Freeze-fracture study of the mechanoreceptive digital corpuscles of mice

Summary The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 ± 517 m^–2 (mean ±s.e.m.) in the P-face and 84 ± 4 m^–2 in the E-face. Particles were 10 ± 1.8 nm in diameter in the P-face and 10 ± 1.5 nm (mean ±s.d.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 ± 224 m^–2 in the P-face and 265 ± 95 m^–2 in the E-face. Particles were 10 ± 1.4 nm in diameter in the P-face and 10 ± 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 ± 283 m^–2 and 1514 ± 514 m^–2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 ± 1.2 nm and 10 ± 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively.The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.     

Fulminant purkinje cell death following axotomy and its use for analysis of the dendritic arborization

Summary Young and adult cats were operated upon and a number of the vermal cerebellar folia were either transected with a vertical incision or isolated by a horizontal cut.In the proximity of the lesion, Purkinje cell bodies and their dendritic trees became stainable with the Fink-Heimer method. Electron microscopy of the silver stained sections show that the argyrophilic Purkinje neurons undergo an electron dense type of degeneration. Stellate cell dendrites adjacent to the degenerating Purkinje trees are normal, suggesting that the cause of cell death is axotomy close to the perikaryon rather than direct injury. The retrograde Purkinje cell degeneration is fulminant since it is evident 6 hours after the lesion.In Fink-Heimer stained sections the entire dendritic tree is impregnated 1–3 days after the lesion. 4–10 days post-operatively, the flattened dendritic tree becomes fragmented and is partially phagocytized. The silver stained arborizations are approximately 280 in width and have an uneven thickness (8–16 ).In longitudinal and horizontal silver stained sections of lesioned cerebellar folia, uninterrupted fields of degenerating Purkinje cell arborizations can be seen, suggesting that the arborizations overlap. The overlap was demonstrated in electron micrographs of single degenerating arborizations surrounded by normal dendritic trees. The degree of overlap varies with the thickness of the arborization and is in the order of 1–2 .This approach indicates that each Purkinje tree occupies an exclusive sheet of molecular layer 8 thick and may overlap for as much as 2 on each side with neighboring trees. The average thickness of the Purkinje tree is approximately 12 .Portions of this work performed by S. Brand are in partial fulfillment of requirements for the degree of Doctor of Philosophy.This work has been funded by NIH grant NS-09904.     

Pyramidal tract of the cat: axon size and morphology

Summary The purpose of this work was to determine the number and morphology of pyramidal tract (PT) axons in the cat, using electron microscopy, modern methods of fixation, and computer-assisted morphometric analysis. Sections taken at the level of the medullary pyramids in three animals were fixed and magnified up to 10,000 x to produce photomicrographs. Morphological data were entered into computer files for analysis by tracing axon perimeters on micrographs mounted on a digitizer tablet. The number of axons per PT averaged 415,000, of which 88% were myelinated and 12% were unmyelinated. 90% of the myelinated axons fell in the diameter range 0.5–4.5 m. Axons larger than 9 m diameter accounted for 1% of the total; the largest were 20–23 m. Myelinated axon mean diameter was 1.98 m; because of the skewed distribution, with many small axons and a few very large axons, median diameter was 1.60 m. Size distribution was relatively uniform throughout the PT cross section, with all sizes represented in all regions. However, the more medial regions had a higher proportion of small fibers than the more lateral regions: mean medial diameter was 1.85 m while mean lateral diameter was 2.09 m. Myelin sheath thickness averaged 7.9% of fiber diameter for axons up to 11 m, but was constant at 0.9 m for larger fibers. Myelinated fibers were distorted from the circular shape in cross section, with a mean circularity index (or form factor) of 0.85, which implies that the fibers could swell about 15% without rupture of the cell membrane. Unmyelinated fibers averaged 0.18 m diameter (range 0.05–0.6 m); the largest unmyelinated axons were larger than the smallest myelinated axons. It is concluded that previous work greatly underestimated the number of axons in the cat pyramidal tract.     

