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. Received: 15 July 1996 / Accepted: 26 August 1996     

A new mechanosensory organ on the anterior spinnerets of the spiderCupiennius salei (Araneae,Ctenidae)

We describe hitherto unknown mechanoreceptors on the anterior spinnerets of the spiderCupiennius salei. These receptors are found at the base of the spigots of the major ampullate glands which produce the dragline used by the spider as a safety thread in various behavioral situations. There are 40–60 mechanoreceptors associated with two spigots of each anterior spinneret. They are likely to provide information on the forces pulling on the dragline and also on its orientation in space. A single sensillum consists of a hole in the cuticle covered by a thin cuticular membrane. It much resembles spider slit sensilla, which are known to detect strains in the exoskeleton. Each sensillum is supplied by two dendrites most likely belonging to two bipolar sensory cells. One of the dendrites ends at the covering membrane and the other more proximally. The sensilla are arranged with their long axes roughly parallel to the circumference of the spigots. External forces, transmitted by the dragline, result in deformation of the central part of the cuticular plate at the base of the spigots and thus in stimulation of the sensilla. This is shown electrophysiologicallly. Considering their morphology, topography, and electrophysiology, these mechanoreceptors are suggested to be important in the sensory control of dragline release by the spider.     

Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium‐binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq‐like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin‐ and dynamin‐like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq‐like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium‐binding regions (EF hands) are conserved. The widespread occurrence of frq‐like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 165–175, 1999     

Synaptic localization of growth‐associated protein 43 in cultured hippocampal neurons during synaptogenesis

Growth‐associated protein 43 (GAP‐43), a novel axonal phosphoprotein, is originally identified as a growth‐cone‐specific protein of developing neurons in vitro. The expression of GAP‐43 is also shown to be up‐regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP‐43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP‐43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP‐43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double‐labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP‐43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double‐labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP‐43 was observed mostly at weak synapsin‐ and synaptotagmin‐positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP‐43 and either synapsin or synaptotagmin expression. These data indicate that GAP‐43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses.

We have investigated the possible role of synapsin I, a nerve terminal-specific protein, in the maturation of neuromuscular synapses in Xenopus cell cultures. Purified synapsin I was loaded into embryonic spinal neurons by injection of the protein into one of the early blastomeres of a Xenopus embryo. At synapses made by synapsin I-loaded neurons, spontaneous synaptic currents occurred with higher frequency and amplitude, and the amplitude exhibited an earlier appearance of a bell-shaped distribution. These characteristics are indicative of more mature quantal secretion. Impulse-evoked synaptic currents also showed a significant increase in amplitude. Using cell manipulation techniques, enhanced transmitter release from synapsin I-loaded neurons was shown to occur at the onset of synaptogenesis, suggesting a presynaptic developmental action of synapsin I prior to synaptic contact. Taken together, these results suggest that endogenous synapsin I may participate in the functional maturation of synapses.     

SynPAnal: Software for Rapid Quantification of the Density and Intensity of Protein Puncta from Fluorescence Microscopy Images of Neurons

Continuous modification of the protein composition at synapses is a driving force for the plastic changes of synaptic strength, and provides the fundamental molecular mechanism of synaptic plasticity and information storage in the brain. Studying synaptic protein turnover is not only important for understanding learning and memory, but also has direct implication for understanding pathological conditions like aging, neurodegenerative diseases, and psychiatric disorders. Proteins involved in synaptic transmission and synaptic plasticity are typically concentrated at synapses of neurons and thus appear as puncta (clusters) in immunofluorescence microscopy images. Quantitative measurement of the changes in puncta density, intensity, and sizes of specific proteins provide valuable information on their function in synaptic transmission, circuit development, synaptic plasticity, and synaptopathy. Unfortunately, puncta quantification is very labor intensive and time consuming. In this article, we describe a software tool designed for the rapid semi-automatic detection and quantification of synaptic protein puncta from 2D immunofluorescence images generated by confocal laser scanning microscopy. The software, dubbed as SynPAnal (for Synaptic Puncta Analysis), streamlines data quantification for puncta density and average intensity, thereby increases data analysis throughput compared to a manual method. SynPAnal is stand-alone software written using the JAVA programming language, and thus is portable and platform-free.     

