Synaptic Physiology and Mitochondrial Function in Crayfish Tonic and Phasic Motor Neurons

Peter V. Nguyen, Leo Marin, and Harold L. Atwood

Department of Physiology, Medical Research Council of Canada Group, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Nguyen, Peter V., Leo Marin, and Harold L. Atwood. Synaptic physiology and mitochondrial function in crayfish tonic and phasic motor neurons. J. Neurophysiol. 78: 281-294, 1997. Phasic and tonic motor neurons of crustaceans differ strikingly in their junctional synaptic physiology. Tonic neurons generally produce small excitatory postsynaptic potentials (EPSPs) that facilitate strongly as stimulation frequency is increased, and normally show no synaptic depression. In contrast, phasic neurons produce relatively large EPSPs with weak frequency facilitation and pronounced depression. We addressed the hypothesis that mitochondrial function is an important determinant of the features of synaptic transmission in these neurons. Mitochondrial fluorescence was measured with confocal microscopy in phasic and tonic axons and terminals of abdominal and leg muscles after exposure to supravital mitochondrial fluorochromes, rhodamine-123 (Rh123) and 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). Mitochondria of tonic axons and neuromuscular junctions had significantly higher mean Rh123 and 4-Di-2-Asp fluorescence than in phasic neurons, indicating more accumulation of the fluorochromes. Mitochondrial membrane potential, which is responsible for Rh123 uptake and is related to mitochondrial oxidative activity (the production of ATP by oxidation of metabolic substrates), is likely higher in tonic axons. Electron microscopy showed that tonic axons contain approximately fivefold more mitochondria per µm2 cross-sectional area than phasic axons. Neuromuscular junctions of tonic axons also have a much higher mitochondrial content than those of phasic axons. We tested the hypothesis that synaptic fatigue resistance is dependent on mitochondrial function in crayfish motor axons. Impairment of mitochondrial function by uncouplers of oxidative phosphorylation, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone, or by the electron transport inhibitor sodium azide, led to marked synaptic depression of a tonic axon and accelerated depression of a phasic axon during maintained stimulation. Iodoacetate, an inhibitor of glycolysis, and chloramphenicol, a mitochondrial protein synthesis inhibitor, had no significant effects on either mitochondrial fluorescence or synaptic depression in tonic or phasic axons. Collectively, the results provide evidence that mitochondrial oxidative metabolism is important for sustaining synaptic transmission during maintained stimulation of tonic and phasic motor neurons. Tonic neurons have a higher mitochondrial content and greater oxidative activity; these features are correlated with their greater resistance to synaptic depression. Conversely, phasic neurons have a lower mitochondrial content, less oxidative activity, and greater synaptic fatigability.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Motor neurons and their neuromuscular synapses possess a spectrum of physiological and morphological attributes that are functionally suited to their natural patterns of electrical activity. In crustaceans, physiological specialization is particularly prominent. Phasic motor neurons have relatively high firing thresholds, produce few impulses (1/h, on average, in the claws of quiescent crayfish) (Pahapill et al. 1985), and generate rapid, powerful muscle contractions (Atwood 1976). Neuromuscular terminals of phasic axons are filiform, often with relatively few synaptic vesicles(Atwood and Johnston 1968) and long, thin mitochondria (Atwood and Wojtowicz 1986; Lnenicka et al. 1986). Conversely, tonic motor neurons have relatively low firing thresholds, produce more impulses (up to 30,000/h) (Bittner and Kennedy 1970), and generate slowly developing muscle contractions that can be sustained for prolonged periods of time (Atwood 1976). Tonic motor terminals are varicose, with large, branched mitochondria (Atwood and Jahromi 1977; Jahromi and Atwood 1974; Lnenicka et al. 1986) and twice the level of the synaptic transmitter glutamate found in phasic neurons (Shupliakov et al. 1995).

Transmission at phasic and tonic neuromuscular synapses is correspondingly dichotomous. Phasic neurons release more transmitter at low frequencies of activation, generating large excitatory postsynaptic potentials (EPSPs), whereas tonic neurons release much less transmitter at low frequencies (Atwood 1976). With repetitive stimulation, phasic synapses show rapid depression (fatigue) of EPSPs, whereas tonic synapses display pronounced facilitation with no depression (Atwood 1976). Many of these physiological differences are also found among motor neurons of mammalian species (Burke and Tsairis 1977; Burke et al. 1973) and lower vertebrates (Kuffler and Vaughan Williams 1953), and it is possible that similar cellular specializations exist to meet functional requirements.

In a previous study (Nguyen and Atwood 1994), we found that crayfish motor axons subjected to higher-than-normal activity levels acquire long-lasting changes in their mitochondria in parallel with increased resistance to synaptic depression. This led us to compare mitochondrial properties and synaptic performance in phasic and tonic axons, which show marked physiological differences.

