Quantitative parameters of synaptic apparatus in the ventral nucleus of the medial geniculate body of the cat

The synaptic apparatus in the ventral nucleus of the medial geniculate body (MGBv) of the cat was examined using electron microscopy and stereological methods, which made it possible to measure the synaptic density. Within 7015 µm² of examined sections, 1586 presynaptic terminal (PST) profiles were found, which corresponds to 226.0·10³ PST per 1 mm² of section surface. The PSP were classified into five groups:RL,RS,F,P, andUT, in accordance with their ultrastructural pattern (dimension of PST profile, dimension and shape of synaptic vesicles, and type of synaptic contact, SC) [18–22]. On the above surface, there were 1012 SC formed by PST of different groups, which corresponds to 144.0·10³ SC per 1 mm² of section surface. TheRL-,RS-,F-,P-, andUT-type PST formed 14.8%, 50.1%, 13.1%, 16.8%, and 5.2% of analyzed SC, respectively. The calculated mean SC numerical density equalled (260.8±54.8)·10⁶ SC per 1 mm³ of fixed MGBv tissue. Among them, 40.2·10⁶ (15.4%) belonged toRL-PST, i.e., to axonal terminals of thecolliculus inferior neurons; 130.2·10⁶ (49.9%) toRS-PST, i.e., mostly to axonal terminals of the auditory cortex neurons; and 33.9·10⁶ (13.0%) toF-PST, i.e., to axons of the GABA-ergic interneurons and neurons of the perigeniculate division of the reticular thalamic nucleus. Group-P PST, i.e., terminal structures of the dendritic arborizations of interneurons, formed 42.7·10⁶ (16.4%) SC per 1 mm³, and 13.8·10⁶ (5.3%) SC belonged toUT-PST, i.e., to terminals of unidentified nature. Among 260.8·10⁶ SC in 1 mm³ of tissue, only 23.8·10⁶ (9% of total number)RL-SC, localized on the relay neurons, are directly involved in the MGBv relay function. All other SC transmit control influences from various structures of the nervous system, and provide adjustment of relay function to the constantly changing environmental conditions and varying status of an orgamism. The mean number of SC, localized on an averaged MGBv relay neuron, was calculated as 9100. Among them, about 1200 SC belong toRL-PST, 5200 SC toRS-PST, 1200 SC toF-PST, 1100 SC toP-PST, and 400 SC toUT-PST.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 208–219, May–June, 1995.     

Genetic linkage between cytological markers and the seed storage protein lociSec2[Gli-R2] andSec3[Glu-R1] in rye

In order to reach a higher accuracy concerning the cytological locations of the rye seed storage protein lociSec2[Gli-R2] andSec3[Glu-R1] located within chromosome arms 2RS and 1RL, respectively, the linkage relationships between the following loci were analyzed: isozyme lociGpi-R1,Mdh-R1, andPgd2, translocationT273W (Wageningen tester set, involving chromosome arms 1RS and 5RL), the telomere C-bands of chromosome arms 1RL (tL1), 2RS (tS2), and 5RS (tS5), and three interstitial C-bands in chromosome arm 1RS (iS1), in the middle of chromosome arm 1RL (iL1), and in the middle of chromosome arm 2RL (iL2), respectively. The data indicated that locusSec3 is located in the distal half of chromosome arm 1RL (between C-bandiL1 and locusPgd2), while locusSec2 is located a short distance (2.9 ± 1.4%) from the telomere C-band of chromosome arm 2RS.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

Synthesis of (2RS,8R,10R)-YM-193221 and an Improved Approach to Tyroscherin,Bioactive Natural Compounds from Pseudallescheria sp.

Short-step syntheses of (2RS,8R,10R)-YM-193221 (1) and tyroscherin (2), which are biologically active compounds isolated from Pseudallescheria sp., were accomplished in six and eight steps from L-tyrosine. The relative stereochemistry of natural YM-193221 was determined to be 8R ^*,10R ^*.     

Synthesis of Both the Enantiomers of 3-Octanol,the Pheromone of Various Species of Ants

(2S,3R,1′S,2′S)-Serricorole (1) and (2S,3R,1′R)-serricorone (2), sex pheromone components of the cigarette beetle (Lasioderma serricorne F.), were synthesized, starting from the enantiomers of methyl 3-hydroxypentanoate. The stereochemistry of the naturally occurring 1 was determined to be 2S,3R,1′S,2′S, and that of 2 to be 2S,3R,1′RS by comparing between the CD spectra of the natural and synthetic samples.     

Mapping seed storage protein loci Sec-1 and Sec-3 in relation to five chromosomal rearrangements in rye (Secale cereale L.)