Early stages of axonal regeneration in the goldfish optic tract: An electron microscopic study

Summary Two hours after the goldfish optic tract was cut, the severed axons in the retinal stump of the tract showed ballooning of the axoplasm and myelin sheath in the region of the cut, with accumulation in the swollen axon of various organelles, including dense cored vesicles. By day 1 the myelin sheath had degenerated back to a node of Ranvier and the tip of the severed axon had formed a myelin-free terminal bulb with a well-organized core of 9–10 nm filaments. By 2 days, such terminal bulbs were often seen to be extended on a neck of cytoplasm a few micrometers in length, presumably indicating axonal outgrowth. In addition, occasional small bundles of axon sprouts were first seen at this time. The sprouts had a diameter of about 2 m and contained a central core of 9–10 nm filaments surrounded by a mantle of cell organelles (smooth endoplasmic reticulum, mitochondria and diverse vesicles), with few if any microtubules. Sprouts within a bundle were separated by fairly uniform 10–15 nm spaces. Beginning at 3 days, significant numbers of microtubules appeared in the sprouts, and there was an increasing proportion of small diameter (0.3 m) sprouts. Thus it was not until 3 days that the sprouts took on the appearance usually considered to be typical of regenerating axons. By 6 days a dense layer of glial cells or macrophages formed a cap over the cut surface of the tract. Penetrating this layer were bundles containing up to 20–30 axon sprouts and also single axons which may have been serving as pioneering fibres to which later-emerging axons would attach. There was no evidence that the regenerating axons were guided by the glial cells. At 6 days astroglia began to separate individual axons within the bundles but oligodendrocytes were still inactive at this time.     

Henneguya pinnae n. sp. aus den Flossen von Ctenopoma kingsleyae Günther (Osteoichthyes,Anabantidae)

Zusammenfassung Henneguya pinnae wird als neue Art beschrieben. Sie wurde im Corium der Flossen von Ctenopoma kingsleyae gefunden. H. pinnae bildet annähernd runde Cysten mit einem Durchmesser bis zu 0,7 mm. Das Plasmodium wird von einem vom Wirt gebildeten Epitheloid eingeschlossen. Die Sporen sind lanzettförmig und sehr schlank. Verdickungen der Sporenschale wurden nur auf Querschnitten in der Region der Polkapseln beobachtet. In diesem Gebiet sind die Sporen etwas abgeflacht, während der Querschnitt sonst annähernd rund erscheint. Länge des Sporenkörpers 10–12 m, Schwanzanhänge 10–15 m, größte Dicke 3–4 m, Polkapsellänge 3 m. Anzahl der Windungen des Polfadens: 5.

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Henneguya pinnae n.sp. from the fins of Ctenopoma kingsleyae Günther (Osteoichthyes, Anabantidae)

Summary A new species, Henneguya pinnae, is described, parasitizing the corium of the fins of Ctenopoma kingsleyae. H. pinnae forms spherical cysts with a diameter up to 0,7 mm. The plasmodium is surrounded by an epitheloid formed by the host. The spores are lanceolate and remarkable slender. Sutural markings are visible in cross-sections in height of the anterior polar capsules only. There the spore is flattened, elsewhere cross sections are nearly spherical. Body length of the spore 10–12 m, tail 10–15 m, diameter 3–4 m, length of the polar capsules 3 m. There are 5 coils of the polar filament.

     

Ultrastructural study of ascending projections to the lateral mammillary nucleus of the rat

Summary We examined the synaptic organization of ascending projections from the pars ventralis of the dorsal tegmental nucleus of Gudden (TDV) and the laterodorsal tegmental nucleus to the lateral mammillary nucleus (LM). The LM neuropil consists of terminals containing pleomorphic synaptic vesicles and forming symmetric synaptic contact, and terminals containing round synaptic vesicles and forming asymmetric synaptic contact. They make up 63% and 37%, respectively, of all axodendritic terminals. All axosomatic terminals contain pleomorphic vesicles and make symmetric contact. Following injection of WGA-HRP into the TDV, many anterogradely labeled terminals and retrogradely labeled cells are found in the LM. Labeled terminals contact mainly proximal (more than 2 m diameter) and intermediate (1–2 m diameter) dendrites. Serial ultrathin sections of the LM show that 55% of axosomatic terminals are labeled anterogradely. Following injection of WGA-HRP into the laterodorsal tegmental nucleus, many anterogradely labeled terminals are found in the LM, but no retrogradely labeled cells are present. Labeled terminals contact mainly distal (less than 1 m diameter) and intermediate dendrites as well as somata. In the LM neurons, 46% of axosomatic terminals are labeled anterogradely. All labeled terminals from these nuclei contain pleomorphic vesicles and make symmetric synaptic contact. These results indicate that almost all axosomatic terminals come from the TDV and the laterodorsal tegmental nucleus, which send inhibitory inputs to the lateral mammillary nucleus.     