Changes in the distribution of calcium calmodulin-dependent protein kinase II at the presynaptic bouton after depolarization

Phosphorylation of synapsin I by CaMKII has been reported to mobilize synaptic vesicles from the reserve pool. In the present study, the distributions of α-CaMKII and of synapsin I were compared in synaptic boutons of unstimulated and stimulated hippocampal neurons in culture by immunogold electron microscopy. CaMKII and synapsin I are located in separate domains in presynaptic terminals of unstimulated neurons. Label for α -CaMKII typically surrounds synaptic vesicle clusters and is absent from the inside of the cluster in control synapses. In contrast, intense labeling for synapsin I is found within the vesicle clusters. Following 2 minutes of depolarization in high K⁺, synaptic vesicles decluster and CaMKII label disperses and mingles with vesicles and synapsin I. These results indicate that, under resting conditions, CaMKII has limited access to the synapsin I in synaptic vesicle clusters. The peripheral distribution of CaMKII around vesicle clusters suggests that CaMKII-mediated declustering progresses from the periphery towards the center, with the depth of penetration into the synaptic vesicle cluster depending on the duration of CaMKII activation. Depolarization also promotes a significant increase in CaMKII immunolabel near the presynaptic active zone. Activity-induced redistribution of CaMKII leaves it in a position to facilitate phosphorylation of additional presynaptic proteins regulating neurotransmitter release.     

Ultrastructure of neurons and synapses in the tentacle gastrodermis of the sea anemone Calliactis parasitica

Little is known about gastrodermal neurons and synapses in the tentacles of sea anemones. Using transmission electron microscopy of serial thin sections of Calliactis parasitica, we have identified both a sensory cell and a ganglion cell with granular vesicles originating from the Golgi complex and have identified four types of synapses in the tentacular gastrodermal nerve plexus. The sensory cell has a recessed apical cilium with a basal body and a perpendicularly oriented centriole, below which are several strands of striated rootlets surrounded by mitochondria. The ganglion cell lacks a cilium and resembles a bipolar neuron, with oppositely directed processes lying parallel to the basally located circular smooth muscle. Both one-way and two-way interneuronal synapses are present with 60- to 90-nm granular vesicles of various densities aligned at the paired electron-dense membranes and fine cross filaments in the intervening 13-nm cleft. Two types of neuroeffector synapses have been located. Dense granular vesicles are present at neuromuscular synapses, whereas less dense vesicles are present at neuroglandular synapses. Most of the synaptic vesicles range from 60 to 120 nm in diameter. Two types of nerve cells and a variety of synaptic loci provide morphological substrates for the spontaneous SS2 conduction pulses in the tentacular gastrodermis of C. parasitica. J Morphol 231:217–223, 1997. © 1997 Wiley-Liss, Inc.     

Statistical independence and neural computation in the leech ganglion

 In this report, the input/output relations in an isolated ganglion of the leech Hirudo medicinalis were studied by simultaneously using six or eight suction pipettes and two intracellular electrodes. Sensory input was mimicked by eliciting action potentials in mechanosensory neurons with intracellular electrodes. The integrated neural output was measured by recording extracellular voltage signals with pipettes sucking the roots and the connectives. A single evoked action potential activated electrical activity in at least a dozen different neurons, some of which were identified. This electrical activity was characterized by a high degree of temporal and spatial variability. The action potentials of coactivated neurons, i.e. activated by the same mechanosensory neuron, did not show any significant pairwise correlation. Indeed, the analysis of evoked action potentials indicates clear statistical independence among coactivated neurons, presumably originating from the independence of synaptic transmission at distinct synapses. This statistical independence may be used to increase reliability when neuronal activity is averaged or pooled. It is suggested that statistical independence among coactivated neurons may be a usual property of distributed processing of neuronal networks and a basic feature of neural computation. Received: 20 September 1999 / Accepted in revised form: 3 March 2000     

Synapses identifiable in the parietal ganglia of the snail Helix lucorum

The synaptic plasticity is a background for learning and memory. Identifiable synapses that are the synapses between individually identifiable neurons are a very convenient model for studying plasticity. Synapses between the interoceptive mechanosensory neurons and the command neurons of the withdrawal behavior were identified in the Helix lucorum brain. It was shown that synaptic plasticity estimated by the dynamics of the elementary postsynaptic potentials elicited by single presynaptic spikes differed from the synaptic plasticity estimated by the dynamics of compound synaptic responses of the same neurons to sensory stimulation. Habituation and heterosynaptic facilitation phenomena are discussed in terms of the dynamics of the elementary postsynaptic potentials.     

