Biomedical subjects

E A Connor

Publications and source records attributed to E A Connor.

16 recordsLinked to original sources

PubMed2000

F-actin is concentrated in nonrelease domains at frog neuromuscular junctions.

To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with beta-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal.

Actins↗

PubMed1998

Stability of frog motor nerve terminals in the absence of target muscle fibers.

Using repeated in vivo imaging, we addressed the role of target muscle fibers in the maintenance of frog motor nerve terminals at synaptic sites. Target-deprived nerve terminals were generated by selective and permanent removal of muscle fibers without damage to the innervation. Individual nerve terminals, stained with the dye FM1-43, were imaged before and again during the subsequent 1-9 months of target deprivation and the stability of the nerve terminal arbors over time was determined. Repeated observation of motor nerve terminals showed that nerve terminals were well maintained at synaptic sites during the first 1-2 months after target loss; the original number of nerve terminal segments was retained at 85% of the synaptic sites after muscle damage. After long periods of target deprivation, 6-9 months, loss or retraction of nerve terminal segments resulted in a reduction in the arbor of most but not all nerve terminals. This apparent nerve terminal destabilization was not a result of illumination or irradiation because a similar decrease in the extent of nerve terminal arbors was not observed at control irradiated neuromuscular junctions. The persistence of many complete target-deprived nerve terminal arbors at synaptic sites long after target degeneration suggested that the cues that confer stability to frog motor nerve terminals likely reside external to muscle fibers and may be associated with the synaptic basal lamina or the terminal Schwann cell. Since the arbors of many target-deprived nerve terminals were eventually reduced, the nonmuscle stabilization cues may not persist indefinitely at target-deprived synaptic sites.

Animals↗

PubMed1997

Developmental regulation of interstitial cell density in bullfrog skeletal muscle.

Denervation of skeletal muscle results in striking connective tissue remodelling in junctional areas of muscle. Since extracellular matrix molecules mediate axonal growth and synaptic differentiation, it is likely that the interstitial cells and matrix molecules that accumulate near synaptic sites after denervation influence the regrowth and regeneration of synaptic connections. The experiments presented here addressed the question of whether the junctional connective tissue in developing bullfrog skeletal muscle was also specialized in its cellular and molecular composition. Denervation responses of muscle, such as extrajunctional sensitivity to acetylcholine, often reproduce the characteristics of developing muscle during synaptogenesis. In developing muscle, the distribution of interstitial cells was nonuniform during the period of muscle fibre birth and synaptogenesis. Interstitial cells were concentrated near synaptic sites as in denervated adult muscle. Unlike denervated adult muscle, there were no junctional accumulations of fibronectin or tenascin, matrix molecules produced by interstitial cells, in developing muscles. These results demonstrate that the junctional connective tissue in developing muscle is identified by a high density of interstitial cells that may play a role in the identification and formation of synaptic sites. Further, the junctional matrix environment of developing muscle is distinct from the matrix remodelling that occurs in response to denervation, suggesting that the matrix production by interstitial cells during development is regulated differently from that after denervation of the neuromuscular junction.

Aging↗

PubMed1997

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Animals↗

PubMed1995

Long-term maintenance of presynaptic function in the absence of target muscle fibers.

Here we have investigated the role of the muscle fiber in the maintenance of presynaptic function at frog motor nerve terminals. Muscle fibers were selectively damaged and prevented from regenerating while leaving the motor innervation intact. Presynaptic activity of the resulting target-deprived nerve terminals was assayed using the fluorescent dye, FM1-43. FM1-43 stains nerve terminals in an activity-dependent fashion in that it incorporates into synaptic vesicles and can be released upon stimulation. We demonstrate that nerve terminals permanently deprived of target for 1 to 5 months maintain the ability to release and recycle synaptic vesicles in response to stimulation. The level of presynaptic function of target-deprived nerve terminals was not detectably different from that of nerve terminals in contact with muscle fibers. The remarkable maintenance of presynaptic function in these target-deprived nerve terminals indicates that the molecular machinery required for vesicular release is maintained in a functional state for long periods without target muscle fibers.

