SPATIAL PATTERNS OF THREADLIKE ELEMENTS IN THE AXOPLASM OF THE GIANT NERVE FIBER OF THE SQUID (LOLIGO PEALII L.) AS DISCLOSED BY DIFFERENTIAL INTERFERENCE MICROSCOPY AND BY ELECTRON MICROSCOPY

The giant nerve fiber of the squid (Loligo pealii L.) has been investigated in situ, and in fresh and fixed preparations, by differential interference microscopy and electron microscopy. A continuous, three-dimensional network, composed of threadlike elements, was disclosed in the axoplasm. The threadlike elements in the axoplasm are twisted as a whole into a steep, right-handed helix. In a peripheral ectoplasmic region, the elements are more parallel to one another and more densely packed than in a central endoplasmic core. The threadlike elements can be resolved into a hierarchy of decreasing order of size. Successive levels of the hierarchy are formed by the association of smaller elements into larger ones. The following levels in the hierarchy of network elements have been distinguished: 1–3-µ-wide threads, 0.1–0.35-µ-wide strands, and 70–250-A-wide unit-filament strands. The differential interference microscope selects, from the network, threads oriented at a specific angle to the long axis of the axon. The specific angle depends upon the orientation of the long axis of the axon relative to the direction of shear. It is postulated that the network configuration is expressed in the solid-state properties of the axoplasm essential for the normal functioning of the nerve fiber.     

EXCITATION OF NERVE FIBERS IN THE SQUID (LOLIGO PEALII)

1. Strength-duration data for the giant fiber of the great stellar nerve of the squid (Loligo pealii) can be approximately described by several mathematical formulations. 2. Excitation time constants for isolated giant fibers are essentially the same as constants of the giant fibers in the intact nerve. 3. The strength-duration curves of the fibers in the intact nerve lie higher on the voltage axis than those of the isolated fibers. It is concluded that the principal effect of other fibers upon the excitation of one fiber in a nerve trunk is that of shunting the stimulating current. 4. Deterioration of the nerve shifts the curve upward and to the left, resulting in shorter time constants. 5. Decreasing interelectrode distance also shifts the curve upward and to the left. 6. Excitation time constants of the giant fibers are larger with plate electrodes than with wire or pore electrodes. 7. The strength-duration curves of the smaller fin nerve fibers lie consistently to the right of, and the time constants are longer than those of the giant fibers.     

IDENTIFICATION OF A CALCIUM-BINDING, BRAIN SPECIFIC PROTEIN IN THE AXOPLASM OF SQUID GIANT AXONS

Abstract— An acidic protein has been isolated from the optic lobes of two cephalopods, Sepia officinalis and Loligo vulgaris. The protein has been obtained in pure form by fractionation with ammonium sulphate and chromatography on DEAE-cellulose and Sephadex G 100. Its apparent molecular weight is 13,000–15,000. Glutamic and aspartic acids account for 35 per cent of the amino acid residues. The protein binds Ca²⁺ ions with an apparent dissociation constant of 2·5 × 10⁻⁵ M at physiological concentrations of KCI. Antibodies have been prepared against the protein purified from Sepia officinalis. By the micro-complement fixation technique it has been shown that the protein is highly concentrated in the nervous system of cephalopods and that the amount in the axoplasm of squid giant axons is eight to nine-fold higher than in the optic lobes of the same animal.     

ON THE CHEMICAL COMPOSITION OF THE AXOPLASM OF SQUID GIANT NERVE FIBERS WITH PARTICULAR REFERENCE TO ITS ION PATTERN

Investigations dealing with the determination of the major chemical constituents of the axoplasm of the giant nerve fiber of the squid are described. Particular emphasis has been placed on determining the components involved in acid-base balance. It was found that 72 per cent of the total solids of axoplasm, representing 13.5 per cent of the wet material, are of relatively low molecular weight (dialyzable) and consist mainly of charged ionic or dipolar constituents. Of the 520 micromoles per gm. of total base, 72 per cent are balanced by organic acids: aspartic acid (65 micro equivalents per gm.), glutamic acid (10 micro equivalents), fumaric and succinic acids (15 micro equivalents), a new polycarboxylic acid (35 micro equivalents), and isethionic acid, a biologically novel sulfonic acid (220 micro equivalents). Besides potassium, sodium, small amounts of calcium, and magnesium there is a considerable fraction of organic (nitrogenous) base. Other features of the chemical composition of squid axoplasm include a relatively high concentration of taurine (100 micro equivalents) and an ultraviolet absorbing substance possibly identical with N-methylpicolinic acid. The distribution of the phosphates, especially the concentration of ATP, has been investigated. Specific techniques elaborated in connection with this study have been described and the biochemical implications of the analytical results are discussed.     