Demonstration and localization of synapsin I in vestibular receptors in the cat: immunocytochemical study

The presence and localization of synapsin I, a neuron-specific phosphoprotein, was investigated in the cat vestibular epithelium, using a rabbit antisynapsin I anti-serum. The staining was performed by immunofluorescence or by a peroxidase-antiperoxidase (PAP) technique. A strong immunoreactivity was observed with both methods. This immunoreactivity appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which form axodendritic synapses with the afferent nerve chalice of type I hair cells, or axosomatic synapses with type II hair cells. Some of the nerve chalices were also labelled; in this case, the immunoreactivity was more evident with PAP staining. These results thus suggest the presence of large amounts of synapsin I in the vestibular efferent nerve endings. These endings are known to be filled with numerous synaptic vesicles. This localization of synapsin I is well correlated with previous work that report a close association between synapsin I and small synaptic vesicles. The presence of synapsin I in sensory endings such as the afferent nerve chalices was unexpected and is under investigation.     

Labial tip sensilla of Blissus leucopterus leucopterus (Hemiptera: Blissidae): Ultrastructure and behavior

The cuticular sensory receptors that are found on the apex of the labium of hemipterans play an important role in their feeding behavior. In this study we describe the ultrastructure, number, and distribution of sensilla on the labium apex of the chinch bug, Blissus leucopterus leucopterus. Each apical field of sensilla on the labium contains 11 uniporous peg sensilla and one sensillum chaeticum. The uniporous peg sensilla are innervated by 4–5 bipolar neurons that send dendrites in the lumen of each peg. Three neurons are associated with each sensillum chaeticum, two neurons have dendrites in the lumen of the sensillum, and the third dendrite ends in a tubular body at the base of the sensillum. Behavioral tests that involve chemical blockage of the sensory receptors show the importance of the labial sensilla in feeding behavior. Both morphological and behavioral evidence indicate that the labial sensilla have a chemosensitive function.     

Tactile learning by a whip spider, <Emphasis Type="Italic">Phrynus marginemaculatus</Emphasis> C.L. Koch (Arachnida,Amblypygi)

The ability of animals to learn and remember underpins many behavioural actions and can be crucial for survival in certain contexts, for example in finding and recognising a habitual refuge. The sensory cues that an animal learns in such situations are to an extent determined by its own sensory specialisations. Whip spiders (Arachnida, Amblypygi) are nocturnal and possess uniquely specialised sensory systems that include elongated ‘antenniform’ forelegs specialised for use as chemo- and mechanosensory feelers. We tested the tactile learning abilities of the whip spider Phrynus marginemaculatus in a maze learning task with two tactile cues of different texture—one associated with an accessible refuge, and the other with an inaccessible refuge. Over ten training trials, whip spiders got faster and more accurate at finding the accessible refuge. During a subsequent test trial where both refuges were inaccessible, whip spiders searched for significantly longer at the tactile cue previously associated with the accessible refuge. Using high-speed cinematography, we describe three distinct antenniform leg movements used by whip spiders during tactile examination. We discuss the potential importance of tactile learning in whip spider behaviour and a possible role for their unique giant sensory neurons in accessing tactile information.     

Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans

Summary The arista, a characteristic appendage of dipteran antennae, consists of 2 short segments at the base and a long distal shaft. A small sensory ganglion, from which arises the aristal nerve, is located proximally in the shaft. The fine structure of the aristal sensory organ was studied in detail in the fruitfly (Drosophila) and for comparison in the housefly (Musca) and the blowfly (Calliphora). In Drosophila, the aristal sense organ consists of 3 identical sensilla that terminate in the hemolymph space of the aristal shaft, and not in an external cuticular apparatus. Each sensillum comprises 2 bipolar neurons and 2 sheath cells; a third sheath cell envelops the somata of all six neurons of the ganglion. The neurons have long slender dendrites with the usual subdivision into an inner and an outer segment. One of the outer segments is highly lamellated and bears small particles (BOSS-structures) on the outside of its cell membrane; the other outer segment is unbranched and has a small diameter. The fine structure of the first dendrite is strongly reminiscent of thermoreceptors known from the antennae of other insects. These thermoreceptors are often coupled with hygroreceptors; however, we can only speculate whether the second dendrite of the aristal organ also has this function. Our present results argue against mechanoreceptive functions, as formerly postulated. The aristal sense organs in Musca and Calliphora are similar to those in Drosophila, but contain more sensilla (12 in Musca, 18 in Calliphora).     