Trypanosoma congolense: the in vitro akinetoplastic induction sensitivity assay

Incubation ofTrypanosoma conglense in diminazene aceturate (Berenil) or isometamidium chloride (Samorin) induced akinetoplastic (AK) forms in vitro. The AK values (expressed in percent) obtained were found to be useful for rapid assessment of relative drug sensitivities. In susceptible clones, AK forms were induced at all drug concentrations tested, whereas in resistant clones they were induced only at higher concentrations. The Berenil-resistant clone exhibited AK values of 0.9%±0.6%–8.9±2% at concentrations of 1–100 g/ml at 4–10 h post-inoculation (p.i.), whereas the Berenil-susceptible clone displayed values of 9.3%±3%–19.2%±5% at 0.1–50 g/ml. Motile trypanosomes were not seen at 100 g/ml at 4 h p.i. or at 10 or 50 g/ml at 10 h p.i. The Samorin-resistant clone showed AK values of 0.5%±0.1%–43%±3% at concentrations of 0.1–100 g/ml at 4 and 10 h p.i., whereas the Samorin-susceptible clone exhibited values of 5.3%±2%–45%±4% at 0.0005–100 g/ml. These results were supported by the findings obtained using a mouse infectivity test.     

Ultrastructure of hair follicle afferent fibre terminations in the spinal cord of the cat

Summary In acute electrophysiological experiments on anaesthetized cats, single identified hair follicle afferent fibres were injected with horseradish peroxidase (HRP). The HRP was injected from an intra-axonal microelectrode in the lumbosacral spinal cord. One to six hours after injection the animals were perfused and the tissue prepared for light and electron microscopy (EM). Axon collateral arborizations containing HRP reaction product were identified in thick sections under the light microscope and the same tissue then cut on the ultramicrotome for EM study. The terminal branches of the collaterals kept their myelin sheaths until they were 0.45–l.0 m in diameter, just before they formed synaptic boutons. Synaptic boutons (1.0–4.0 m in diameter) were usually of theen passant variety and made contact with dendrites. The contacts were asymmetrical (Type I) and contained round, clear synaptic vesicles of 35–60 nm diameter. Both the non-myelinated portion of the terminal axon and the synaptic boutons received axo-axonic contacts. These axo-axonic boutons contained clear (agranular) vesicles irregular in profile.MRC research student.     

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: Whole-cell patch-clamp studies

We have performed whole-cell patch-clamp studies on dispersed seccretory cells of the rat mandibular gland to determine how -adrenergic stimulation causes fluid secretion. When the pipette contained a high K⁺ solution, the resting membrane potential averaged –33 mV±1.1 (SEM,n=34) and the clamped cell showed strong outward rectification. We monitored K⁺ and Cl^– currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and –80 mV, respectively. Isoproterenol (1–2 mol/l) caused increases in the clamp current at 0 mV (the K⁺ current) and at –80 mV (the Cl^– current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca²⁺ ionophore, A23187. The -adrenergic antagonist, phentolamine (1–2 mol/l), had no effect on the response, but the -adrenergic antagonist, propranolol (10 mol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mol/l), forskolin (1 or 20 mol/l) or cholera toxin (2.5 g/ml). However, increasing the Ca²⁺-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that -adrenergic agonists activate Cl^– and K⁺ channels by raising cytosolic Ca²⁺. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca²⁺ via the phosphatidylinositol cycle.This project was supported by the National Health and Medical Research Council of Australia     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Vasopressin studies in the rat