In the present study, we addressed the question: is there an inverse correlation between the functional activity of a neuron's mitochondria and fatigability of its synapses? This possibility is suggested by morphological observations: terminals of tonic axons have a larger mitochondrial volume (Lnenicka et al. 1986), as do the preterminal axons (Case and Lnenicka 1992). The hypothesis that higher mitochondrial content is linked to a greater ability to sustain transmission during recurrent impulse activity is consistent with the commonly held tenets that electrical activity and synaptic transmission in the nervous system require metabolic energy, and that provision of ATP by mitochondria should be coupled to ongoing levels of electrical activity (Erecinska and Silver 1989; Wong-Riley 1989). To date, no study has yet demonstrated a tight correlation between mitochondrial oxidative activity and synaptic fatigability in single living neurons in situ, although evidence for high oxidative activity has been reported for chemically fixed neurons that are presumed to be electrically active in vivo (Mjaatvedt and Wong-Riley 1988; Sickles and Oblak 1984). Conversely, low oxidative activity has been reported for neurons that receive predominantly inhibitory synaptic input and are presumed to be less active in impulse generation (Mjaatvedt and Wong-Riley 1988).

A convenient qualitative measure of mitochondrial oxidative activity in living cells is rhodamine-123 (Rh123) fluorescence. This cationic lipophilic fluorochrome is taken up selectively into living mitochondria (Chen 1989). The primary driving force for Rh123 uptake is the mitochondrial membrane potential (Vmt) (Chen et al. 1981; Davis et al. 1985; Emaus et al. 1986; Farkas et al. 1989; Johnson et al. 1981; Maro et al. 1982). Rh123 fluorescence is linearly related to Vmt (Emaus et al. 1986) and is reduced both by treatment with drugs that depolarize Vmt and by anaerobic conditions (Chen et al. 1981; Johnson et al. 1981). Thus it is generally accepted that Rh123 is a mitochondrion-specific dye that can be used qualitatively to assess relative mitochondrial oxidative activity.

In the present study, we sought to correlate mitochondrial function with synaptic fatigability in living phasic and tonic motor axons of the crayfish. Mitochondrial oxidative activity, as measured by confocal imaging of mitochondria-specific Rh123 fluorescence, was higher in tonic motor axons than in phasic axons. Tonic axons and their terminals also contain a higher density of mitochondria. The greater resistance to synaptic fatigue seen in tonic axons was highly dependent on oxidative metabolism. These results support the hypothesis that mitochondrial oxidative activity can critically influence synaptic fatigability in motor neurons.

Preliminary accounts of this work have been reported briefly in abstract form (Nguyen and Atwood 1992) and noted in a review article (Atwood and Nguyen 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Animals

Freshwater crayfish (Procambarus clarkii) were obtained from a supplier in Louisiana (Atchafalaya) and were kept at 16°C in tanks filled with dechlorinated tap water. Animals were fed carrots and lentils twice weekly. All experiments were performed on crayfish measuring 4.5-6 cm in length (rostrum to telson) and weighing ~2.5-5 g. The preparations examined were the phasic (deep) abdominal extensor muscles (Nguyen and Atwood 1994; Parnas and Atwood 1966), the tonic (superficial) abdominal flexor muscles (Kennedy and Takeda 1965), and the main extensor muscle (carpopodite extensor) of the walking leg (Atwood and Cooper 1995).

Materials

Unless otherwise indicated, the standard physiological solution used in all experiments was a modified van Harreveld's crayfish saline solution (van Harreveld 1936) composed of (in mM) 205.3 NaCl, 5.3 KCl, 13.5 CaCl2, 2.5 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

The following metabolic inhibitors were dissolved in crayfish solution at the specified concentrations: dinitrophenol (DNP), 0.8 mM; sodium azide (NaN3), 20 mM; and iodoacetate (IAA), 0.1 mM. Chloramphenicol (CAP) was dissolved in dimethylsulfoxide (DMSO) and diluted with crayfish solution to a final drug concentration of 0.25 mM (in 1% DMSO). Acute perfusion of tonic flexor preparations with 1% DMSO (in crayfish saline) did not affect EPSP amplitudes before and during tetanic stimulation. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was dissolved in 95% ethanol and diluted to a final effective drug concentration of 15 µM in 2% ethanol. The latter did not affect EPSP amplitudes of tonic abdominal flexor axon 6 during tetanic stimulation.

Rh123 was dissolved in crayfish solution at a concentration of 13 µM (5 µg/ml). Supplementary experiments on nerve terminal fluorescence utilized the supravital fluorescent dye 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp) (Magrassi et al. 1987) at a concentration of 5 µM. In other work, this dye was found to be taken up by mitochondria of crayfish axons (Harrington and Atwood 1995) and released from these structures by metabolic inhibitors, as is Rh123 (Bradacs and Atwood 1993). This dye was more resistant to bleaching than Rh123 and gave better images of nerve terminals.

All solutions were stored at 10°C after adjustment of their pH values to 7.4. The source of all reagents (except IAA: ICN) was Sigma Chemical (St. Louis, MO).

Microscopic observations

Dissected abdominal or leg muscles with their attached nerves were stained with Rh123 for 10 min at 10°C, or with 4-Di-2-Asp for 1 min at 20°C, and then viewed under confocal microscopy for measurements of relative mitochondrial fluorescence after the dye-containing solution had been rinsed out. For the phasic abdominal extensors, only the posterior branch of the nerve root in segment 3 was imaged. Images of tonic axons in the superficial abdominal flexor nerve root of segment 3 were collected from the distal half of this nerve. In general, confocal images were collected from secondary branches of motor axons, situated ~0.5-2 mm proximal to synaptic terminals (Fig. 1A).


