Norepinephrine uptake in aging adrenergic nerve terminals

Norepinephrine (NE) uptake has been studied in the adult (3-6 months) and aging (5 yr) chick iris, in which there is a discrete population of norepinephrine containing nerve terminals. although total accumulation of 3H-NE in the iris does not change with age, there is a decline in Na+-dependence, temperature-sensitivity, ouabain-and inhibitor -sensitivity of uptake. The results indicate either a loss of active, carrier-mediated NE uptake during aging, or a change in the biochemical and pharmacological characteristics of this uptake. changes in the composition of the organ and in the sites of accumulation of NE are considered.     

Axonal uptake of horseradish peroxidase isoenzymes during Wallerian degeneration

Horseradish peroxidase isoenzymes were applied around crushed mouse hypoglossal nerves to study the influence of electrical charge on the uptake and ultrastructural distribution of macromolecules in axons distal to an injury. Both isoenzymes tested (Sigma type IX, cationic and type VII, anionic) were readily taken up into axons and moved in a distal direction along the nerve. Samples taken 1-3 mm below the crush showed that reaction products from both enzymes covered the inner surface of the axonal plasma membrane and were attached to organelles, particularly microtubules and neurofilaments. However, reaction product in the Schwann cell basement lamina and in the endoneurial collagen was much more dense with cationic peroxidase than with the anionic isoenzyme. Our study shows that both cationic and anionic macromolecules can move into axons distal to a nerve lesion. It can be assumed that also other agents can be taken up into axons and that 'wound substances' in this way may influence the processes by which axons are destroyed during Wallerian degeneration.     

The action of inhibitory drugs on nerve terminals in crayfish muscle

Summary The presynaptic inhibitory effects of -amino-butyric acid (GABA) and related drugs on the excitatory and inhibitory terminal potentials were studied using extracellular microelectrodes.Triphasic conducted action potentials recorded from the excitatory nerve fiber in the vicinity of the terminal were altered to monophasic positive potential changes after application of inhibitory drugs. These drugs thus can block conduction near to the terminal.Diphasic potential changes recorded near to the excitatory terminal (e.n.t.p.s) were made monophasic positive after application of inhibitory drugs. Neural presynaptic inhibition had the same effect. Also during drug induced inhibition therefore the point of block of conduction in the excitatory terminal shifted centrally.The potentials recorded from the inhibitory nerve terminal were not or very little affected by applied GABA.GABA in concentrations that blocked excitatory transmission did not change the frequency of spontaneous excitatory potentials.It is concluded that presynaptic inhibition by GABA and related drugs mimicks in all respects known neural presynaptic inhibition. The probable mechanism of this inhibition is a conductance increase without much potential change in the excitatory nerve terminal.

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Zusammenfassung Die hemmenden Effekte von GABA und verwandten Drogen auf die Potentiale der erregenden und hemmenden Nervenendigungen wurden mit Hilfe von extracellulären Mikroelektroden-Ableitungen untersucht.Von erregenden Nervenfasern in der Nähe der Endigung wurden triphasische Aktionspotentiale abgeleitet. Nach der Gabe von hemmenden Drogen wurden die Potentiale monophasisch positiv; diese Substanzen könnten also einen Block der Fortleitung des Aktionspotentials herbeiführen.Nahe der erregenden Endigungen abgeleitete diphasische Potentiale (e.n.t.p.) wurden nach Applikation von hemmenden Drogen kleiner und monophasisch positiv. Präsynaptische Hemmung durch Reiz des hemmenden Nerven hatte dieselbe Wirkung. Es verschiebt sich also auch während der Hemmung durch Drogen die Stelle, an der die Fortleitung des Aktionspotentials aufhört, zentralwärts.Von der hemmenden Nervenendigung abgeleitete Potentialänderungen wurden durch GABA nicht oder sehr wenig beeinflußt.GABA in Konzentrationen, bei denen die erregende synaptische Übertragung unterbrochen war, veränderte nicht die Frequenz der spontanen erregenden Potentiale.Die Befunde zeigen, daß die präsynaptische Hemmung durch GABA und verwandte Substanzen von der präsynaptischen Hemmung über den hemmenden Nerven nicht unterschieden werden kann. Wahrscheinlich ist der Mechanismus dieser Hemmung eine Erhöhung der Leitfähigkeit der Zellmembran der erregenden Nervenendigung, ohne daß dabei das Membranpotential sich wesentlich verschiebt.