Summary In previous experiments single i.v. injections of 0.2–10 U ADH were made in alcohol anesthetized rats and the amount of extra water reabsorbed during the antidiuretic phase (urine deficit) was measured. The dose response curve resembled a saturation curve with a fairly linear rise up to 1–1.5 U. When 4–6 U were injected the urine deficit was not appreciably greater while 75% of the ADH injected appeared in the urine during antidiuresis. It was concluded that during a single injection of ADH 1–1.5 U were bound almost instantaneously at receptors of the tubular wall and inactivated during the slow process of water reabsorption, while the excess ADH was excreted in the urine. It was estimated that 1 U ADH was needed for the reabsorption of approximately 5 cm³ water. The time required for this process is short at a high rate of reabsorption and vice versa.In the present investigation the single i.v. injections were repeated with 20 U. The higher dose permitted the separate determination of ADA in 3 consecutive samples of the urine collected during the antidiuretic phase. The result fully confirmed the working hypothesis e.g.:1. The antidiuretic activity (ADA) obtained with 20 U Pitressin was not greater but even (though not significantly) smaller than that obtained previously with 5 U Pitressin or 1 U Tonephin.2. 95±15% of the ADA injected appeared in the urine. This means that the difference between the 20 U injected and the 18.5–19 U appearing in the urine after deduction of the 1–1.5 U ADH supposedly bound at tubular pore sites was too small to be detected with our bioassay.3. Under the assumption that 1 U Pitressin was used up for the reabsorption of approximately 4 cm³ water a vasopressin-water-equivalent in the order of 1 mole vasopressin for 10⁸ mole water reabsorbed, could be calculated.4. The amount of vasopressin excreted by the kidney follows an exponential function with a half life of 5 min.5. The vasopressin clearance is approximately 1.0 cm³/min · rat and lies within the range of inulin clearance (1.2 cm³/min · rat). It is suggested that elimination of excess vasopressin proceeds by a simple filtration process.6. Calculating on a weight basis the ADH-requirement of the 200 times heavier human kidneys leads to the value 200–300 U. Using a vasopressin-water-equivalent of 4–5 cm³ water per 1–1.5 U (action time 50 min) it can be predicted that the human kidney must lose approximately 261 water per day under the condition of a complete lack of vasopressin. This agreement with the actual observations in diabetes insipidus patients supports the belief that some of the concepts worked out in the alcohol anesthetized rat are valid under circumstances other than the strict conditions of this preparation.

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Zusammenfassung In früheren Experimenten wurden Einzelinjektionen von 0,2–10 E ADH in Alkohol narkotisierte Ratten gemacht und die Wassermenge bestimmt, die während der antidiuretischen Phase rückresorbiert wurde. Die Dosis-Antwort-Kurve hatte den Charakter einer Sättigungskurve mit einem praktisch linearen Anstieg bis 1–1,5 E. Der antidiuretische Effekt war bei einer Injektion von 4–6 E nicht deutlich größer als bei der kleineren Dosis, gleichzeitig fanden sich 75% der injizierten ADH-Menge im Urin der antidiuretischen Phase. Es wurde geschlossen, daß während einer Einzelinjektion von ADH 1–1,5 E fast augenblicklich von Receptoren der Tubuluswand gebunden und dann während des langssamen Prozesses der Wasserresorption inaktiviert werden, während das überschüssige ADH im Urin ausgeschieden wird. Schätzungsweise war 1 E ADH erforderlich, um 5 cm³ Wasser rückzuresorbieren. Die für den Resorptionsvorgang erforderliche Zeit war relativ kurz bei hoher Resorptionsrate und umgekehrt.In den vorliegenden Untersuchungen wurden die Einzelinjektionen mit 20 E wiederholt. Die höhere Dosis erlaubte, die antidiuretische Aktivität (ADA) in drei aufeinanderfolgenden Urinportionen der antidiuretischen Phase getrennt zu bestimmen. Das Resultat bestätigte die Arbeitshypothese.1. Die mit 20 E Pitressin erzielte ADA von 4 cm³ war nicht größer, sondern (nicht signifikant) kleiner als diejenige, die in den früheren Versuchen mit 5 E Pitressin oder 1 E Tonephin gefunden wurden.2. 95±15% der injizierten ADA erschienen im Urin. Das heißt, die Differenz zwischen den 20 injizierten E und den 18,5–19 E, die nach Abzug der vermutlich an der Tubuluswand gebundenen 1–1,5 E ausgeschieden wurden, war zu klein, um mit unserem Bioassay entdeckt zu werden.3. Unter der Annahme, daß 1 E Pitressin verbraucht wurde für die Resorption von ungefähr 4 cm³ Wasser, kann ein Vasopressin-Wasser-Äquivalent von ungefähr 1 Mol Vasopressin für 10⁸ Mol Wasser errechnet werden.4. Die durch die Niere ausgeschiedene Vasopressin-Menge folgt einer e-Funktion mit einer Halbwertszeit von 5 min.5. Die Vasopressin-Clearance beträgt ungefähr 1,0 cm³/min · Ratte und liegt somit wenig unter der Inulin-Clearance (1,2 cm³/min · Ratte). Diese Tatsache legt die Vermutung nahe, daß das überschüssige Vasopressin durch einfache Filtration ausgeschieden wird.6. Wenn man unter Berücksichtigung der unterschiedlichen Nierengewichte den ADH-Bedarf der 200mal schwereren Menschenniere schätzt, so kommt man auf 200–300 E. Setzt man ein Vasopressin-Wasser-Äquivalent von 4–5 cm³ pro 1–1,5 E ADH bei einer Wirkzeit von 50 min in Rechnung, so sollte die menschliche Niere ca. 261 Wasser pro Tag bei völligem Fehlen von ADH verlieren. Die Übereinstimmung mit dem wirklichen Wert könnte dafür sprechen, daß ein Teil der Resultate, die an der Alkohol-narkotisierten Ratte gewonnen wurden, nicht nur unter den strikten Bedingungen dieses Präparates gelten.