Animals↗

PubMed1994

Retrograde signaling in the formation and maintenance of the neuromuscular junction.

The neuromuscular junction is characterized by precise alignment between the nerve terminal and the postsynaptic apparatus formed by the muscle fiber. Organization of the neuromuscular junction during embryonic development, growth, and maintenance is coordinated by signals exchanged between motor neurons and their target muscle fibers. Identification of proteins such as agrin, likely to represent neuronal agents that direct the organization of the postsynaptic apparatus, has focused attention on characterization of proteins that mediate retrograde signals that regulate the organization and function of the nerve terminal. The results of these studies implicate a role for both adhesive and diffusible signals in coordinating the development, maturation, and maintenance of the motor nerve terminal. The diversity of molecules identified to date that appear to play a role in these processes implies a considerable level of redundancy in the transduction pathway. However, studies of early nerve-muscle interactions suggest that a common feature of many of these retrograde agents is activation of a protein kinase coupled with an increase in cytosolic Ca2+ concentration. While the molecular signals that regulate growth and maintenance of neuromuscular junctions are less well understood it seems likely that similar adhesive and diffusible factors will be involved.

Animals↗

PubMed1994

Synaptic activity and connective tissue remodeling in denervated frog muscle.

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.

Acetylcholine↗

PubMed1991

Molecular alterations in the perijunctional region of frog skeletal muscle fibres following denervation.

The anatomical distribution of a frog skeletal muscle antigen was studied using immunofluorescence microscopy and a monoclonal antibody 3B6 that was produced against denervated skeletal muscle. In innervated muscles, the monoclonal antibody 3B6 stain was associated with the inner surface of the muscle plasma membrane at the endplate and myotendinous junction. After denervation, the monoclonal antibody 3B6 stain extended from the endplate laterally around the perimeter of muscle fibres and longitudinally well beyond the endplate for a total length of 600-1000 microns. The monoclonal antibody 3B6 stain thus forms a cylindrical structure centred on the endplate. This observation shows that denervation produces a non-homogeneous molecular change in skeletal muscle fibres: an antigen that is present in high concentrations at innervated endplates appears in restricted perijunctional regions of denervated muscle fibres. It further suggests that perijunctional regions of denervated muscle fibres differ from the remaining non-endplate regions in molecular composition and possibly also in function.

Animals↗

PubMed1987

Cell accumulation in the junctional region of denervated muscle.

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.

Animals↗

PubMed1985

Influence of the extracellular ionic environment on ganglionic fast excitatory postsynaptic currents.

The influence of changing the extracellular sodium or calcium concentration as well as the addition of strontium or magnesium on characteristics of nicotinic fast excitatory postsynaptic currents (EPSCs) has been studied in voltage-clamped sympathetic ganglion B cells of the bullfrog. Lithium substitution for sodium influenced both the EPSC size and decay time course in a concentration-dependent manner. In those cells exposed to either a 50% or 100% lithium-substituted solution, the EPSC decay was faster than that of control EPSCs. With a 50% replacement of lithium for sodium, the EPSC size at -520 mV was similar to control values. However, with a 100% substitution, the EPSC size was significantly reduced below control values although the voltage dependence of the decay tau, the shape of the peak EPSC-voltage relationship, or the EPSC reversal potential was not changed by replacing lithium for sodium. The change in EPSC size and decay tau in the lithium solution was due to the presence of lithium and not simply the consequence of a reduction in the external sodium concentration; as with a 50% substitution of sucrose or mannitol for sodium chloride the EPSC decay was slowed. EPSC size at -50 mV and the voltage dependence of tau was similar to control values when 50% of the sodium was replaced by sucrose. The peak EPSC-voltage relationship was linear in cells exposed to either the control or the 50% sucrose-substituted solution, although the EPSC reversal potential was shifted to a more negative voltage with 50% sucrose substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Alteration of the fast excitatory postsynaptic current by barium in voltage-clamped amphibian sympathetic ganglion cells.