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FIG. 1. Confocal microscopy of mitochondria in single motor axons. A: schematic diagram of a motor axon to the abdominal extensor muscles, showing location of region scanned for confocal microscopy. Scale bar: longitudinal distances between different branches. Relative diameters of branches are not drawn to scale. B: longitudinal optical sections (left) were collected from a living motor axon in situ and then projected in the verticalZ-axis to generate a 2-dimensional composite "through-view" image of the superficial regions of the axon's interior (right). C1: confocal micrograph of rhodamine-123 (Rh123)-stained tonic flexor nerve root, showing axon 6 and other smaller motor axons. Solid arrows point to edges (axolemma) of axon 6. Boxed area was magnified to produce micrograph at right. C2: mitochondrial fluorescence was measured by outlining individual fluorescent profiles (white outlines, with white open arrows) inside an axon and computing the average pixel value within each outlined area. A fluorescent profile that was clearly separated from another profile by a resolvable dark region was treated as a discrete mitochondrial segment. Micrographs are projections of 5 consecutive optical sections (total depth of field: ~5 µm). D: average mitochondrial pixel values measured from axon 6 over 60 min of saline superfusion following staining with Rh123. Bars: SDs derived from 36 mitochondrial segments per time point. Scale bar: C1, 10 µm; C2, 2 µm.

The methods used here for confocal microscopic measurement of Rh123 and 4-Di-2-Asp fluorescence in crayfish axons were identical to those described in Nguyen and Atwood (1994). Briefly, preparations were viewed with a Nikon Optiphot epifluorescence microscope adapted for confocal optics (BioRad MRC-600). A ×40 (numerical aperture 0.55) Nikon water-immersion objective and a blue excitation filter set were used to view axonal dye fluorescence. Optical sectioning was performed to collect 7-10 consecutive images of 0.8 µm thickness along the vertical Z-axis of the preparation (Fig. 1B). All images represented longitudinal optical sections through motor axons (Fig. 1, B and C).

For comparison of mean mitochondrial pixel values, the microscope's gain and black level settings were kept at 80-95% and 22-35% of maximum levels, respectively (Nguyen and Atwood 1994). These settings yielded mean mitochondrial pixel values of 140-165 and 70-90 (background subtracted) for tonic and phasic abdominal axons, respectively. Photobleaching of Rh123 was not a significant factor in these experiments, because precautions were taken to minimize repeated and prolonged exposure of axons to the incident laser beam. The use of identical ranges of gain and black level settings allowed for the comparison of pixel values between different samples, and in the case of the leg extensor axons, measurements could be made simultaneously from pairs of axons lying side by side in the same preparation. Thus the significantly lower pixel values obtained for mitochondria in phasic axons were not the result of differences in manual gain or black level settings.

The average fluorescence intensities of mitochondria in motor axons were calculated with the use of the STATS function of the CM software (BioRad). Mean pixel values were computed from composite projections of seven sections in a Z series collected from an axonal region. Thus a two-dimensional "through-view" of a longitudinal interior section of an axon was used to generate pixel values for mitochondrial profiles within the axon (Fig. 1C). Fluorescent organelles were individually outlined by hand (Fig. 1C), with the use of the STATS function, and the mean pixel value within each enclosed area was computed. Mean background pixel values were subtracted from individual mitochondrial values. All reported pixel values represent means obtained by averaging the values for individual mitochondria from each set of experiments.

A Mann-Whitney two-tailed rank sum test was used to compare mean mitochondrial pixel values among motor axons.

Electron microscopy

Representative sections of phasic and tonic axons and neuromuscular junctions were obtained for confirmation of mitochondrial content. The mitochondrial numerical densities (number of mitochondria per µm2 of axonal cross-sectional area) of representative tonic abdominal flexor and phasic abdominal extensor motor axons were compared by processing nerve roots for electron microscopy and counting the number of mitochondria present in cross-sectional profiles of axons observed in individual sections. Nerve roots were prefixed in situ for 1 h (at room temperature) in 2.5% glutaraldehyde and 0.5% formaldehyde (in 0.1 M cacodylate buffer, pH 7.4), and processed according to previously established procedures for crayfish material (Lnenicka et al. 1986). Transverse sections of motor axons were cut, mounted, viewed, and quantitatively assessed as described previously in Nguyen and Atwood (1994).

A Student's unpaired t-test was used for statistical comparisons of mitochondrial densities in phasic and tonic axons.

Electrophysiological methods

PHASIC MOTOR AXON. The abdominal deep (phasic) extensor musculature was dissected as previously described (Parnas and Atwood 1966). The second nerve root of the third abdominal segment contains six phasic motor axons (5 excitors, one inhibitor) innervating the deep extensors of segments 3 and 4; only one excitor (axon 3) (after Parnas and Atwood 1966) supplies muscle L1 in segment 4. This innervation pattern allows the synaptic responses of axon 3 to be examined unambiguously in segment 4 (Mercier and Atwood 1989). A suction electrode (inner tip diameter 20 µm) was used to stimulate the nerve root in segment 3, whereas conventional microelectrodes (resistances 15-20 MOmega ) were used to penetrate fibers of muscle L1 in segment 4 for intracellular recording of evoked EPSPs.

Initial EPSP amplitudes were recorded at 0.1 Hz (mean of 8 sweeps) while the preparation was superfused (at 2 ml/min) with crayfish saline containing half (6.7 mM) the normal calcium concentration and 5 times (12.3 mM) the normal magnesium concentration. This modification was employed to attenuate contractions of muscle L1, which would otherwise dislodge the recording microelectrode (Mercier and Atwood 1989). The level of NaCl was adjusted to compensate for osmolality changes.