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This work was supported by Contract AF 61 (052)-947 of the USAF School of Aerospace Medicine, European Office of Aerospace Research (OAR), U.S. Air Force and the Deutsche Forschungsgemeinschaft.     

A pharmacologic study on the histamine releasing effect of atracurium and other muscle relaxants in rat isolated ileum

In this study, the effects of histamine, antihistamines (terfenadine and mepyramine), 5-hydroxytryptamine, and muscle relaxants, atracurium, vecuronium and gallamine, on the tone and contractility of rat ileum were studied and compared in vitro.The aim of the present investigation was to measure, pharmacologically, the histamine releasing effect of muscle relaxants, e.g. atracurium, vecuronium and gallamine, by comparing their contractile response in the absence and presence of antihistamines and comparing their mechanical responses with those produced by histamine and 5-hydroxytryptamine (5-HT).The results showed that the antihistamines, triludan (terfenadine) and mepyramine produced opposite effects in rat ileum. Terfenadine (0.1–20 M) produced concentration-dependent contractions in the rat ileum, whereas mepyramine (0.1–10 M) relaxed the muscle, e.g. by 1.2 g tension. Atracurium (0.5–500 M), vecuronium (0.2–200 M), and gallamine (0.1–7.0 M) produced marked contractions (1.5–4.0 g tension) in rat ileum, and these contractions were markedly reduced by mepyramine (1.3 M) or terfenadine (5 M), implicating histamine release in the generation of these contractions. However, there was some residual contraction which was not blocked by mepyramine, but by 5-HT antagonist, methysergide (1 M), indicating that a mechanism other than histamine release may be responsible for the residual contraction, i.e. release of other mediators such as 5-HT, prostaglandins, or calcium. 5-HT (0.5–500 M) and histamine (0.5–500 M) produced contractions in the rat ileum, but 5-HT was more effective than histamine in producing these contractions. Similarly, gall amine was more effective than atracurium and vecuronium in contracting the rat ileum. Since very high concentrations of muscle relaxants were used, it is suggested that in clinical concentrations, the histamine releasing effect of muscle relaxants was minimal, except that of gallamine, which may release histamine event at very low concentrations. The results are discussed in terms of pharmacologic and immunologic implications of drug reactions at the rat intestinal smooth muscle.     

Presence of an osmiophilic feltwork distinct from the Golgi apparatus in spinal ganglion cells of the rat

Summary Rat dorsal root ganglia were osmicated for 48 h at 42 ° C in a 2% unbuffered aqueous solution of osmium tetroxide. In addition to the osmiophiliccis-element of the Golgi apparatus, the osmium stained a cytoplasmic structure which, although present in the perikaryon of all ganglionic neurons, was well developed in type B₁ cells. In photographic stereopairs of 1–2 m thick sections examined with the electron microscope at 100 kV, these osmiophilic elements appeared as long, wavy thread-like elements with fusiform enlargements. Their average diameter was smaller than that of the thinner cisternae of the smooth endoplasmic reticulum. Occasionally branched, these osmiophilic structures were oriented in all directions of space. When examined in 1.5-7 m thick sections with a high voltage electron microscope, they formed an osmiophilic feltwork which extended from the juxtanuclear to the subplasmalemmal regions of the perikaryon. It was not connected with the osmiophiliccis-element of the Golgi apparatus. In sections of glutaraldehyde-fixed tissues impregnated with the double impregnation technique or postfixed with reduced osmium, the osmiophilic tubules were clearly distinguished from microtubules, cisternae of endoplasmic reticulum or endocytic structures.     

