RESUMEN
The mechanosensory neurons of arachnids receive diverse synaptic inputs in the periphery. The function of most of these synapses, however, is unknown. We have carried out detailed electron microscopic investigations of the peripheral synapses at sensory neurons in the compound slit sense organ VS-3 of the spider Cupiennius salei. Based on the localization of discrete presynaptic vesicle populations, it is possible to discriminate at least four different synapse types, containing either: (1) small round, electron-lucent vesicles 32 nm in diameter; (2) large round, clear 42-nm vesicles; (3) a mixture of small and large clear, round vesicles, similar in size to those in Type 1 and Type 2 synapses, respectively, and granular and dense-core vesicles; or (4) clear, round 37- to 65-nm vesicles. Combined immunocytochemical labeling at the light and the electron microscopic level suggests that gamma-aminobutyric acid (GABA) is the transmitter in many of the 32-nm vesicle synapses, and glutamate in many of the 42-nm ones. Based on vesicle type and particular synaptic configuration, various forms of presumed efferent synaptic contacts are distinguishable with the sensory neurons, the surrounding glia, and between the putative efferent fibers themselves. These include simple unidirectional synapses, reciprocal synapses, serial synapses, and convergent as well as divergent dyads. These various synaptic microcircuits are suited to serve a variety of functions. Among these are direct postsynaptic inhibition or excitation of the mechanosensory neurons, and disinhibition or sensitization via presynaptic inhibition or excitation. The observed synaptic configurations are compared with those at the crustacean muscle receptor organ. They reveal a remarkable complexity of synaptic microcircuits at spider sensilla and suggest manifold possibilities for subtle, efferent control of sensory activity.
Asunto(s)
Vías Eferentes/ultraestructura , Mecanorreceptores/ultraestructura , Neuronas/ultraestructura , Nervios Periféricos/ultraestructura , Terminales Presinápticos/ultraestructura , Arañas/anatomía & histología , Sinapsis/ultraestructura , Vesículas Sinápticas/ultraestructura , Animales , Vías Eferentes/metabolismo , Ácido Glutámico/análisis , Inmunohistoquímica , Mecanorreceptores/fisiología , Microscopía Electrónica , Neuronas/metabolismo , Nervios Periféricos/metabolismo , Terminales Presinápticos/clasificación , Arañas/fisiología , Sinapsis/clasificación , Ácido gamma-Aminobutírico/análisis , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The single-electrode voltage-clamp technique requires sharp glass-capillary microelectrodes, whose electrical properties often limit the capabilities of the recording system. Here, we describe a rapid and simple way of coating fine microelectrodes with Dricote and Vaseline that improves their performance during voltage-clamp. The coating prevented clogging of the tips, improved the capacitance compensation of the electrodes, helped to seal the electrode tips into cell membranes and allowed visualization of the tips under saline solution. This new coating method led to greatly improved recordings and better characterization of the transduction and voltage-activated currents in an isolated preparation of spider mechanosensory neurons.
Asunto(s)
Vidrio , Microelectrodos , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Animales , Conductividad Eléctrica , Mecanorreceptores/fisiología , Sistema Nervioso/citología , Fenómenos Fisiológicos del Sistema Nervioso , Neuronas/fisiología , ArañasRESUMEN
Neurophysiological and behavioral work often requires that various laboratory stimulators be feedback-stabilized. We describe the design and performance of a versatile electronic controller that can be used to extend and flatten the frequency response of commercially available stimulating devices. The design includes flexible proportional-integral-derivative control action and active second-order, high-pass compensation. As an example application of this controller to 3 different electro-mechanical vibrator/transducer combinations demonstrates that the useful frequency response can be extended by more than a decade as compared with the uncontrolled device.