Mechanosensory afferents innervating the swimmerets of the lobster

The mechanosensory innervation of the lobster (Homarus americanus) swimmeret was examined by electrophysiologically recording afferent spike responses initiated by localized mechanical stimulation of the caudal surface of the swimmeret. Two functional groups of subcuticular hypodermal mechanoreceptors innervate the swimmeret. Afferents of one group innervate the small discrete "ridges" of calcified cuticle lining the margins of both swimmeret rami. Putative ridge receptors are bipolar sensory neurons responding phasically to deformation of the ridge cuticle with the number and frequency of impulses produced dependent on stimulus strength and velocity. Afferents of the second group, which innervate substantial areas of hypodermis underlying the soft, flexible cuticular regions of the swimmeret, were designated "wide-field" hypodermal mechanoreceptors. These neurons have multiterminal receptive fields and respond phaso-tonically to cuticular distortion. The response properties of both types of hypodermal mechanoreceptors imply that they are activated during the characteristic beating movements of the swimmerets.     

Sex-specific antennal sensory system in the ant <Emphasis Type="Italic">Camponotus japonicus</Emphasis>: structure and distribution of sensilla on the flagellum

The antennae are a critically important component of the ant’s highly elaborated chemical communication systems. However, our understanding of the organization of the sensory systems on the antennae of ants, from peripheral receptors to central and output systems, is poorly understood. Consequently, we have used scanning electron and confocal laser microscopy to create virtually complete maps of the structure, numbers of sensory neurons, and distribution patterns of all types of external sensilla on the antennal flagellum of all types of colony members of the carpenter ant Camponotus japonicus. Based on the outer cuticular structures, the sensilla have been classified into seven types: coelocapitular, coeloconic, ampullaceal, basiconic, trichoid-I, trichoid-II, and chaetic sensilla. Retrograde staining of antennal nerves has enabled us to count the number of sensory neurons housed in the different types of sensilla: three in a coelocapitular sensillum, three in a coeloconic sensillum, one in an ampullaceal sensillum, over 130 in a basiconic sensillum, 50–60 in a trichoid-I sensillum, and 8–9 in a trichoid-II sensillum. The basiconic sensilla, which are cuticular hydrocarbon-receptive in the ant, are present in workers and unmated queens but absent in males. Coelocapitular sensilla (putatively hygro- and thermoreceptive) have been newly identified in this study. Coelocapitular, coeloconic, and ampullaceal sensilla form clusters and show biased distributions on flagellar segments of antennae in all colony members. The total numbers of sensilla per flagellum are about 9000 in unmated queens, 7500 in workers, and 6000 in males. This is the first report presenting comprehensive sensillar maps of antennae in ants.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines

The pectines of scorpions are ventral bilateral appendages supporting 10⁴–10⁵ chemosensory sensilla called pegs. Each peg contains 10–18 sensory neurons, some of which show ultrastructural evidence of axo-axonic synapses with other sensory neurons in the same sensillum. In extracellular recordings from single-peg sensilla, individual sensory units can be distinguished by impulse waveform and firing frequency. Cross-correlation analysis of impulse activity showed that at least two of these units, types `A1' and `A2', are inhibited during the 100-ms period immediately following activity of a third unit, type `B'. This interaction between sensory units in a single sensillum also occurs in surgically isolated pectines, indicating that it does not involve efferent feedback from the central nervous system. Other sensillar neurons appear to have excitatory interactions. Thus, in scorpion pectine, chemosensory information undergoes some form of processing within individual sensilla prior to its relay to the CNS, making this an unusually accessible preparation for study of first-order chemosensory processing events. Accepted: 12 April 1997     

Pattern of synaptic connections in the pineal organ of the ayu,Plecoglossus altivelis (Teleostei)

Summary Synaptic connections were studied by means of electron microscopy in the sensory pineal organ of the ayu, Plecoglossus altivelis, a highly photosensitive teleost species. Three types of specific contacts were observed in the pineal end-vesicle: 1) symmetrically organized gap junctions between the basal processes of adjacent photoreceptor cells; 2) sensory synapses endowed with synaptic ribbons, formed by basal processes of photoreceptor cells and dendrites of pineal neurons; 3) conventional synapses between pineal neurons, containing both clear and dense-core vesicles at the presynaptic site. Based on these findings, the following interpretations are given: (i) The gap junctions may be involved in an enhancement of electric communication and signal encoding between pineal photoreceptor cells. (ii) The sensory synapses transmit photic signals from the photoreceptor cells to pineal nerve cells. (iii) The conventional synapses are assumed to be involved in a lateral interaction and/or summation of information in the sensory pineal organ. A concept of synaptic relationships among the sensory and neuronal elements in the pineal organ of the ayu is presented.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany     

Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior

The role of histamine as a fast neuro-transmitter of imaginai insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdc ^JK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdc ^p211 , hdc ^p217 , and hdc ^p218 , variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.Abbreviations ERG electroretinogram - WT wild type