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Central inhibitory microcircuits controlling spike propagation into sensory terminals.

The phenomenon of afferent presynaptic inhibition has been intensively studied in the sensory neurons of the chordotonal organ from the coxobasal joint (CBCO) of the crayfish leg. This has revealed that it has a number of discrete roles in these afferents, mediated by distinct populations of interneurons. Here we examine further the effect of presynaptic inhibition on action potentials in the CBCO afferents and investigate the nature of the synapses that mediate it. In the presence of picrotoxin, the action potential amplitude is increased and its half-width decreased, and a late depolarizing potential following the spike is increased in amplitude. Ultrastructural examination of the afferent terminals reveals that synaptic contacts on terminal branches are particularly abundant in the neuropil close to the main axon. Many of the presynaptic terminals contain small agranular vesicles, are of large diameter, and are immunoreactive for gamma-aminobutyric acid (GABA). These terminals are sometimes seen to make reciprocal connections with the afferents. Synaptic contacts from processes immunoreactive for glutamate are found on small-diameter afferent terminals. A few of the presynaptic processes contain numerous large granular vesicles and are immunoreactive for neither GABA nor glutamate. The effect that the observed reciprocal synapses might have was investigated by using a multicompartmental model of the afferent terminal.

Action Potentials↗

Structure and function relationship in the abdominal stretch receptor organs of the crayfish.

The structure/function relationship in the rapidly and slowly adapting stretch receptor organs of the crayfish (Astacus leptodactylus) was investigated using confocal microscopy and neuronal modeling methods. Both receptor muscles were single muscle fibers with structural properties closely related to the function of the receptors. Dendrites of the rapidly adapting neuron terminated in a common pile of nerve endings going in all directions. Dendrites of the slowly adapting neuron terminated in a characteristic T shape in multiple regions of the receptor muscle. The slowly adapting main dendrite, which was on average 2.1 times longer and 21% thinner than the rapidly adapting main dendrite, induced larger voltage attenuation. The somal surface area of the slowly adapting neuron was on average 51% larger than that of the rapidly adapting neuron. Variation in the neuronal geometry was greatest among the slowly adapting neurons. A computational model of a neuron pair demonstrated that the rapidly and the slowly adapting neurons attenuated the dendritic receptor potential like low-pass filters with cut-off frequencies at 100 and 20 Hz, respectively. Recurrent dendrites were observed mostly in the slowly adapting neurons. Voltage signals were calculated to be propagated 23% faster in the rapidly adapting axon, which is 51% thicker than the slowly adapting axon. The present findings support the idea that the morphology of the rapidly and the slowly adapting neurons evolved to optimally sense the dynamic and the static features of the mechanical stimulus, respectively.

Abdomen↗

Characteristic expression patterns of allatostatin-like peptide, FMRFamide-related peptide, orcokinin, tachykinin-related peptide, and SIFamide in the olfactory system of crayfish Procambarus clarkii.

The olfactory system plays important roles in various crustacean behaviors. Despite numerous studies on different aspects of the olfactory neural pathway, only the decapod-tachykinin-related peptide (decapod-TRP) has been identified as a neuromodulator in this processing to date. To establish the functions of other related neuropeptides, we initially performed cDNA cloning of FMRFamide-related peptide (FaRP) and allatostatin (AST)-like peptide from the crayfish Procambarus clarkii, followed by in situ hybridization (ISH) analysis of these peptides, along with decapod-TRP, orcokinin, and crustacean-SIFamide. Cloned FaRP cDNA encodes seven copies of C-terminal RN(F/Y)LRFamide-containing peptide, whereas AST-like peptide cDNA comprises 29 copies of AST-like peptide (-YXFGLamide) and three additional putative peptides. ISH analysis of the brain revealed specific expression of crustacean-SIFamide mRNA in most projection neurons (cell cluster 10), and predominant localization of other mRNAs to interneurons. The data suggest that the crustacean-SIFamide neuropeptide is involved in output of the deutocerebrum to the protocerebrum. Double-fluorescence ISH data further disclose that, in cluster 9, orcokinin is coexpressed in decapod-TRP-specific interneurons, whereas AST-like peptide-containing cells do not overlap with orcokinin-expressing cells. On the other hand, FaRP-expressing cells overlap with both orcokinin- and AST-like peptide-specific cells. In cluster 11, where signals for AST-like peptide are absent, a number of interneurons express both decapod-TRP and orcokinin, emphasizing a close relationship between these two factors with regard to olfactory processing, and possibly tactile and/or visual sensory systems. These characteristic expression patterns of neuropeptides support their distinct involvement in the modulation of olfactory processing.