Serotoninergic synapses on ependymal and hypendymal cells of the rat subcommissural organ

Summary The nervous input to the subcommissural organ (SCO) of the rat has been investigated with Falck—Hillarp fluorescence histochemistry and electron microscopical techniques. Previous fluorescence histochemical observations of a dense plexus of serotoninergic nerve fibres in relation to the basal SCO were confirmed. Electron microscopically, unmyelinated fine varicose axons ranging in size from 0.1–0.6 m were observed to penetrate into the SCO hypendyma. Boutons and presynaptic varicosities filled with a diversity of round and elongated clear vesicles, and occasional large dense cored vesicles establish asymmetric (Gray's type I) synaptic contacts with the basal processes and somata of the SCO ependymal and hypendymal cells. A typical varicosity in synaptic contact with an SCO cell contains a population of approximately 85% clear, elongated vesicles 45 × 60 nm in diameter, 15% clear, round vesicles 50 nm in diameter, and 1–2% large dense cored vesicles with a vesicle diameter of about 85 nm and a dense core diameter of 50–55 nm. The mean length of the postsynaptic membrane specialization was found to be 0.5 m.Experiments with specific neurotoxic drugs revealed that the nerve terminals in synaptic contact with the SCO cells are identical to the fibres of the serotoninergic plexus identified fluorescence histochemically. Thus, an intraventricular injection of either 5,6-dihydroxytryptamine or 5,7-dihydroxytryptamine induced typical degenerative changes in most of the boutons in synaptic contact with the SCO cells, and also a disappearance of the yellow fluorescent nerve plexus. It is concluded that the SCO of the rat receives a dense plexus of serotonin-containing nerve fibres which form typical synaptic contacts with the specialized ependymal cells of the SCO and that these fibres may constitute the only direct nervous input to the organ.The degeneration of the serotoninergic synapses elicited a long-lasting, pronounced increase in the secretory activity of the SCO. Despite long survival times after the treatment with neurotoxic drugs, we found no evidence of regenerative restitution of the serotoninergic innervation nor normalization of the secretory activity of the SCO. The observed inverse relationship between secretory activity and serotoninergic innervation is in line with previous observations which indicate that the 5-hydroxytryptamine input to the SCO ependymal and hypendymal cells exerts a powerful inhibition on their protein synthetic machinery.Presented in part at the symposium Organization and Function of Central Catecholamine Neurons, December 10, 1975, Lund, Sweden.     

The ciliary ganglion of the cat: a light and electron microscopic study

The ciliary ganglia of eight healthy adult cats were studied by light and electron microscopy. The ganglion, measuring about 2 mm in length, was consistently found to be attached to the branch from the oculomotor nerve supplying the inferior oblique muscle. The number of neurons varied from 2773 to 3794 after applying Abercrombie's correction. The mean of average somal diameter of the neurons was 36.5 m (SD = 5.0 m) and the mean of somal cross-sectional area was 904.2 m² (SD = 262.8 m²). The mean of average nuclear diameter was 13.9 urn (SD = 1.8 m) and the mean of nuclear cross-sectional area was 142.2 m² (SD = 37.1 m²). The mean of the aspect ratios of the soma and nucleus were 1.2 (SD = 0.1) and 1.1 (SD = 0.1) respectively. The frequency distributions of these parameters were all unimodal. Under the light microscope, the Nissl granules in the neurons were prominent and were distributed peripherally, perinuclearly or randomly in the cytoplasm. Under the electron microscope, the rough endoplasmic reticulum showed a similar pattern of distribution in the cytoplasm. In some neurons, glycogen-like granules were present; these were either distributed randomly throughout the cell, or aligned in single rows in relation to sub-surface cisterns and between the cisterns of smooth and rough endoplasmic reticulum. Most of the dendrites were short protrusions from the cell body; some contained glycogen-like granules. Occasionally, the dendritic protrusions were electron-dense. All the synapses encountered were axodendritic. In most axon terminals, the synaptic vesicles were spherical and measured 30–50 nm in diameter; in some, they were flattened, measuring 50 nm by 20 nm. Some axon terminals containing either spherical or flattened synaptic vesicles also contained large dense-cored vesicles that measured 80–100 nm, while their dense core measured 40–60 nm.     

