their own nerve branches (Figs. 1, 2, 3). 2. Proprioceptors. Each of the seven leg joints is supplied with at least one set of the well-known internal joint receptors, slit sensilla (single slits and lyriform organs), and long cuticular hairs. In addition, we found previously unnoticed “hair plates” on both sides of the coxa, near the prosoma/coxa joint; they are deflected by the articular membrane during joint movements (Fig. 4). 3. Sensory cells and innervation. CoS-impregnation shows that each slit of the slit sense organs — be it a single slit or several slits in a lyriform organ — is innervated by two bipolar sensory cells (Fig. 6). We also confirm previous reports of multiple innervation in the internal joint receptors and in the long joint hairs and cuticular spines. Most of the ascending nerve branches run just beneath the cuticle for at least a short distance (Fig. 5); hence they are convenient sites for electrophysiological recordings of sensory activity even in freely walking spiders.

Matched transfer characteristics of single units in a compound slit sense organ

The transfer characteristics of the lyriform slit sense organ HS8 on the leg tibia ofCupiennius salei Keys. (Fig. 1) were investigated by electrophysiological experiments. The organ is made up of 7 slits. It was stimulated by deflecting the metatarsus toward the fixed tibia. The responses of the constituent slits were recorded individually. 1. The step responses of the 2nd to the 4th slits can be described bypower functions (t−k), where (t) is the time and (k) is a constant between 0 and 1 (Figs. 6, 8). This means that the sensilla take a position between a frequency independent system (displacement receptor) and a first order differentiator (velocity receptor). The standard deviations of the receptor responses coincide intra-individually and inter-individually (Fig. 5). 2. Thelinearity of the system was demonstrated using the second slit as an example: a) The slit responds proportionally to step amplitudes from 0.1° to 0.4°; (k) varies only slightly from 0.32 to 0.38 (Fig. 6). b) For stimulus frequencies of 0.01 Hz to 8 Hz the transfer functions calculated from the step response and those estimated from sinewave stimulation match well (Fig. 7). c) For stimulus frequencies of 0.01 Hz to 100 Hz the number of spikes/stimulus sinewave is nearly the same, no matter whether calculated from the step responses or measured by sinewave stimuli (Fig. 12). 3. Comparing the results for the 3 slit sensilla investigated (of a total of 7) the following conclusions are drawn: a) Thehigh-pass characteristic (exponentk) of the sensilla increases from the 2nd to the 4th slit, while the relative gain decreases (Fig. 8). b) Thebandwidth of the sensilla decreases according to decreasing sensitivity from slit 2 to slit 4, since the maximal stimulating frequency which can be encoded unequivocally (three spikes/stimulating sinewave) is less than 20–30 Hz for all the slits investigated (Fig. 12). c) Thefrequency-dependent threshold curves confirm the calculated transfer functions. For a stimulus frequency of 1 Hz the thresholds of the 2nd to the 4th slit are ca. 0.025°, 0.25° and 1.25° (deflection of the metatarsus), at 100 Hz 0.01°, 0.05° and 0.125° (extrapolated). From 0.01 Hz to 100 Hz no tuning of different slits to different “resonant frequencies” was seen (Figs. 11, 12). d) The characteristic curves (input amplitude vs. output-spike frequency) of the 2nd to 4th slit show an overlap of the amplitude ranges to which the sensilla respond; their linear parts, however, are linked up. This results in an extension of both the effective linear amplitude range and the range of precise detection of amplitude changes. The upper and lower limits of the linear parts of the characteristic curves depend on the stimulus frequency (Figs. 9, 10, 14, Eq. 6). 7. The receptor system can be described as consisting of a linear part with high-pass character plus a nonlinear rectifying part with a threshold. The linear part precedes the nonlinear one (Figs. 15, 16).