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

Selective uptake and anterograde transport of horseradish peroxidase by hippocampal granule cells

Introduction of horseradish peroxidase into the ventriculocisternal system results in selective labeling of the granule cells of the dentate gyrus and their axons, the mossy fibers. This labeling pattern is not seen after direct injections of horseradish peroxidase into the dorsal hippocampus. The density of the granule cell labeling appears to be related to their proximity to the site of highest horseradish peroxidase concentration. The combined distribution of horseradish peroxidase in the granule cells and mossy fibers strongly suggests that the latter element is labeled as the result of anterograde transport of horseradish peroxidase taken up by the granule cell perikarya or dendrites. This labeling was found in the absence of injury to the hippocampus, suggesting that neuronal damage is not necessary for anterograde transport horseradish peroxidase to occur.     

Presynaptic effect of gamma-aminobutyric acid on the inhibitory nerve and nerve terminals in the crayfish neuromuscular junction

Experiments were carried out in voltage-clamped fibres of the opener muscle of the first walking leg or claw of small crayfish. Repetitive discharges in the inhibitory nerve innervating the muscle were induced by adding serotonin (10(-6) mol/l) and forskolin (10(-4) mol/l) to the superfusate. Rates of nerve discharge were determined by recording nerve evoked inhibitory postsynaptic currents (IPSCs) in the voltage-clamped muscle fibre. Subsequently, the effect of gamma-aminobutyric acid (GABA) on the rate of IPSCs in normal and Cl- -deficient superfusate was investigated. In normal superfusate GABA (10(-5) mol/l) abolished the IPSCs whereas in Cl- -deficient superfusate GABA (10(-4) mol/l) enhanced the rate of IPSCs. Moreover, in Cl- -deficient superfusate the rate of asynchronous quantal release of inhibitory transmitter could be enhanced by GABA. The results indicate that in the crayfish neuromuscular junction the inhibitory axon is supplied with GABA receptors which may affect (a) axonal excitation and (b) quantal output at the inhibitory axon terminals.     

Synaptic transmission and intracellular sodium: ionophore induced sodium loading of nerve terminals

Application of the sodium ionophore, Monensin, to crustacean neuromuscular preparations induced a potentiation of the synaptic potential which subsided after removal of Monensin. The rate and extent of potentiation were influenced by the concentration of extracellular sodium, and potentiation occurred when Monensin was applied in calcium-free solutions. Some features of long-lasting facilitation induced by repetitive stimulation of the motor axon were duplicated by Monensin application. The results support the hypothesis that long-lasting facilitation of transmitter release can result from increased intracellular sodium.     

The uptake of horseradish peroxidase and its subsequent redistribution by guinea pig gallbladder in vivo

Horseradish peroxidase (HRP) was injected into the emptied gallbladder of guinea pigs at laparotomy. The fate of the HRP was observed by light and electron microscopy over the following 2 hr. Within 5 min. HRP appeared in pits and apical vesicles either as a rim of increased electron density or more evenly distributed throughout the pit or vesicle. The pits and vesicles were more frequently seen at the edge of the cell apex. From 15 min. HRP was identified in the basolateral cell space in increasing quantities with spill over through the basement membrane into the lamina propria: by 30 min. and 2 hr, little further change was observed. The quantity of nonspecific electron density in epithelial cell multivesicular bodies and residual bodies made the assessment of intracellular handling of the HRP impossible. Dilution of the HRP in the lamina propria made the identification of the eventual fate of the HRP difficult to determine.     

Atypical olfactory glomeruli contain original olfactory axon terminals: an ultrastructural horseradish peroxidase study in the rat

The labelling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity has been studied in the rat. In such conditions, atypical glomeruli, previously described according to their high acetylcholinesterase content, display a strong tracer accumulation. The course of afferent olfactory fibres could be followed along the lateral and dorsal surface of the olfactory bulbs. The primary olfactory axons ending in atypical glomeruli have been identified with horseradish peroxidase in electron microscopy. They differ significantly from classical olfactory terminals owing to the presence of large dense-cored vesicles accompanying small clear ones. Moreover, the olfactory terminals do not gather in dark nodules as they do classically in olfactory glomeruli. The study demonstrates that a subset of olfactory neuroreceptors displaying original ultrastructural characteristics projects selectively into atypical olfactory glomeruli. Ultrastructural features indicate that olfactory information processing taking place in the neuropil might be similar to that which occurs in typical glomeruli. Considered together, the atypical olfactory neuroreceptors, glomeruli and acetylcholinesterase-containing centrifugal fibres could constitute a new olfactory subsystem. This hypothesis is discussed by taking into account previous demonstration of other olfactory subsystems devoted to the processing of olfactory cues of fundamental biological importance.     