Barium-induced alterations in fast excitatory postsynaptic currents (e.p.s.cs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. In the presence of 2-8 mM barium, e.p.s.c. decay was prolonged and in many cells the e.p.s.c. decay phase deviated from a single exponential function. The decay phase in these cases was more accurately described as the sum of two exponential functions. The frequency of occurrence of a complex decay increased both with increasing barium concentration and with hyperpolarization. Miniature e.p.s.c. decay also was prolonged in barium-treated cells. E.p.s.c. amplitude was not markedly affected by barium (2-8 mM) in cells voltage-clamped to -50 mV whereas at -90 mV there was a progressive increase in peak size with increasing barium concentration. In control cells the e.p.s.c.-voltage relationship was linear between -20 and -100 mV; however, this relationship became progressively non-linear with membrane hyperpolarization in barium-treated cells. The e.p.s.c. reversal potential was shifted to a more negative value in the presence of barium. There was a voltage-dependent increase in charge movement during the e.p.s.c. in barium-treated cells which was not present in control cells. We conclude that the voltage-dependent alteration in e.p.s.c. decay time course, peak amplitude and charge movement in barium-treated cells is due to a direct postsynaptic action of barium on the kinetics of receptor-channel gating in postganglionic sympathetic neurones.

Action Potentials↗

PubMed1984

Comparison of cholinergic activation and desensitization at snake twitch and slow muscle fibre end-plates.

Characteristics of receptor-channel activation and desensitization have been compared at voltage-clamped snake slow and twitch fibre end-plates maintained in an isotonic potassium propionate solution. Miniature end-plate current (m.e.p.c.) decay was slower and less voltage dependent at slow fibre end-plates than at twitch fibre end-plates. The peak m.e.p.c. amplitude versus voltage relationship and reversal potential were similar at the two end-plate types. Acetylcholine-induced noise and m.e.p.c.s were recorded at slow fibre end-plates. At most slow fibres the spectral density was not adequately fitted by a single Lorentzian function. Rather, the observed spectral density was greater at high frequencies than the values predicted using the m.e.p.c. decay rate. The noise could be well described by the sum of two Lorentzian functions, one of which corresponded to a single Lorentzian function with the corner frequency determined by the m.e.p.c. decay rate. The shape of the carbachol concentration-peak end-plate current relationship was similar at both slow and twitch fibre end-plates. However, for all concentrations tested, the peak carbachol-induced end-plate current (e.p.c.carb.) value was markedly less at slow fibre end-plates than at twitch fibre end-plates. The onset of desensitization was determined using two methods. The first concerned analysis of the time course of decay of the e.p.c.carb. from a peak value during the sustained application of agonist. The second involved a double-perfusion technique in which a 'desensitizing' dose was applied for varying intervals before the application of a second 'test' dose of carbachol. With both methods the development of desensitization at both end-plate types was dependent on carbachol concentration and duration of exposure. At each end-plate type the time course of desensitization onset often exhibited two components; one with a time constant of seconds and a slower component having time constants in the range of tens to hundreds of seconds. The slope of the relationship between carbachol concentration and equilibrium desensitization at slow and twitch fibre end-plates was close to two, suggesting that two molecules of agonist are probably bound during the development of desensitization. However, for all concentrations tested, desensitization developed more rapidly and to a greater extent at twitch fibre end-plates than at slow fibre end-plates. The voltage dependence of the 3 min steady-state desensitization produced by 108 microM-carbachol was very similar (approximately -0.0250 mV-1) at both fibre types.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1983

Procaine alters fast excitatory postsynaptic current decay in amphibian sympathetic ganglia.

1--The effects of procaine (25-200 microM) on the fast excitatory postsynaptic currents (e.p.s.cs) of bullfrog sympathetic ganglion B cells were studied with a two-microelectrode voltage clamp system. 2--Procaine decreased peak e.p.s.c. size with no measurable decrease in quantal content. 3--The peak e.p.s.c.-voltage relationship was linear in control cells, but showed a marked nonlinearity in cells treated with procaine so that peak e.p.s.c. size decreased progressively at hyperpolarized values of membrane potential. The e.p.s.c. reversal potential was not altered in procaine. 4--Although the e.p.s.c. decay time course was well described by a single exponential in control cells, the decay phase often became complex in the presence of procaine. The decay phase consisted of two components in the presence of procaine which became more obvious with increasing concentration and membrane hyperpolarization. 5--In control cells, the e.p.s.c. time constant of decay increased with membrane hyperpolarization. In the presence of procaine, the first time constant of decay, tauf, increased with hyperpolarization up to -40mV, but then decreased with hyperpolarization between -40 and -100mV. 6--We conclude that procaine has two sites of action at postganglionic sympathetic neurones: (1) it reduces the number of activatable receptor-channel complexes and (2) procaine blocks open synaptic channels. Blockade of open channels became more important with hyperpolarization.