For measurements of synaptic depression, axon 3 was stimulated at 5 Hz, and EPSPs were recorded periodically during a 20-min stimulation period. The mean of 16 sweeps was measured at each time point; all recordings were made from the most medial fibers of muscle L1 (Mercier and Atwood 1989). "Final" EPSP amplitudes were measured after 20 min, and were used as an indicator of relative synaptic fatigue. All EPSPs were corrected for nonlinear summation (Martin 1955), assuming a reversal potential of +11.5 mV (Onodera and Takeuchi 1975).

In experiments in which metabolic inhibitors were used, the preparation was superfused with a solution containing the inhibitor for 15 min before EPSP measurements.

TONIC MOTOR AXON. The abdominal superficial (tonic) flexor musculature (Kennedy and Takeda 1965) was dissected via a ventral approach. The muscles of the left side of abdominal segment 3 were removed, together with the posterior and anterior exoskeletal ribs and the overlying soft ventral exoskeleton. The superficial tonic flexor nerve root, containing only six tonic flexor motor axons (Wine et al. 1974), was cut near its point of emergence from the abdominal nerve cord. Tonic axon 6 is the largest flexor efferent supplying the superficial flexors, and produces the largest extracellularly recorded action potential in the superficial third root (Kennedy and Takeda 1965; Wine et al. 1974). This axon has a more intermittent pattern of impulse production than the other axons in this nerve root, and thus is less "tonic" in its activity (Gillary and Kennedy 1969; Kirk and Glantz 1981). Nevertheless, it exhibits resistance to synaptic depression, as do the other axons in this nerve root, and thus differs markedly from the phasic axons supplying the fast-contracting deep abdominal muscles. A suction electrode (inner tip diameter 10 µm) was used to stimulate the nerve root and recruit axon 6 (which has the lowest stimulation threshold). An extracellular electrode (inner tip diameter 15 µm) recorded presynaptic spikes produced by axon 6, and was positioned along the nerve root distal to the stimulating electrode. This configuration allowed confirmation of the exclusive recruitment of axon 6 (largest size and shortest latency). An intracellular microelectrode was used to record EPSPs evoked by axon 6 at sites along the three most lateral muscle fibers, which are heavily innervated by this axon (Velez and Wyman 1978).

The mean initial EPSP size evoked by axon 6 was measured by averaging 24 sweeps (stimulation frequency 1 Hz) accumulated over a period of 4 min just before tetanic stimulation. After initial EPSP measurement, axon 6 was stimulated at 10 Hz for 20 min, and EPSPs were recorded continuously during this period. Preparations were superfused during EPSP recordings. Drug applications were made as for the phasic motor axon.

The effective input resistance (rin) of tonic abdominal flexor muscle fibers was measured by passing current pulses (duration 200 ms) with an intracellular microelectrode and recording the resultant change in membrane potential with a second intracellular microelectrode (Lnenicka and Atwood 1985). These two electrodes (filled with 3 M KCl) were situated in the central region of a muscle fiber, with an interelectrode separation of <100 µm.

For all electrophysiological experiments, values of EPSPs are reported as means ± SE. Student's unpaired t-test was used for statistical comparisons of measured parameters.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Mitochondrial fluorescence

Stability of mitochondrial fluorescence after treatment of axons with Rh123 was assayed in the tonic abdominal flexor axons. Numerous Rh123-stained organelles aligned parallel to the longitudinal axis of the axon were observed (Fig. 1). Most of these were elongate, as are mitochondria seen in electron micrographs and in Nomarski differential interference contrast microscopy of crayfish axons (Case and Lnenicka 1992). The fluorescent organelles seen in confocal micrographs were concentrated just inside the axonal membrane. This is where most of the mitochondria occur in crayfish and lobster motor axons, as shown by electron microscopy (Atwood et al. 1989; Forman et al. 1987) (see Fig. 6). Thus the morphology and spatial distribution of these fluorescent organelles confirm their identities as mitochondria.


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FIG. 6. Tonic axons and neuromuscular junctions contain more mitochondria per unit cross-sectional area than their phasic counterparts. A: electron micrograph of a tonic flexor axon viewed in cross section. Arrowheads point to some of the mitochondria located in axoplasm (Axp) just inside axonal membrane (Axm). Scale bar: 1 µm. B: phasic extensor axons viewed in cross section. Note that scale bar for this micrograph is 2 µm. Thus cross-sectional area of larger axon is several times that of tonic axon in A. Area of boxed region is approximately twice that of tonic axon in A, and is shown in C. Arrowheads in C point to some mitochondria in axoplasm. Scale bar in C: 1 µm. D: cross sections of axon branches of paired motor axons of leg extensor muscle, illustrating higher mitochondrial content of tonic axon. E: example of a tonic terminal with synapses and many mitochondria. F: example of paired phasic and tonic nerve terminals, showing characteristic difference in size and mitochondrial content. Arrowheads: mitochondria. Arrows: synapses. Scale bars in D-F: 1 µm.