Animals↗

Adult neurogenesis: a common strategy across diverse species.

Adult neurogenesis, the generation of new neurons from adult precursor cells, occurs in the brains of a phylogenetically diverse array of animals. In the higher (amniotic) vertebrates, these precursor cells are glial cells that reside within specialized regions, known as neurogenic niches, the elements of which both support and regulate neurogenesis. The in vivo identity and location of the precursor cells responsible for adult neurogenesis in nonvertebrate taxa, however, remain largely unknown. Among the invertebrates, adult neurogenesis has been particularly well characterized in freshwater crayfish (Arthropoda, Crustacea), although the identity of the precursor cells sustaining continuous neuronal proliferation in these animals has yet to be established. Here we provide evidence suggesting that, as in the higher vertebrates, the precursor cells maintaining adult neurogenesis in the crayfish Procambarus clarkii are glial cells. These precursor cells reside within a specialized region, or niche, on the ventral surface of the brain, and their progeny migrate from this niche along glial fibers and then proliferate to form new neurons in the central olfactory pathway. The niche in which these precursor cells reside has many features in common with the neurogenic niches of higher vertebrates. These commonalities include: glial cells functioning as both precursor and support cells, directed migration, close association with the brain vasculature, and specialized basal laminae. The cellular machinery maintaining adult neurogenesis appears, therefore, to be shared by widely disparate taxa. These extensive structural and functional parallels suggest a common strategy for the generation of new neurons in adult brains.

Animals↗

Distribution of ultrastructural tracers in crustacean axons.

Ruthenium red and horseradish peroxidase were used to compare the uptake of exogenous molecules into crayfish motor axons and their sheaths in severed and intact peripheral nerves. Both tracers penetrated the axonal sheath and were subsequently seen lining small vesicles and tubules in the axoplasm. Tracer appeared to enter the axon via pinocytotic vesicles. There were no perceptible quantitative or qualitative differences in ruthenium red uptake between intact and severed axons. However, counts of tracer-filled vesicles in axons exposed to peroxidase showed that at least three times as much tracer penetrated the severed as opposed to the intact axons.

Animals↗

Fine structure of synaptic vesicles in two types of nerve terminals in crayfish stretch receptor organs: influence of fixation methods.

Crayfish stretch receptor organs were used. The standard procedure consisted of primary fixation with a 1.6% glutaraldehyde solution containing either buffer which was 60% hyposmotic or buffer which was isosmotic with the physiological solution (440 milliosmols), washing with isosmotic buffer, and postfixation with an isosmotic 1% osmium tetroxide solution. Under these conditions, we encountered two types of nerve terminals; small-vesicle terminals (SVTs) containing small elongated vesicles (300 approximately 330 A) and large-vesicle terminals (LVTs) containing larger round vesicles (430 A). Their location and physiological evidence suggest that SVTs and LVTs are inhibitory and excitatory, respectively. A hyperosmotic primary fixation solution due to increased glutaraldehyde concentration gave results similar to the standard procedures, while a hyperosmotic primary fixation solution due to increased buffer concentration caused shrinkage of the nerve terminal. A hyperosmotic buffer wash produced elongation of vesicles in SVTs and LVTs, while washing with hypoosmotic buffer rendered vesicles in both types round. Direct fixation with isosmotic osmium tetroxide yielded less elongated vesicles in SVTs and irregularly round vesicles in LVTs. However, under all conditions, vesicles in SVTs were smaller than those in LVTs. These results suggest that the consistent morphological difference in vesicles between SVTs and LVTs is size rather than shape, and it is important to standardize the osmolarity of primary fixation and washing solutions when discussing the differences of vesicle shape in various kinds of synapses.

Adaptation, Physiological↗

Trans-glial channels in ventral nerve roots of crayfish.