Summary Linkage relationships were established between the secalin loci, Sec 1 (40-K gamma and omega secalins, homologous to the wheat gliadins) and Sec 3 (HMW = high-molecular-weight secalins, homologous to the wheat HMW glutenin subunits), and five chromosomal rearrangements involving chromosome 1R of rye (Secale cereale L.). These were: interchanges T273W (1RL/5RS), T306W (1RS/5RL), and T850W (1RS/ 4RL), Robertsonian centromere split Rb1RW and the interchanged Robertsonian split Rb2R/248W. The analysis established the linkage relationships between the secalin loci and the breakpoints of the rearrangements, in addition to the quantitative effects of the rearrangements on the linkage. Sec-1 is located in the satellite at a position at least 2.5 cMorgan from the proximal border of the terminal C-band, and about 30 cMorgan from the nucleolar organizing region (NOR). The locus is also physically closer to the terminal C-band than to the NOR, but not as much as corresponds with the map distances. Similarly, the physical distance between Sec-3 and the centromere is greater than corresponds with the recombination frequency (0%–9%). Although overall recombination in 1RL remains the same, recombination between the centromere and Sec-3 is greatly reduced in the Robertsonian split combined with the interchange. This is not the case with the single Robertsonian split.     

Glucose utilization and growth response to protein hydrolysates byFusobacterium species

Glucose uptake was measured in the supernatants of 18 strains ofFusobacterium species cultured in BM medium. Some species, such asF. nucleatum andF. necrophorum, used between 25% and 48% of the glucose in the medium, but the terminal pH remained near neutral. By contrast, strains ofF. mortiferum andF. necrogenes used on average over 90% of the available glucose in the medium and produced a predictably low acidic pH. Strains ofF. varium used between 86% and 91% of the glucose present but produced a near neutral pH of between 5.8 and 5.9. The metabolic fate of glucose inF. varium was, therefore, examined in more detail. Glucose stimulated the growth of this species, and [¹⁴C]glucose was incorporated into the metabolic end products and various cellular components. Protein hydrolysates, tested for their growth-promoting effects onFusobacterium species, produced two general growth response patterns. Most species grew prolifically on trypticase, proteose peptone, and yeast extract, but poorly in casamino acids and vitamin-free casamino acids. Growth in bactocasitone was poor, but for three species,F. necrophorum, F. varium, andF. nucleatum, there was an approximately linear growth response up to 0.5%. These results suggest a major role for nitrogen metabolism but do not preclude glucose as an energy source in at least some species ofFusobacterium.     

Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Electron microscopic study of terminal degeneration in the anterodorsal thalamic nucleus of the cat

Summary The ultrastructure and synaptic organization of the anterodorsal nucleus (AD) of the thalamus were investigated under normal and experimental conditions. The large glomeruli are composed of an extensive central dendrite, probably arising from a projecting neuron, and of various terminal boutons. Besides the typical small bouton (RS) filled with round vesicles, two specializations of the large bouton (RL) containing round vesicles are found. The larger one (RL₁) is characterized by a looser arrangement of synaptic vesicles and many mitochondria; it undergoes dark degeneration following unilateral lesions of the mamillary body in both AD. The intermediate type (RL₂) is distinguished by a denser arrangement of vesicles; it undergoes dark degeneration following lesions of the midline nuclei of the thalamus. The two types of terminal boutons (F₁ and F₂) which contain flattened or pleomorphic vesicles do not degenerate following lesions of the mamillary body and midline nuclei; they belong to Golgi type II interneurons.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Fusion of isolated synaptic vesicles as a model of the terminal stage of regulated exocytosis

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10^–7 to 10^–5 M Ca²⁺ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Ca²⁺, Sr²⁺, and Ba²⁺, were compared. ATP induced membrane fusion both in the presence and in the absence of Ca²⁺, and the effects of ATP and Ca²⁺ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Ca²⁺ but also ATP. The intensity of Ca²⁺-dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Ca²⁺-regulated membrane fusion is possible without the involvement of the entire SNARE complex.Neirofiziologiya/Neurophysiology, Vol. 36, No. 4, pp. 272–280, July–August, 2004.This revised version was published online in April 2005 with a corrected cover date.     