We collected confocal microscopic images every 20 min for 1 h after Rh123 treatment, and computed mitochondrial pixel values. For three tonic flexor preparations, the range of mean mitochondrial pixel values was 140-164, with means ± SE ranging from 21 to 27. A total of 36 mitochondrial profiles (representing whole mitochondria or mitochondrial segments) was analyzed at each time point, and two different regions of the tonic motor nerve root were alternately imaged over the 60-min period. Mean mitochondrial pixel values did not fluctuate appreciably over this time period, indicating stability under these conditions (Fig. 1D).

Effects of metabolic inhibitors on mitochondrial fluorescence in tonic axons

Prestained tonic flexor axons were viewed under confocal microscopy before, during, and after acute exposure to DNP, CCCP, azide, IAA, and CAP. In prestained tonic flexor axons, DNP treatment led to loss of organelle fluorescence. The normally punctate pattern of organelle staining seen before drug exposure (Fig. 2A) was replaced by a diffuse blur of high-intensity fluorescence present throughout the axoplasm (Fig. 2B). This pattern is consistent with that reported in other cell types following acute exposure to mitochondrial poisons, and represents the release of Rh123 into the surrounding axoplasm, consequent to dissipation of Vmt (Johnson et al. 1981). Soon thereafter, much of the diffuse fluorescence disappeared from the axon, and a much smaller number of punctate fluorescent profiles remained (Fig. 2C). An identical pattern of drug-induced release of Rh123 from prestained mitochondria was observed for two other inhibitors of oxidative metabolism (15 µM CCCP and 20 mM NaN3). Experiments with 4-Di-2-Asp have shown the same effect in leg extensor motor axons (Bradacs and Atwood 1993; H. Bradacs and H. L. Atwood, unpublished observations), confirming the similar behavior of the two supravital fluorochromes.


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FIG. 2. Effects of dinitrophenol (DNP) on mitochondrial Rh123 fluorescence in tonic flexor axons. A: projection of 5 optical sections collected from tonic axon 6, 10 min after staining and just before introduction of 0.8 mM DNP. Arrows: edges of axon 6. B: micrograph taken from same longitudinal region, 3 min after addition of DNP. Fluorescence inside axons increased as a result of drug-induced release of dye from axonal mitochondria. Five optical sections are projected. C: ~10 min after DNP addition and saline superfusion, much of the released rhodamine was eliminated from the axon. Fewer fluorescent intracellular organelles are evident than in A. Micrograph consists of 6 optical sections. D: after DNP washout and drug-free saline superfusion for 15 min, preparation was restained with Rh123. Punctate appearance and fluorescence of some axonal mitochondria were partially restored. Picture represents a projection of 6 optical sections. Total depth of field for each micrograph was ~4-5 µm. Scale bar: 10 µm. E: summary of effects of drugs on mitochondrial fluorescence in tonic flexor axon 6. For saline-treated preparations, mitochondrial pixel values were measured 10 min after staining. For chloramphenicol (CAP) and iodoacetate (IAA), values were obtained after 40 min of drug superfusion. For DNP, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and azide, pixel values were measured after 10, 5, and 15 min, respectively, of drug superfusion. Numbers above error bars: numbers of mitochondrial profiles sampled (2 animals per group).

The efficacy of the inhibitors in promoting the loss of mitochondrial fluorescence varied: DNP and CCCP strongly attenuated organelle fluorescence (P < 0.002 for comparisons with saline controls, Fig. 2E), but azide's effects were less robust. The weaker effects of azide may have stemmed from relatively lower permeability for this drug and the slower depolarization of Vmt during the ensuing respiratory blockade (Zubay 1983).

After washout, continuous superfusion of the preparation with drug-free saline for 15 min was undertaken to test for reversibility of the inhibitors' effects. Following restaining with Rh123, mitochondrial fluorescence was only partially restored in DNP-, CCCP-, and azide-treated preparations (Fig. 2D). Mitochondrial pixel values measured after restaining were only 56 ± 17% (SE; CCCP), 72 ± 21% (DNP), and 69 ± 20% (azide) of predrug values. This recovery of mitochondrial fluorescence may represent partial restoration of Vmt.

In contrast to the respiratory poisons, IAA and CAP (inhibitors of glycolysis and mitochondrial protein synthesis, respectively) had no effect on mitochondrial fluorescence in prestained axons (Fig. 2E). Tonic axons exposed to IAA or CAP for up to 1 h showed no significant alterations(P > 0.01) in Rh123 fluorescence.

The collective results (Fig. 2E) demonstrate that the fluorescent organelles seen in tonic flexor axons are mitochondria, and that metabolic inhibitors cause them to release Rh123, in agreement with previously reported effects of these agents on Vmt.

Mitochondrial fluorescence is higher in tonic than in phasic motor axons

The average mitochondrial pixel value in living tonic flexor axons was significantly higher (142 ± 20, 216 mitochondrial segments from n = 3 animals) than that measured from phasic extensor axons (67 ± 15, 252 mitochondrial segments from n = 3 animals, P < 0.01, Mann-Whitney rank sum test, Fig. 3). Mitochondria in tonic axons sometimes displayed localized fluorescent varicosities. These might represent sites of high oxidative activity ("hot spots") within a mitochondrion (Chen 1988). Mitochondria of phasic extensor axons showed fewer fluorescent hot spots (Fig. 3).