The sheath around the roots of the sixth abdominal ganglion in the ventral nerve cord of the crayfish consists of concentric layers of thin glial processes alternating with wide clefts containing filamentous connective tissue. Regions of each glial lamella are perforated by single, short, tubular channels: the trans-glial channels. In thin plastic sections examined in the electron microscope, the channels appear as slits that are 240 A wide and 450-550 A long which traverse glial lamellae less than 1,500 A thick. Branched tubular channels cross glial sheets that are thicker than 1,500 A. The thickest glial wrap is adaxonal; it closely encapsulates individual axons and its cell membrane is separated from the axolemma by a collagen-free space of only 150 A. The adaxonal glial cytoplasm contains unique, three-dimensional networks of interconnected tubules. Separate tubular lattices occur along these thicker processes. In replicas of freeze-fractured sheaths, the outer half of the plasma membrane belonging to the thin glial sheets exhibits many volcano-like protrusions which represent cross fractures through the necks of trans-glial channels. Corresponding depressions on the inner half of these membranes are sites where the plasma membrane invaginates to form the channels. Although some channels are randomly dispersed, others are lineraly positioned in restricted areas across successive glial layers. The number of channels is far more readily appreciated in replicas than in thin sections. The average frequency of channels is 16 per mu2 (range 8 to 33) in normal roots and does not differ significantly from the average of 13 per mu2 in proximal stumps of roots fixed three to four weeks after the roots were cut. The channels are not precisely aligned from one glial layer to the next but do appear to coincide approximately with the adaxonal tubular lattice. The combination of trans-glial channels and adaxonal tubular lattices may provide a complex conduit that could facilitate a rapid, passive flow of electrolytes and nutrients across the nerve sheath to the axonal surface. Horseradish peroxidase solutions bathing the ventral roots enter the trans-glial channels, extracellular clefts and finally the tubular lattices. This distribution supports the proposed role of the channels in a rapid extracellular passage of solutes. The channel profiles have a range of forms consistent with the supposition that they are not static but continually reforming. There are indications that, proximal to the cut, the areas of glial plasma membrane with channel profiles contain more junctional complexes between regenerating cells than between glial cells of normal sheaths. The channel profiles and aggregates of particles belonging to junctions are closely associated when they occupy the same region of the membrane.

Animals↗

The retina and retinal projection on the lamina ganglionaris of the crayfish Pacifastacus leniusculus (Dana).

The retinular cell morphology and ommatidia arrangement in the crayfish retina are studied. Each ommatidium contains eight receptor cells (R1-R8). Seven of them (R1-R7) contribute to a large spindle-shaped rhabdom with orthogonal layers of microvilli. Distal to the rhabdom of R1-R7, the 8th receptor cell forms a separate rhabdom with horizontal microvilli. The cell is four-lobed, is devoid of screening pigments, and forms a thin axon projecting past the first optic neuropile (lamina ganglionaris) and has a terminal in the second (medulla externa), indicating a separate function of the receptor. The axons from the eight receptors of one ommatidium project to different synaptic compartments (cartridges) in the lamina. A pattern is described where eight axons from three adjacent ommatidia join in one cartridge; and conversely, the axons from one ommatidium split and join to three cartridges. The seven axons from R1-R7 terminate in two levels of the lamina plexiform layer (epl 1 and epl 2), four in the distal and three in the proximal part. Among the monopolar ganglion cells, two types are found with lateral branches restricted to either of the two receptor terminal layers (M3 in epl 1 and M4 in epl 2) and axons terminating in the second optic neuropile. A correlation between the two orthogonal channels for e-vector discrimination and the two levels of terminals within the lamina is suggested. The retina is divided into dorsal and a ventral part with a mirror symmetry axis horizontally in the eye.

Animals↗

Formation of hemi-desmosomes during regeneration of crayfish nerve root sheath as studied with freeze-fracture.