Architecture and synaptic relationships in the intermediolateral nucleus of the thoracic spinal cord of the rat: HRP labelling, catecholamine histochemistry and electron microscopic studies

Summary The organization of the intermediolateral nucleus (IML) of the thoracic spinal cord was examined using glyoxylic acid-induced fluorescence histochemistry, retrograde horseradish peroxidase (HRP) labelling and electron microscopy. In serial sections of T2, it was found that the distribution of catecholamine nerve terminals was intimately related to the neuronal perikarya of IML. Potassium permanganate fixation and 5-hydroxydopamine treatment revealed small dense-cored vesicles in axon varicosities with or without synaptic specializations. A gelatinous region, composed of small diameter dendrites and unmyelinated axons, formed a narrow longitudinal bundle in the centre of the nucleus. The population of the axon varicosities in the IML was 0.17 ± 0.02/m² in 75 nm sections. The average size of the axon varicosities with flat synaptic vesicles was 1.44 ± 0.05 m² and that of varicosities with spherical vesicles was 0.97 ± 0.02 m². After HRP injection into the superior cervical ganglion, ipsilateral IML neurons were labelled in T1–T3 segments of the spinal cord. Axon varicosities with flat and others with spherical synaptic vesicles synapsed on the dendrites labelled by HRP. Among axon varicosities synapsing on the preganglionic sympathetic neurons, 74.8 ± 7.1% at axo-somatic synapses and 46.0 ± 6.7% at synapses on proximal dendrites contained flat synaptic vesicles.     

The paratrigeminal nucleus. I. Neurons and synaptic organization

Summary The paratrigeminal nucleus, a diffuse collection of neurons on the lateral medullary surface, lies embedded in the fibres of the restiform body, ascending spinocerebellar tract and the spinal tract of the trigeminal nerve. Its rostrocaudal extent is approximately 1500–2000m in the rat, 2800–3500 m in the rhesus monkey, and 5000–6000/m in the human, where it is a well-defined nucleus. In Golgi preparations the neuronal somata are generally fusiform, ranging from 8–15 m in width and 15–25/m in length. Electron microscope studies show that their polar dendritic processes are thick and long and they intertwine in bundles among islands of cells or myelinated fibres. The perikarya have a high nucleus to cytoplasm ratio and the scanty cytoplasm is conspicuous for its paucity of organelles and its plethora of polysomal arrays. The neuropil is complex and contains a heterogeneous population of axonal varicosities displaying markedly different axoplasmic structures. Many different types of large granular vesicle-containing axons prevail. These axons engage in axo—somatic, axo—dendritic, axo—spinous and axo—axonic synapses with the processes of the cells within the nucleus.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Involvement of GABAB Receptors in Presynaptic Inhibition of Fibers of the Descending Projections of the Spinal Cord in the Frog Rana Ridibunda

Isolated spinal cord preparations from Rana Ridibunda frogs were used for studies of the effects of the GABA_B receptor agonists (–)-baclofen (50 and 100 M) and GABA (4–8 mM) and the specific GABA_B receptor antagonist 2-hydroxysaclofen (100 M) on the transmission of signals from fibers of the ventral columns monosynaptically connected with motoneurons in segments 9 and 10. These experiments showed that (–)-baclofen (50 and 100 M) produced significant and dose-dependent suppression of excitatory postsynaptic potentials (EPSP) in motoneurons and ventral root potentials evoked by stimulation of fibers of the ipsi- and contralateral ventral columns. The inhibitory effect of (–)-baclofen (100 M) on descending EPSP was 35–50% blocked by the GABA_B receptor antagonist 2-hydroxysaclofen (0.2 mM). The inhibitory effect of GABA (4–8 mM) on descending EPSP was 60% blocked by the GABA_A receptor antagonist picrotoxin (0.05 mM). (–)-Baclofen (50 M) and GABA (4 and 6 mM) were found to have inhibitory effects on ventral root potentials evoked by stimulation of the ipsi- and contralateral ventral columns. The data obtained here, as well as data obtained by pharmacological analysis and conditioning by stimulation of the ipsi- and contralateral ventral columns, are regarded as a significant argument supporting the existence of GABA_B receptor-mediated presynaptic inhibition of descending fibers connected monosynaptically to spinal cord motoneurons in the frog Rana Ridibunda.