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FIG. 3. Mitochondrial fluorescence in tonic abdominal flexor axons is higher than in phasic abdominal extensor axons. Note presence of elongated mitochondria in tonic axon 6 (A), some of which appear to have localized varicosities (fluorescent "hot spots"). In contrast, the mitochondria visible in phasic axons (B) were less numerous and showed fewer bright spots. Micrographs were taken from different animals, but with same microscope settings and at same time (10 min) after staining. Each picture is a projection of 5 optical sections (depth of field ~4 µm). Scale bar in B (10 µm) also applies to A. Numbers above error bars in C: numbers of mitochondrial profiles sampled from 3 animals.

To compare mitochondrial fluorescence of phasic and tonic axons in the same preparation under identical conditions, we also measured fluorescence intensities of individual mitochondria in paired phasic and tonic axons of the leg extensor muscle after treatment with Rh123 or 4-Di-2-Asp (Fig. 4). Individual mitochondria were, on average, more fluorescent in the tonic axon, but the measurements overlapped, suggesting a wider range of values and higher maximum fluorescence in the tonic axon. The distributions of fluorescence values for the phasic and tonic axons were highly significantly different (Kolmogorov-Smirnov 2-sample test, P < 0.001).


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FIG. 4. Comparison of mitochondrial fluorescence in paired phasic and tonic axons of leg extensor muscle. A: confocal micrograph illustrating differential fluorescence of the 2 axons. Arrows: single mitochondrion in each axon. Scale bar: 10 µm. Sheath around axons contains a number of very bright nonlinear structures, presumed to be mitochondria within the glial cells. B: histograms showing distribution of measurements for individual mitochondria in the paired axons after treatment with Rh123 and 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). Measurements from tonic axon extended to higher values and had a wider range than measurements from phasic axon. For both histograms, distributions of measurements for phasic and tonic axons were highly significantly different (Kolmogorov-Smirnov 2-sample test, P < 0.001).

The difference in brightness of individual mitochondria could be due in part to a difference in size, because larger mitochondria would be expected to accumulate more dye than smaller ones. Rh123 is known to be accumulated within the matrix of mitochondria, without substantial binding to mitochondrial membranes (Emaus et al. 1986). To check the likely importance of this factor, we measured and compared cross-sectional areas of individual mitochondria from electron micrographs of abdominal axons. The means ± SE for phasic and tonic axons were0.0149 ± 0.00095 µm2 and 0.0185 ± 0.0016 µm2, respectively. The values were significantly different (P < 0.003). However, the percentage difference between the means is only 20%, which represents a difference in diameter of ~11%. The percentage difference in mean fluorescence for abdominal axons is 112%. Thus the difference in mitochondrial size can account for only a small part of the difference in fluorescence.

These studies show consistently that mitochondrial fluorescence is higher in tonic than in phasic abdominal and leg motor axons. The results suggest that the sampled mitochondria in tonic axons have a higher mean membrane potential and thus a higher overall oxidative activity than those in phasic axons.

Comparative observations on fluorescence of neuromuscular junctions

Observations on relative fluorescence of axons with Rh123 and 4-Di-2-Asp were extended to neuromuscular junctions in the tonic flexor and phasic abdominal extensor muscles and the leg extensor muscle. In abdominal muscles, several axons contribute to junctional regions, so the identities of nerve terminals associated with a particular axon could not be specified. Nevertheless, striking overall differences were observed in junctional terminals of the two muscles (Fig. 5). In the tonic abdominal flexor muscle, the terminals were more varicose, and much brighter, than those in the phasic abdominal extensor muscle. Individual mitochondria could not readily be observed in tonic endings, but it is known from electron microscopy of other preparations that they are larger and more complex than in phasic endings (Lnenicka et al. 1986) (see also Fig. 6). In phasic terminals, individual mitochondria could readily be observed in the thin terminals (Fig. 5B); they were slender in form, in accordance with previous electron microscopic work on other preparations (Lnenicka et al. 1986).


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FIG. 5. Neuromuscular junctions of tonic flexor axons (A) and phasic extensor axons (B) stained with 4-Di-2-Asp. Preparations were exposed to 5 µM dye in crayfish solution for 1 min before viewing with confocal microscopy; settings on microscope were same throughout. A: typical varicose boutons of tonic flexor axons, showing brightly fluorescing synapse-bearing nerve terminals (NT) overlying the muscle fibers (MU). B: axon (AX) branches and synaptic terminals (NT and up-arrow  indicating contained mitochondria) of phasic extensor muscle L1. Axons run diagonally across the muscle's surface (MU), from bottom right corner to top left corner. Mitochondria can be seen within axons and filiform nerve terminals. Fluorescence is less intense than in tonic axons and terminals. Scale bar: 10 µm. C: neuromuscular junctions of phasic (P) and tonic (T) axons of leg extensor muscle, showing typical filiform phasic terminals and varicose, less extensive tonic terminals. Tonic terminals always fluoresce more brightly than phasic terminals. Scale bar: 10 µm.

In the leg extensor muscle, it was relatively easy to follow phasic and tonic axon branches to their respective neuromuscular junctions (Fig. 5C). Terminals of the two axons could be unambiguously identified on single muscle fibers. It was strikingly apparent that phasic terminals were slender and relatively nonvaricose, and much less fluorescent than tonic terminals. These observations are consistent with those in abdominal muscles. Thus the difference in fluorescence observed in axons appears to be mirrored at the neuromuscular junction, with tonic junctions showing greater overall fluorescence than phasic junctions. This difference in fluorescence likely reflects the greater numbers and sizes of mitochondria in tonic junctions, as well as a possible difference in fluorescence of individual mitochondria.