The multilamellate glial sheath of mixed nerve roots of the sixth abdominal ganglion of crayfish contains numerous hemi-desmosomes which appear to attach glial lamellae to material in adjacent extracellular clefts. These junctions, which have been described in detail in an earlier report (Shivers and Brightman, '76), are irregular in shape, punctuate and may be as large as 1 mum in diameter. Surgical interruption of sixth ganglion nerve roots results in regeneration of motor axons and their multilamellate glial sheaths. As the glial processes grow and re-establish a highly organized axon sheath, hemi-desmosomes appear. These junctions are present at the advancing edge of glial processes as well as on their lateral margins. Developing hemi-desmosomes are characterized as a diffuse aggregation of 120-130 A intramembrane particles which are present three weeks following nerve section. As growth and reorganization of the sheath proceeds, the intramembrane particles appear to aggregate and form irregular clusters of varying dimensions. Regenerating nerves freeze-cleaved 8 to 16 weeks following surgery exhibit junctional particle aggregates similar to those in normal unoperated nerve roots. Origin of the intramembrane particles which comprise the junctional aggregated in unknown. Perhaps they are synthesized de novo by the regenerating glial cells or, they may be remnants of complexes which became dispersed following surgery. This is the first report of a freeze-fracture study of hemi-desmosome plasticity in an invertebrate nervous system.

Animals↗

The distribution of 3H-leucine labeled protein in the retinula cells of the crayfish retina.

The synthesis and distribution of 3H-leucine labeled protein was studied under conditions of diurnal lighting in the retinula cells of the crayfish retina with both light and electron microscopic autoradiography. Times ranging from two minutes to seven days after an intracardiac injection were analyzed. Quantification of the electron microscopic autoradiograms revealed that labeling of the cytoplasm was greater than the rhabdome at 2, 5, and 30 minutes and reached a peak at 12 hours. The rhabdome showed increasing activity after 5, 30, and 60 minutes, also reaching a peak at 12 hours. Radioactive label in cytoplasmic multivesicular bodies was higher than activity measured in either the total cytoplasm or rhabdome at all times except two minutes. Two temporally different microvillar labeling patterns were seen under diurnal lighting conditions. (1) Microvilli forming the slightly enlarged distal tip of the rhabdome retained their radioactivity at 1, 3, and 7 days, when labeling of the rest of the rhabdome microvilli was decreasing. (2) In the remainder of the microvilli, labeling at 1 and 12 hours appeared as a gradient which declined toward the proximal end of the rhabdome. This gradient subsequently reversed itself, showing heavier proximal labeling at three days. In a second experiment, labeling patterns in light and dark adapted rhabdomes were compared. In the dark, a distinct gradient of activity was observed with radioactivity concentrated distally and declining toward the proximal end of the rhabdome. A more even distribution of label was present in the light adapted eye, but a slight distal-proximal gradient was still present. The dark adapted rhabdomes had more radioactivity per unit area than those exposed to light.

Adaptation, Ocular↗

Ultrastructural basis of impulse propagation failure in a nonbranching axon.

The morphological basis of intermittent conduction failure in the excitor axon innervating the crayfish opener and stretcher muscles was investigated using the electron microscope. The connective tissue component of the sheath surrounding the axon was found consistently to be thinner in the region at which blocking occurs than in control regions located one cm either proximally or distally, at which blocking does not occur. Otherwise in these regions differences in the width of the periaxonal spaces, the length or width of the mesaxons, the density of mitochondria, the width of the adaxonal glial cell layer, or the structure of lamination of the sheath are not observed. Because of the thinner connective tissue component of the sheath in the joint region, neighboring axons are distributed more densely around the excitor, and the volume of the extracellular space is reduced. The possibility that the reduced extracellular space might allow excessive accumulation of potassium during repetitive discharge, causing conduction block, is discussed. Alternative mechanisms consistent with this morphology are also considered.

Animals↗

Ultrastructural specificity of synaptic sites in nerve terminals mediating both presynaptic and postsynaptic inhibition.

The ultrastructural basis of presynaptic inhibition of the excitatory efferent axon innervating the opener muscle of the crayfish walking leg was studied using the electron microscope. The inhibitory efferent axon terminals form axoaxonal synapses onto excitor terminals in very close proximity to sites of excitatory transmitter release. There is no evidence that these synapses are located at regions of low safety factor of action potential conduction in the excitor. In the area of axoaxonal synaptic connections, the inhibitory axon also forms inhibitory neuromuscular junctions on the opener muscle. Synaptic vesicles are clustered in active zones bordered postsynaptically by extensive invaginations, or junctional folds, in the sarcolemma. These results are consistent with the hypothesis that the primary effect of presynaptic inhibition is to shunt depolarization-secretion coupling at specific sites of transmitter release in the target cell, rather than to block impulse propagation into a more generalized population of nerve terminals distal to the axoaxonal synapse. Further physiological implications of this structural arrangement are considered.

Animals↗