The ventral lateral geniculate nucleus,pars lateralis of the rat

Summary The synaptic organization of the pars lateralis portion of the ventral lateral geniculate nucleus is similar to that of other thalamic nuclei. There are four types of synaptic knobs (RL, RS, F1, F2). RL knobs are large and irregularly shaped, contain round synaptic vesicles and make multiple asymmetrical junctions. They are found primarily in synaptic islands making contact with gemmules, spines, small dendrites, and other synaptic profiles containing pleiomorphic synaptic vesicles (F2). Smaller RS knobs contain round vesicles and make asymmetrical junctions with the same type of elements as RL knobs, with the exception of the F2 profiles, but are seldom found in synaptic islands. F1 knobs contain flattened synaptic vesicles and form symmetrical junctions with F2 knobs, gemmules, spines, and small-medium dendrites in synaptic islands, throughout the neuropil, and on the proximal dendrites and soma of the largest type of neuron. F2 knobs are irregularly shaped, contain pleiomorphic synaptic vesicles and make symmetrical junctions primarily with gemmules and spines in synaptic islands. They are postsynaptic to RL and F1 knobs. Occipital decortication indicates that cortical terminals are of the RS type. Bilateral enucleation indicates that retinal terminals are of both the RL and RS type. The large amount of geographic overlap of retinal and cortical terminals on gemmules, spines, and small dendrites found in the neuropil outside of synaptic islands logically would maximize axonal sprouting between these two sources.We would like to thank Mr. Peter Rossetti for his excellent technical assistance on a major portion of this project, Ms. Judith Strauss for photographic assistance, and Ms. Nancy Wood for typing. Supported by grants NS 10579, NS 08724, 5 S01 RR 05402, and 2 T01 GM 00326     

The fine structure of the synaptic membrane adhesions on Octopus synaptosomes

Summary Synaptosomes (nerve-ending particles), derived by homogenization and centrifugation fromOctopus andEledone brains, have been examined after OsO₄-fixation and PTA-staining, to determine the structure of the synaptic apparatus which holds together the synaptosomes and their postsynaptic processes. Both synaptic membranes are well-defined, with branching processes passing from the presynaptic membrane into the cytoplasm of the synaptosome, where synaptic vesicles apparently adhere to them. Small projections, with occasional web-like extensions, are seen along the cytoplasmic surface of the postsynaptic membrane. In transverse and oblique views of the cleft, bars are seen between the synaptic membranes. In frontal view, this part of the synaptic apparatus has a lattice arrangement of quadrilateral and pentagonal facets.A possible interpretation of these findings is discussed, and the functions of the synaptic apparatus are considered in the light of this.I am grateful to ProfessorsJ. Z. Young, F. R. S. andE. G. Gray for their advice and encouragement, and to Mr.S. Waterman for skilled photography.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Morphological differences between excitatory and inhibitory nerve terminals in cockroach coxal muscles

Two morphologically distinct types of neuromuscular junction on the coxal leg muscles of the cockroach, Periplaneta americana, which have been physiologically described as innervated by fast, slow and inhibitory nerve fibers, have been found. In one type of neuromuscular junction the axon terminal contains many round clear synaptic vesicles and contacts several sarcoplasmic extensions from the muscle fiber. The muscle processes adhere to the axon terminal for a short distance (short contact or SC type). The axon terminal of the other type of neuromuscular junction directly contacts the muscle fiber and no extensions of the muscle fiber are formed. The contact region is comparatively long (long contact or LC type). The nerve terminal contains many polymorphic synaptic vesicles. From a correlation of the present morphological findings and the previous physiological results, it may be suggested that the SC type of nerve terminal represents both fast and slow nerve terminals and the inhibitory terminal is of the LC type.     

Inheritance of evolved glyphosate resistance in <Emphasis Type="Italic">Conyza canadensis</Emphasis> (L.) Cronq.

N-(phosphonomethyl)glycine (glyphosate) resistance was previously reported in a horseweed [Conyza (=Erigeron) canadensis (L.) Cronq.] population from Houston, DE (P ₀ ^R ). Recurrent selection was performed on P ₀ ^R , since the population was composed of susceptible (5%) and resistant (95%) phenotypes. After two cycles of selection at 2.0 kg ae glyphosate ha^–1, similar glyphosate rates that reduced plant growth by 50%, glyphosate rates that inflicted 50% mortality in the population, and accumulations of half of the maximum detectable shikimic acid concentration were observed between the parental P ₀ ^R and the first (RS₁) and second (RS₂) recurrent generations. In addition, RS₁ and RS₂ did not segregate for resistance to glyphosate. This suggested that the RS₂ population comprised a near-homozygous, glyphosate-resistant line. Whole-plant rate responses estimated a fourfold resistance increase to glyphosate between RS₂ and either a pristine Ames, IA (P ₀ ^P ) or a susceptible C. canadensis population from Georgetown, DE (P ₀ ^S ). The genetics of glyphosate resistance in C. canadensis was investigated by performing reciprocal crosses between RS₂ and either the P ₀ ^P or P ₀ ^S populations. Evaluations of the first (F₁) and second (F₂) filial generations suggested that glyphosate resistance was governed by an incompletely dominant, single-locus gene (R allele) located in the nuclear genome. The proposed genetic model was confirmed by back-crosses of the F₁ to plants that arose from achenes of the original RS₂, P ₀ ^P , or P ₀ ^S parents. The autogamous nature of C. canadensis, the simple inheritance model of glyphosate resistance, and the fact that heterozygous genotypes (F₁) survived glyphosate rates well above those recommended by the manufacturer, predicted a rapid increase in frequency of the R allele under continuous glyphosate selection. The impact of genetics on C. canadensis resistance management is discussed.