Asunto(s)
Artrópodos/fisiología , Mecanorreceptores/fisiología , Neurofisiología/instrumentación , Estimulación Física/instrumentación , Animales , Retroalimentación , VibraciónRESUMEN
A site-directed antibody was used immunocytochemically to measure the distribution of sodium channels in the tissues of a spider mechanoreceptor organ. The VS-3 slit sense organ contains 7-8 pairs of bipolar sensory neurons; these neurons are representative of a wide range of arthropod mechanoreceptors. Sensory transduction is thought to occur at the tips of the dendrites and to cause action potentials that are regeneratively conducted to the cell bodies, although it has not been possible to confirm this by direct intracellular recordings from the dendrites. Wholemount preparations were labelled by immunofluorescence and thin sections were immunogold labelled, using an antibody to the highly conserved SP19 sequence of the voltage-activated sodium channel. Labelling for sodium channels was found in the neurons and in their surrounding glial cells. Both cytoplasm and membranes were labelled, but immunogold particles were clearly aligned along cell membranes, indicating that the majority of labelling represented membrane-bound sodium channels. Channel density in the dendrites was similar to the axons and higher than in the cell bodies, supporting the idea of active conduction in the sensory dendrites. Labelling in glial cell membranes was indistinguishable from the neighboring neurons, suggesting a significant role for sodium channels in the functions of these supporting cells.
Asunto(s)
Mecanorreceptores/metabolismo , Canales de Sodio/metabolismo , Arañas/metabolismo , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Dendritas/metabolismo , Dendritas/fisiología , Dendritas/ultraestructura , Femenino , Inmunohistoquímica , Mecanorreceptores/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Neuroglía/fisiología , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Neuronas Aferentes/ultraestructura , Neuropéptidos/farmacología , Canales de Sodio/farmacología , Canales de Sodio/ultraestructuraRESUMEN
Using antisera directed against octopamine (OA), we identified and mapped octopamine-immunoreactive (OA-ir) neurons and their projections in the fused, central ganglion complex of wandering spiders, Cupiennius salei. Labeled cell bodies are concentrated in the subesophageal ganglion complex (SEG) where they are arranged serially in ventral, midline clusters. OA-ir processes from these cells project dorsally. Some neurites end close to segmental septa; others merge into longitudinal tracts connecting the neuromeres. Labeled collaterals leaving these tracts project into peripheral neuropil. In the brain, OA-ir somata were found only in the two cheliceral hemiganglia, where a cluster of 4-5 relatively large cells (soma diameter 25 microns) lies next to a group of small somata (diameter < 10 microns). Neurites originating from the large somata descend into the SEG and merge into longitudinal tracts. The central body of the brain contains profuse ascending projections. Except for fine varicosities that are confined to the roots of nerves, we found no OA-ir fibers leaving the central nervous system (CNS). Within the CNS, however, OA-ir varicosities are concentrated in neuropil and near hemolymph spaces. This distribution suggests that OA acts as a neurotransmitter and/or local neuromodulator at central synapses, while it is also released into the hemolymph and presumably acts hormonally at peripheral sites. Using high-pressure liquid chromatography measurements, the hemolymph was in fact found to contain 12-40 nM of free octopamine.
Asunto(s)
Sistema Nervioso/anatomía & histología , Neuronas/citología , Octopamina/análisis , Arañas/anatomía & histología , Animales , Artrópodos/anatomía & histología , Femenino , Ganglios/anatomía & histología , Ganglios/citología , Inmunohistoquímica , Sistema Nervioso/citología , Especificidad de la EspecieRESUMEN
Tilly Edinger (1897-1967) was a vertebrate paleontologist interested in the evolution of the central nervous system. By combining methods and insights gained from comparative neuroanatomy and paleontology, she almost single-handedly founded modern paleoneurology in the 1920s while working at the Senckenberg Museum in Frankfurt am Main. Edinger's early research was mostly descriptive and conducted within the theoretical framework of brain evolution formulated by O. C. Marsh in the late 19th century. Nevertheless, she became immediately known in 1929 after publishing an extensive review on "fossil brains." Reconstructing evolutionary history from the fossil record instead of from the comparative analysis of living forms allowed her to identify the sequence of neural innovations within several mammalian lineages. Anti-Jewish terrorism forced Edinger to leave Nazi Germany in 1939. After finding refuge first in England, she continued her career at Harvard's Museum of Comparative Zoology. There she documented the occurrence of gross neural correlates of specialized behavior in several vertebrate lineages, and identified parallel evolution in mammalian sulcation patterns. Her insight that neural innovations need not be "correlated" with either nonneural innovations or with evolutionary "success" led her to reject Marsh's theory of progressive increase in brain size over time and other "anthropocentric" understandings of brain evolution. Edinger's research, her insistence on a stratigraphic and evolutionary framework for interpretation, and her massive compilations of paleoneurological literature established her as the leading definer, practitioner, and chronicler of her field.