Mitochondrial content in phasic and tonic motor axons and presynaptic terminals

For comparison of mitochondrial numbers in phasic and tonic axons, the nerves containing these axons were fixed and processed for electron microscopy, and the numbers of mitochondria per µm2 of cross-sectional area were calculated. The sample electron micrographs of Fig. 6 show that tonic flexor motor axons contained more mitochondria per µm2 than phasic extensor motor axons. Statistical comparison of samples confirmed the difference (P < 0.001, unpaired t-test, Table 1). Additional observations on the main axons and secondary branches of leg extensor motor axons also indicated a substantial phasic-tonic difference (Fig. 6D), and agreed with observations by Case and Lnenicka (1992). Thus the higher mean mitochondrial number in tonic axons complements the higher relative oxidative activity of some of these mitochondria. The observations support the hypothesis that the total mitochondrial oxidative capacity of the tonic axons is considerably greater than that of the phasic axons.

 
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TABLE 1. Mitochondrial content in tonic and phasic abdominal motor axons

Electron microscopic observations of phasic and tonic neuromuscular junctions in the extensor muscle, where they could be readily identified, confirmed the relatively greater mitochondrial content of the tonic terminals. In fact, the disparity in mitochondrial content was often greater than in the preterminal axons (Fig. 6F). In an additional recent study of these terminals, based on eight serial reconstructions from electron micrographs, the percentage volume for mitochondria in tonic terminals was 16%, and for phasic terminals the percentage volume was 6% (King et al. 1996).

Differential synaptic physiology

The synaptic responses of tonic abdominal flexor axon 6 and phasic abdominal extensor axon 3 were examined during acute in situ stimulation. At a stimulation frequency of 0.1 Hz, tonic axon 6 produced a mean initial EPSP amplitude of 0.7 ± 0.1 mV in lateral fibers (n = 5 animals). During10-Hz stimulation for 20 min ("tetanic" stimulation), EPSPs potentiated rapidly and attained a plateau amplitude of 2.6 ± 0.2 mV after 10 min of stimulation (Fig. 7A). This elevated EPSP amplitude was maintained with no decline for the remaining 10 min of stimulation. Potentiated EPSPs in axon 6 could be maintained without significant depression during 1 h of stimulation (not shown). Thus the neuromuscular junctions of axon 6 showed a remarkable ability to sustain transmission without fatigue over extended periods.


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FIG. 7. Synaptic physiology of crayfish tonic and phasic abdominal motor neurons. A: tetanic stimulation of tonic flexor axon 6 elicits facilitation without depression. Initial excitatory postsynaptic potentials (EPSPs) produced by 1-Hz stimulation of axon 6 (1st 3 points on graph, 0 time point of bottom EPSP traces) were small in size, but facilitated rapidly during 10-Hz stimulation. Enhanced EPSP amplitudes recorded during 10-Hz stimulation persisted throughout the 20-min period of tetanic stimulation. Scale bars: vertical, 2 mV;horizontal, 2 ms. SE bars (n = 5 animals) are shown for only final EPSPs measured after 20 min of stimulation. Bars on initial EPSP points in graph are smaller than thickness of graph points. Sample EPSP traces shown here were recorded from 1 muscle fiber in 1 preparation. B: stimulation of phasic extensor axon 3 (20-min, 5-Hz stimulation) produces marked synaptic fatigue. "Gap" between 2 and 6 min on graph resulted from stimulation-induced muscle contractions that precluded intracellular EPSP measurements. Scale bars: vertical, 10 mV; horizontal, 10 ms. n = 5 Animals for graph. Traces were recorded from 1 muscle fiber of 1 preparation.

In phasic extensor axon 3, stimulation at 0.1 Hz yielded a mean initial EPSP amplitude of 16.2 ± 5.0 mV (n = 5 animals). After initial EPSPs had been measured in low-calcium, high-magnesium crayfish solution, stimulation was continued at 5 Hz in standard crayfish saline solution. In accordance with a previous study (Mercier and Atwood 1989), 5-Hz continuous stimulation of axon 3 depressed EPSPs (Fig. 7B): mean EPSP amplitudes after 10 and 20 min of stimulation were 5.2 ± 1.3 mV and 1.0 ± 0.3 mV, respectively. A transient facilitation of EPSP size occurred over the first 2-4 min of stimulation in some preparations, as observed in other crayfish phasic motor synapses (Lnenicka and Atwood 1985). Thus, unlike tonic flexor axon 6, phasic extensor axon 3 showed marked synaptic fatigue during continuous stimulation at 5 Hz.

Effects of metabolic inhibitors on synaptic physiology

TONIC FLEXOR AXON 6. Acute exposure to 0.8 mM DNP significantly increased initial EPSP amplitude during 1-Hz stimulation (Fig. 8A). Mean initial EPSP amplitude at 1 Hz was 2.1 ± 0.5 mV (n = 4), which was significantly larger than the mean for drug-free controls (0.7 ± 0.1 mV, P < 0.05). Another mitochondrial uncoupler, CCCP (15 µM), also evoked an increase in mean initial EPSP size (1.0 ± 0.3 mV, n = 4). Sodium azide (20 mM) did not significantly change initial EPSP size (0.8 ± 0.1 mV, n = 4, P < 0.5, Fig. 8B). The elevated initial EPSP amplitude evident after DNP and CCCP treatment is likely attributable to an uncoupler-induced release of mitochondrial and nonmitochondrial intraterminal calcium (Akerman and Nicholls 1981; Heinonen et al. 1984; Jensen and Rehder 1991; Nicholls and Akerman 1981), which would increase transmitter release (Alnaes and Rahamimoff 1975; Glagoleva et al. 1970).