THE SELECTIVE ACTION OF NICOTINE ON THE CENTRAL NERVOUS SYSTEM OF THE SQUID,LOLIGO PEALII

1. In specimens of freshly hatched squid, Loligo pealii, nicotine acts upon the cerebral ganglia alone. 2. After 1 minute in the nicotine solution 1:500,000, the latent period for the mantle spasm is independent of the time spent in the solution. 3. The mantle spasm is conditioned by a chemical reaction, since the temperature coefficient of the process has a magnitude of about 2.8. 4. The velocity of the process which brings about the mantle spasm varies as the cube root of the concentration of the nicotine.     

COMPARATIVE STUDIES IN SYNAPTOSOME FORMATION: THE PREPARATION OF SYNAPTOSOMES FROM THE HEAD GANGLION OF THE SQUID, LOLIGO PEALII

Abstract— Relatively high concentrations of ACh have been found in the head ganglion of the squid ( Loligo pealii ) and the identity of the ACh has been verified by ion-exchange chromatography. Following homogenization in media iso-osmotic with sea water about 40 per cent of the ACh survives in particle-bound form. Experiments using media of varying osmolarity suggest that this bound ACh is osmotically sensitive. A study has been made of the subcellular fractionation of squid head ganglion using sucrose homogenates. A rapid and novel method is described for the preparation of a synaptosome fraction freed from mitochondria. This preparation contains synaptosomes of well-preserved morphology with occluded cytoplasm and a high specific content of ACh. The synaptosomes are osmotically sensitive and when suspended in water they burst, releasing cytoplasmic constituents and ACh-containing synaptic vesicles. The synaptic vesicles can be separated from other sub-synaptic constituents by density gradient centrifugation.     

ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE MEMBRANES OF THE SQUID NERVE FIBER

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO₃)₂. Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.     

ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg ( s.e.m. ), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μ m in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270-420 μ m in diameter) and its separate components ( s.e.m .; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2-3 times higher in the sheaths (5-13 pmol-mg⁻¹) than in the axoplasm (2-4 pmol mg⁻¹), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107-222 μ m ) than in the axon (2-5 μ m ). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long-lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.     

A CALCIUM ACTIVATED PROTEASE IN SQUID AXOPLASM

Evidence for a protease in squid axoplasm which is selectively activated by Ca²⁺ and blocked by SH-inhibitors is presented. This protease appears to be particularly effective in degrading squid neurofilament proteins, but also extensively degrades various other major protein components in axoplasm.     

ELECTROPHYSIOLOGY OF EXTRAOCULAR PHOTORECEPTORS IN THE SQUID LOLIGO FORBESI(CEPHALOPODA: LOLIGINIDAE)

The electrophysiology of extraocular photoreception in the myopsidsquid, Loligo forbesi Steenstrup 1856 has been examined. Extracellulargenerator potentials were evoked by white light flashes. Intracellularrecordings from extraocular photoreceptor cells in the parolfactorybodies of the squid demonstrated that they had resting potentialsaround –40 mV, and were depolarised by flashes of white,but not red light (>650 nm). The evoked depolarisation consistedof a transient component, followed by a steady plateau component.The amplitude of depolarisation increased with the logarithmof the light intensity and was maintained for the duration ofthe light stimulus. Action potentials were seen in some recordingsand these increased in frequency during light flash stimulation. (Received 11 February 1997; accepted 10 May 1997)     

CALCIUM-CONTAINING ELECTRON-DENSE STRUCTURES IN THE AXONS OF THE SQUID GIANT SYNAPSE

Following the Oschman and Wall technique, electron-dense structures (EDS) were found on unstained, unosmicated membranes of squid giant synapse axons. These densities contain high concentrations of calcium and phosphorus as identified by energy dispersive X-ray analysis. Based on the signal strength, the quantity is significantly greater than that of other regions of the membrane or tissue spaces. The calcium EDS occur as plaques or globules along the axonic membrane, and small globules are found between sheath cell processes. EDS also occur at the synaptic site. These densities were correlated with the opacity change seen in giant axons. It is proposed that these structures represent sites where the calcium-binding protein found by other investigators has become nearly saturated with calcium.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