Asunto(s)
Encéfalo/anatomía & histología , Fósiles , Neuroanatomía/historia , Paleontología/historia , Alemania , Historia del Siglo XIX , Massachusetts , Neurociencias/historiaRESUMEN
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.
Asunto(s)
Mecanorreceptores/fisiología , Rótula/inervación , Arañas/fisiología , Transmisión Sináptica/fisiología , Animales , Mecanorreceptores/anatomía & histología , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Umbral Sensorial/fisiología , Transducción de Señal/fisiología , Canales de Sodio/fisiología , Arañas/anatomía & histologíaRESUMEN
Julia Barlow Platt was a comparative embryologist and neurobiologist who was primarily interested in segmentation of the head in vertebrates. She was born on September 14, 1857 in San Francisco, California. Platt grew up in Burlington, Vermont, attended the University of Vermont and began graduate studies at Harvard University. Her nine years as a graduate student were spent on two continents with some of the most influential comparative zoologists of the time. Platt's remarkable scientific accomplishments over a ten year period include a description of axial segmentation currently used in the staging of chick embryos and the first description of a separate anterior head segment in Squalus embryos. Her most controversial study identified ectodermal cells in Necturus embryos that gave rise to head cartilage and dentine, a discovery which was the impetus for the reassessment and modification of the germ layer concept. She was one of the first women to 'matriculate' at a German university and receive a Ph.D. degree. Platt played a pioneer role in opening opportunities for other women who followed her. Platt was one of the first women neuroscientists. Among her contributions, she distinguished dorsolateral placodes, epibranchial placodes, and the first stages of lateral line organs in Necturus, and she described nerve fibers originating in the spinal cord and extending to the notochord in Branchiostoma (= Amphioxus). After receiving a Ph.D. degree in Freiburg, Germany in 1898, Platt was unable to secure a suitable teaching position and, as a result, her scientific career came to an end. She retired to Pacific Grove, California, where she pursued civic duty with the same vigor and energy she had dedicated to scientific research. We provide a sketch of her remarkable life and work as a comparative embryologist, neuroscientist and civic leader.
Asunto(s)
Embriología/historia , Neurociencias/historia , Historia del Siglo XIX , Historia del Siglo XX , Estados UnidosRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Mecanorreceptores/fisiología , Mecanorreceptores/ultraestructura , Neuronas Aferentes/fisiología , Órganos de los Sentidos/inervación , Arañas/fisiología , Potenciales de Acción/fisiología , Animales , Dendritas/fisiología , Microscopía Electrónica , Microtúbulos/fisiología , Estimulación FísicaRESUMEN
Ludwig Mauthner was only 19 years old when he published his discovery of the colossal fibers in the spinal cord of fishes which now bear his name. Based on Mauthner's works, archival material, and contemporary sources we provide a summary of his life and work as neuroanatomist and ophthalmologist in imperial Austria. In the years 1859-1863 Mauthner published four papers on the structure of the central nervous system in vertebrates. His first report on fishes contains the original description of a 'colossal myelinated nerve fiber' on each side of the central canal, extending through the entire spinal cord. Another, more general, treatise on 'the morphological elements of the nervous system' (published in 1863) summarizes his neurohistological studies of various vertebrates. It includes a classification of nerve cells based on their (histochemical) reaction to carmine. The main findings were soon shown to be artefactual; the paper had a long-range impact, however, because it provoked fruitful controversy among contemporary neuroanatomists. Mauthner published several monographs and numerous articles in ophthalmology, a newly developing branch of medicine that he chose for his later career. After abruptly resigning from a professorship at Innsbruck University, he opened a private practice in Vienna and continued lecturing in his field. He became a noted eye-surgeon, was elected Assistant Director of the Vienna 'Allgemeine Poliklinik', and in 1894 became Professor and Chair of Ophthalmology at the University of Vienna. Mauthner unexpectedly died on the night following the formal announcement of his appointment.