The effect of polarizing current on action potential and transmitter release in crayfish motor nerve terminals

Summary Potential changes were recorded from the terminal regions of crayfish motor nerve fibres by means of extracellular microelectrodes. Near to the recording site, a nerve branch was cut and sucked into a close fitting glass capillary electrode. Nerve action potentials were recorded through this electrode; in addition, current could be passed which de- or hyperpolarized the nerve and the terminal.Hyperpolarization of the terminal led to an increased average amplitude of the EPSP (up to 400% of control), which was due to an increased probability of release of quanta of transmitter. The average amplitude of the EPSP rose steeply with increasing current strength up to –6 A, larger hyperpolarizing current had little more effect. Part of this facilitatory effect of hyperpolarization was instantaneous, the full effect, however, developed only within 10 min after hyperpolarization. Similarly, the EPSP returned to the control amplitude only 10 min after the hyperpolarizing current was switched off. During hyperpolarization, the action potentials recorded from the nerve and from the terminal were prolonged by about 100% and increased in amplitude. The frequency of spontaneous synaptic potentials was reduced (down to 12% of control) during hyperpolarization.Depolarization of the terminal had little effect on the EPSP and on terminal potential changes, probably because small current strengths led to block of conduction proximal to the terminal. The frequency of spontaneous synaptic potentials was increased during depolarization of the terminal.The effects of polarizing current on the crayfish motor nerve terminal are very similar to the respective effects on cholinergic vertebrate terminals indicating a very similar mechanism of release of transmitter. Part of the facilitatory effect of hyperpolarization should be due to the prolongation of the action potential, in addition a mobilization of transmitter must be assumed. Possible relations between synaptic facilitation due to repetitive stimulation and hyperpolarization of the terminal are discussed.     

Effect of lithium on veratridine-induced quantal and non-quantal release from inhibitory nerve terminals in crayfish muscle

Muscle fibres of small crayfish were voltage clamped and superfused for about 10 min with Li⁺ saline (Na⁺ replaced by Li⁺) which contained 5 mmol/l glutamate to desensitize excitatory postsynaptic receptors. Then 100 mol/l veratridine were added to the superfusate which caused strong asynchronous quantal release of inhibitory transmitter. However, in the presence of Li⁺ strong inhibitory quantal release was only transient. It could be activated a second time by removal of Li⁺ and readministration of Na⁺. From the total of 0.7 to 1.1 million quanta released by veratridine only about 30–35% could be released in Li⁺ saline. The voltage clamp DC-currents recorded during veratridine-induced quantal release suggested that a nonquantal release component is additionally involved. This non-quantal release component was most prominent during the period of quantal release in Li⁺ superfusate while it was less obvious during the second enhancement of quantal release in normal saline. Together with previous results (Martin and Finger 1988) it may be concluded that quantal release, but not non-quantal release, is decreased by Li⁺ in the nerve terminals.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Synaptic integration in excitatory and inhibitory crayfish motoneurons

The passive integrative properties of two crayfish abdominal motoneurons, the fast flexor inhibitor (FI) and a posterior, ipsilateral fast flexor excitor (FE), were studied electrophysiologically and through simulations with multicompartment models of their electrotonic structures. Responses of the models to simulated giant neuron input were quite similar to the motoneurons' responses to giant neuron stimulation, which suggests that differences in the electrotonic structures and the sites of synaptic input to the two cells can account in large part for differences in their responses to a common input. A full action potential created in the initial axon compartment of the FI model produced attenuated potentials in the adjacent integrating segment compartment and contralateral soma compartment. These potentials are similar in amplitude and time course to attenuated antidromic action potentials recorded in the corresponding regions of the FI neuron. A location of the spike initiation zone of the FI at the initial axon segment is consistent with this result. The responses of FI to ipsi- and contralateral inputs are different. Shock of a single abdominal second root produced a larger, faster rising excitatory postsynaptic potential in the ipsilateral FI soma than in the contralateral soma. Second root shock also caused the contralateral FI to produce an action potential either alone or before the ipsilateral FI neuron. Responses of the FI model to ipsilateral and contralateral inputs differ in the same way as the cell's responses. Inputs to the FI model that are ipsilateral to the soma compartment produce larger responses there than do contralateral inputs. Conversely, those contralateral inputs produce larger responses in the initial axon compartment than do ipsilateral inputs. This difference results from the long integrating segment that connects the soma compartment to the initial axon compartment. These results can account for the FI responses to lateralized inputs. Unlike the responses of FIs, the soma responses of contralaterally homologous FEs to ipsilateral and contralateral second root shocks were similar in waveform and amplitude, with the ipsilateral root producing the larger response. This result is consistent with theoretical results from the FE model simulations. We conclude that a smaller size, larger input resistance and shorter membrane time constant allow the FE to respond to giant neuron input before the FI, and so help to achieve the proper timing of flexor contraction and relaxation during a tailflip.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological effects of VIth nerve section in the kitten as revealed by horseradish peroxidase

The effects of the chronic section of the VIth nerve have been studied in kittens with the help of the horseradish peroxidase intra-axonal retrograde tracing method. Two months following the section of the VIth nerve, only 50% of the abducens motoneuron population was left. However, shapes, somata sizes and cell distribution through the nucleus of the remaining neurons were similar to those shown by controls. Functional implications of the findings are discussed.     