Animals↗

PubMed1983

Kinetic analysis of atropine-induced alterations in bullfrog ganglionic fast synaptic currents.

The concentration- and voltage-dependent effects of atropine on the fast excitatory post-synaptic current (e.p.s.c.) of bullfrog sympathetic ganglion cells have been analysed and fitted to a kinetic scheme of open-channel blockade. Atropine (1-75 microM) reduced the peak e.p.s.c. amplitude without altering the quantal content or the reversal potential. The e.p.s.c. decay was complex in the presence of atropine, being well fitted by two exponential components. With increasing concentrations of atropine the time constant of the fast component, tau 2, decreased and the time constant of the slow component, tau 1, increased. Over the voltage range -30 to -100 mV tau 2 exhibited little or no voltage dependence and tau 1 increased with hyperpolarization. The amount of charge moved during the e.p.s.c. was reduced as a function of atropine concentration. Driving functions, which represented the rate of channel opening, were derived from e.p.s.c.s both from control and atropine-treated cells. The characteristics of the driving functions did not vary with membrane voltage, but the driving functions were shorter in duration from atropine-treated than control cells. The area under the driving function decreased as a function of atropine concentration. The decay time constants and the amplitude ratio of the exponential components were used to calculate the closing, blocking, and unblocking rate constants, alpha, G, and F. alpha and F remained constant with increasing atropine concentration, but G declined significantly. The voltage dependence of the equilibrium constant, G/F, implied that the transient blocking site for atropine is halfway through the ionic channel. The sequential model does not predict the concentration-dependent decrease in the blocking rate, G, or in the charge moved during an e.p.s.c. We conclude that atropine has an action more complex than simple channel blockade in sympathetic ganglion cells.

Animals↗

PubMed1983

Analysis of fast excitatory postsynaptic currents in bullfrog parasympathetic ganglion cells.

Excitatory postsynaptic currents (EPSCs), miniature excitatory postsynaptic currents (MEPSCs), and acetylcholine-induced current fluctuations (noise) have been studied in voltage-clamped bullfrog parasympathetic ganglion cells of the atrial septum. EPSCs were also recorded from voltage-clamped sympathetic B cells and, in general, it was found that the basic properties of the EPSCs are similar in both parasympathetic and sympathetic B cells. For parasympathetic cells, the EPSC reached a peak amplitude of several nanoamperes within 3 msec and decayed exponentially. For 31 cells voltage-clamped to -50 mV (22 to 23 degrees C), peak amplitude was -4.3 +/- 1.4 nA (mean +/- SD) and the decay time constant, tau, was 5.6 +/- 1.0 msec. tau was independent of EPSC amplitude at a set voltage but increased with hyperpolarization, the coefficient of voltage dependence being -0.0070 +/- 0.0025 mV-1 in 13 cells (21 to 23 degrees C). The EPSC amplitude-voltage relationship was linear between -30 and -90 mV. The reversal potential, determined by interpolation, was -4.0 +/- 6.7 mV (n = 11). The EPSC tau had a Q10 equal to 2.9. Blocking the acetylcholinesterase with methane sulfonyl fluoride (MSF) pretreatment prolonged EPSC decay but decreased EPSC amplitude. In addition, EPSC decay after MSF treatment deviated from a single exponential function. MEPSCs exhibited decay characteristics very similar to those of EPSCs recorded at the same voltage and temperature. Acetylcholine-induced current fluctuations were well described by a single Lorentzian function with the estimated mean channel open time (tau noise) very similar to the EPSC decay time constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1980

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells.

Excitatory postsynaptic currents (EPSCs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range --90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/-0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.

Animals↗