Asunto(s)
Neuroanatomía/historia , Oftalmología/historia , Animales , Austria , Encéfalo/citología , Reacción de Fuga , Peces/anatomía & histología , Historia del Siglo XIX , Neuronas/citología , Médula Espinal/citología , Vertebrados/anatomía & histologíaRESUMEN
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.
RESUMEN
In spiders, retrograde cobalt staining was used to clarify the distribution and detailed innervation of the three types of proprioceptors in the tibio-metatarsal leg joint: internal joint receptors, lyriform slit sense organs, and cuticular spines and hairs. The axons of all these receptors run in just two lateral, ascending nerves, which had previously been associated only with the internal receptors. Each nerve contains several hundred axons ranging in diameter from 0.1 micron to ca. 10 micron. Each slit of the four tibial lyriform organs is innervated by two bipolar sensory neurons. The lateral nerves are entirely sensory and run just beneath the cuticle, a convenient site for electrophysiological recording. We demonstrate simultaneous nerve and muscle recordings from intact spiders; these, in combination with selective sensory ablations, show that a resistance reflex in the flexor metatarsi muscles is elicited by internal joint-receptor units.
Asunto(s)
Mecanorreceptores/fisiología , Propiocepción , Reflejo/fisiología , Arañas/fisiología , Animales , Extremidades , Femenino , Mecanorreceptores/citología , Arañas/anatomía & histologíaRESUMEN
1. Intracellular microelectrodes recorded the receptor potential and receptor current in the neurons of spider slit sense organs during mechanical stimulation of the slits. 2. Mechanical stimulation produced two patterns of action potential discharge, corresponding to the two groups of neurons described previously by electrical stimulation. 3. Tetrodotoxin eliminated the action potentials and revealed a receptor potential with both static and adapting components. Voltage clamp gave an inward receptor current with a similar time course. 4. Replacement of sodium ions in the bath reversibly eliminated the receptor current, indicating that it is carried by sodium ions. However, this effect was comparatively slow, suggesting that the tips of the sensory dendrites lie in a chemically restricted environment.
Asunto(s)
Mecanorreceptores/fisiología , Neuronas/fisiología , Canales de Sodio/fisiología , Sodio/fisiología , Arañas/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica , Electrofisiología , Femenino , Mecanorreceptores/efectos de los fármacos , Microelectrodos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Estimulación Física , Canales de Sodio/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
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+.
Asunto(s)
Cationes/metabolismo , Canales Iónicos/fisiología , Mecanorreceptores/fisiología , Neuronas Aferentes/fisiología , Animales , Femenino , Litio/metabolismo , Técnicas de Placa-Clamp , Rubidio/metabolismo , ArañasRESUMEN
Chelicerates constitute a basic arthropod group with fossil representatives from as early as the Cambrian period. Embryonic development and the subdivision of the segmented body region into a prosoma and an opisthosoma are very similar in all extant chelicerates. The mode of head segmentation, however, has long been controversial. Although all other arthropod groups show a subdivision of the head region into six segments, the chelicerates are thought to have the first antennal segment missing. To examine this problem on a molecular level, we have compared the expression pattern of Hox genes in the spider Cupiennius salei with the pattern known from insects. Surprisingly, we find that the anterior expression borders of the Hox genes are in the same register and the same relative segmental position as in Drosophila. This contradicts the view that the homologue of the first antennal segment is absent in the spider. Instead, our data suggest that the cheliceral segment is homologous to the first antennal segment and the pedipalpal segment is homologous to the second antennal (or intercalary) segment in arthropods. Our finding implies that chelicerates, myriapods, crustaceans, and insects share a single mode of head segmentation, reinforcing the argument for a monophyletic origin of the arthropods.
Asunto(s)
Expresión Génica , Genes Homeobox , Cabeza/crecimiento & desarrollo , Arañas/crecimiento & desarrollo , Animales , Evolución Molecular , Datos de Secuencia Molecular , Arañas/embriologíaRESUMEN
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.