SITE OF BIOSYNTHESIS OF BRAIN-SPECIFIC PROTEINS IN THE GIANT FIBRE SYSTEM OF THE SQUID

The synthesis of brain-specific proteins has been examined in perikaryal and axonal regions of the giant fibre system of the squid. After in vitro incubation of stellate ganglia, stellate nerves and isolated giant axons with radioactive amino acids, the labelled soluble proteins have been extracted from the giant fibre lobe, the axoplasm and the axonal sheath of the giant axon and have been separated by gel electrophoresis on a continuous system. In addition, they have been challenged with antisera prepared against the cephalopod brain-specific proteins L1 and L2 and the resulting precipitate has been resolved by sodium dodecyl sulphate-gel electrophoresis. Synthesis of these two proteins appears to be restricted to the giant fibre lobe, while an additional discrete protein band (L5) also becomes clearly labelled in the isolated giant axon. Radioactive components migrating in the region of the L1 and L2 proteins are synthesized in the isolated giant axon. They can be distinguished from tbese proteins on the basis of electrophoretic and immunochemical criteria.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

EFFECTS OF NERVE STIMULATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—In vitro experiments were performed in order to determine whether nerve stimulation would affect the RNA metabolism of an identified giant neurone (R2) in the abdominal ganglion of Aplysia californica. The electrophysiological activity of the neurone was continuously monitored with an intra- or extracellular microelectrode. The mere presence of an intracellular microelectrode inside the neurone had no significant effect on the incorporation of tritiated nucleosides into the RNA of the giant neurone. Prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, produced a marked increase in the amount of labelled RNA in the nucleus as well as in the cytoplasm. Electrophoresis studies suggested that this increase in labelling might concern RNA with molecular weights corresponding to ribosomal as well as to non-ribosomal RNA.     

垂体前叶内神经纤维可能参与ACTH分泌的调节

我们建立了垂体组织块短时温育并施加电场刺激的离体实验体系,运用此方法并结合放射免疫测定激素含量,观察了大鼠垂体前叶内神经纤维对促肾上腺皮质激素（ＡＣＴＨ）释放的影响。结果表明,电场刺激能够促使垂体前叶ＡＣＴＨ释放显著增加,刺激参数为强度３０ｍＡ,波宽０．５ｍｓ,频率１０Ｈｚ。这个效应可为温育液中加入河豚毒素（ＴＴＸ）和藜芦碱所阻断,但ＴＴＸ不能阻断精氨酸加压素（ＡＶＰ）诱发的ＡＣＴＨ分泌。同样参数的电场刺激对分散培养的大鼠垂体前叶细胞ＡＣＴＨ的分泌没有显著作用。以上结果说明,我们所用参数的电场刺激产生的效应是兴奋了垂体前叶内的神经纤维,而非直接刺激腺细胞所致。上述结果提示：垂体前叶激素分泌的调节除了传统的体液途径之外,还可能存在直接的神经控制。     

INCORPORATION OF LABELLED PHOSPHATE INTO PHOSPHOLIPIDS IN SQUID GIANT AXONS

Inorganic phosphate labelled with ³²P was applied to giant axons excised from squid (Loligo pealeii) by addition of ³²Pi to the bathing solution, by injection into the axon, or by addition to axoplasm which had been separated from the sheath. The preparations were kept at 10 to 25° for various times up to 4 hr. When ³²P_i was supplied by way of the bathing solution, axoplasm and sheath were usually separated at the end of incubation before extraction of the lipids. Lipids were extracted with chloroform-methanol and resolved by paper chromatography. The lipids which became labelled appeared to be the same in sheath and axoplasm. They were identified by cochromatography with known lipids and by chromatography of products formed from them by mild alkaline hydrolysis. They included phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, and probably somelysophosphatidylethanolamine. Some labelled components remained unidentified. Phosphatidylcholine was apparently present, but did not become significantly labelled either in sheath or in axoplasm, or in a squid's stellate ganglion. There was no evidence that separation from the sheath impaired the capacity of the axoplasm for lipid synthesis.     

CLEAVAGE FURROW FORMATION IN A TELOLECITHAL EGG (LOLIGO PEALII) : I. Filaments in Early Furrow Formation

Formation of the first cleavage furrow in the telolecithal egg of Loligo was studied with the electron microscope. Before the actual furrow forms, a dense filamentous band develops below the plasma membrane from membrane-bounded dense bodies which appear to be Golgi-derived. The egg surface is thrown into a number of longitudinal folds which parallel the furrow and eventually become incorporated into it. These longitudinal folds contain a network of tubules and vesicles. Frequently, multivesiculate bodies are associated with the furrow and possibly give rise to the network of tubules and vesicles. Apparently part of the membrane between the two new blastomeres is derived from the surface of the longitudinal folds. The theory of furrow formation by contraction is discussed in light of the filamentous band.