Tonic depolarization of excitatory nerve terminals in crayfish muscle by high concentrations of extracellular potassium

At voltage-clamped fibres of the claw opener muscle of small crayfish, spontaneous quantal release of excitatory transmitter elicited by raising extracellular K+ to 100 mM was investigated. On application of the high K+ concentration, the rates of quantal release increased to n = 10,000-25,000 quanta/s within 10 s, and thereafter declined exponentially, either with a single (tau congruent to 15-40 s) or with two (tau 1 congruent to 15-40 s, tau 2 greater than 70 s) time constants. The total number of quanta released per trial ranged from s = 200,000 to 800,000 quanta. The results were derived by means of the fluctuation analysis technique.     

Excitability changes in crayfish motor neurone terminals

1. Changes in the post-activation excitability of crayfish motor nerve terminals were used to measure afterpotentials that might be related to facilitation of transmitter release.2. The refractory period is followed by a period of supernormal excitability in which the threshold of nerve terminals drops to about 70% of its pre-activation level at about 15 msec following an impulse. The threshold returns exponentially to its pre-activation level with a time constant of about 25 msec at 13 degrees C. Such a supernormal excitability is rarely seen in pre-terminal nerve branches or in the main axon.3. Following a brief high-frequency tetanus the peak of the supernormal excitability is greater than that following a single impulse. At low temperature this peak is reduced and delayed, and the decay rate of the supernormal excitability is prolonged with a Q(10) of about 2.5.4. Depolarization of nerve terminals decreases, and hyperpolarization increases, the magnitude of the post-activation supernormal excitability.5. The magnitude of the supernormal excitability depends on the external potassium concentration, but not on sodium. In low calcium the peak supernormal excitability is often reduced. High calcium concentration and manganese ions have no effect, but cobalt abolishes the supernormal excitability, and its effects are only slightly reversible. Both cobalt and manganese reversibly block neuromuscular transmission.6. Strophanthidin has no effect on the post-activation supernormal excitability, but proteolytic enzymes reduce or abolish it, and hyperosmotic solutions also affect it.7. It is suggested that the action potential is followed by a depolarizing afterpotential in nerve terminals which is caused by a transient increase in the potassium concentration around the terminals. There is no evidence that afterpotentials in nerve terminals are related to facilitation in any way.     

Localization of the spinal nucleus of accessory nerve in rat: a horseradish peroxidase study

The spinal nucleus of the accessory nerve (SNA) comprises the group of somata (perikarya) of motor neurons that supply the sternocleidomastoid and trapezius muscles. There are many conflicting views regarding the longitudinal extent and topography of the SNA, even in the same species, and these disagreements prompted the present investigation. Thirty Sprague-Dawley rats (15 males, 15 females) were used. The SNA was localized by retrograde axonal transport of horseradish peroxidase. Longitudinally, the SNA was found to be located in the caudal part (caudal 0.9-1.2 mm) of the medulla oblongata, the whole lengths of cervical spinal cord segments C1, C2, C3, C4, C5 and rostral fourth of C6. In the caudal part of the medulla oblongata, the SNA was represented by a group of perikarya of motor neurons lying immediately ventrolateral to the pyramidal fibres that were passing dorsolaterally after their decussation. In the spinal cord, the motor neuronal somata of the SNA were located in the dorsomedial and central columns at C1, in the dorsomedial, central and ventrolateral columns at C2 and in the ventrolateral column only at C3, C4, C5 and rostral quarter of C6. The perikarya of motor neurons supplying the sternocleidomastoid were located in the caudal part (caudal 0.9-1.2 mm) of the medulla oblongata ventrolateral to the pyramidal fibres that were passing dorsolaterally after their decussation. They were also located in the dorsomedial and central columns at C1, in the dorsomedial, central and ventrolateral columns at C2 and only in the ventrolateral column at the rostral three-quarters of C3. The perikarya of motor neurons supplying the trapezius muscle were located in the ventrolateral column only in the caudal three-quarters of C2, the whole lengths of C3, C4 and C5, and in the rostral quarter of C6.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.     

Cervical cord neurons labeled by horseradish peroxidase application to the sciatic nerve in the rat

After horseradish peroxidase (HRP) application to the proximal cut end of the sciatic nerve in rats aged 3-10 days, HRP-labeled neuronal cell bodies were found ipsilaterally in the ventrolateral region of the ventral horn in the cervical enlargements of the spinal cord. Such labeled neurons were occasionally seen in rats aged 15 days, but not seen at all in rats aged 60-90 days. The labeling was presumably the result of a retrograde transneuronal axonal transport of HRP applied to the sciatic nerve.