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FIG. 8. Effects of metabolic inhibitors on synaptic transmission in tonic flexor axon 6. A and B: DNP and sodium azide (NaN3) both induce a transient facilitation of EPSPs, followed by pronounced depression. Dashed lines: levels of initial EPSP sizes measured before 10-Hz tetanic stimulation. Final EPSPs measured after 20 min of tetanic stimulation were significantly smaller than initial EPSPs for both drug treatments. C: sample EPSP traces recorded during stimulation. Numbers: time (in min) of measurement. Rec.: recovery of EPSP amplitude 20 min after drug washout. Traces were recorded from 1 muscle fiber of 1 preparation in each case. Scale bars: 2 mV, 4 ms. D: final EPSP sizes produced by axon 6, expressed as percentages of initial EPSP sizes. Stimulation for 20 min at 10 Hz in inhibitors of oxidative ATP synthesis (DNP, NaN3, and CCCP) induced a dramatic and significant decline in final EPSP amplitudes. CAP and IAA, inhibitors of mitochondrial protein synthesis and glycolysis, respectively, did not produce significant depression in axon 6. IAA did slightly reduce degree of tetanic potentiation seen in axon 6 [compare with saline controls (SAL)]. Numbers above error bars: numbers of animals tested.

These same inhibitors were tested for their effects on synaptic transmission during repetitive stimulation at 10 Hz. In 0.8 mM DNP, the EPSPs of axon 6 facilitated rapidly (within 1 min) to a peak mean value of 4.7 ± 0.2 mV (Fig. 8A). This potentiation was extremely short lived, lasting only 3 min. EPSP amplitudes rapidly declined to pretetanus values after 4 min of 10-Hz stimulation. Thereafter, a gradual rundown of EPSPs was consistently observed. After 20 min of stimulation, mean EPSP amplitude was significantly lower (0.2 ± 0.2 mV) than that measured in drug-free saline controls (2.6 ± 0.2 mV, P < 0.05). A similar effect has been reported for the crayfish opener muscle's tonic motor axon (Lang and Atwood 1973).

Like DNP, both CCCP (not shown) and azide (Fig. 8B) induced a transient tetanic potentiation of EPSPs, followed by rapid depression. The mean final EPSP amplitudes measured after 20 min of stimulation in these drugs were significantly lower than those measured in drug-free controls(P < 0.01).

For all three mitochondrial inhibitors, the drug-induced depression of EPSPs seen during stimulation was reversed after washout (Fig. 8C). For DNP, EPSP amplitudes measured at 1-Hz stimulation frequency 20 min after drug washout averaged 146 ± 31% (n = 4) of those measured before 10-Hz stimulation. For CCCP and azide, these values were 141 ± 20% and 133 ± 23%, respectively. Preparations stimulated and then left in DNP without washout remain depressed (Acosta-Urquidi 1980; Lang and Atwood 1973).

These results suggest that mitochondrial oxidative phosphorylation is required for maintenance of synaptic transmission during high-frequency stimulation. The normally fatigue-resistant motor synapses of the tonic axon are transformed into highly fatigable synapses similar to those of a phasic axon by treatments that affect mitochondrial oxidative function.

In sharp contrast, IAA (an inhibitor of glycolysis) and CAP (a mitochondrial protein synthesis inhibitor) had no significant effects on the synaptic stamina seen in tonic axon 6. Mean EPSP amplitude after 20 min of 10 Hz stimulation in 0.1 mM IAA was 302 ± 66% (n = 4) of that recorded before tetanic stimulation (Fig. 8D). This was not significantly different from the increase observed in drug-free controls (386 ± 38%, P > 0.2). Mean initial EPSP amplitude was unaffected by IAA (0.9 ± 0.2 mV vs. 0.7 ± 0.1 mV for controls, P > 0.1). CAP (0.25 mM) did not attenuate the EPSP potentiation evoked during tetanic stimulation, but did slightly increase the mean final EPSP amplitudes measured after 20 min of tetanic stimulation (445 ± 84% of initial EPSP, n = 5, compared with 386 ± 38% for drug-free controls, P > 0.2, Fig. 8D). Overall, these findings show that acute application of inhibitors of glycolysis and mitochondrial protein synthesis had very little effect on synaptic stamina of tonic axon 6.

Measurements of effective muscle fiber rin were made to assess postsynaptic changes. Both 0.8 mM DNP (n = 15 cells) and 20 mM azide (n = 16) increased rin of flexor muscle fibers (1,003 ± 209 KOmega and 893 ± 142 KOmega , respectively) compared with drug-free saline controls (656 ± 65 KOmega , n = 16, P > 0.2 for an unpaired t-test comparison with both DNP- and azide-treated cells). Conversely, CCCP (15 µM) induced a significant 53% decrease in rin (307 ± 65 KOmega , n = 15, P < 0.05). DNP, CCCP, and azide also depolarized the membrane potentials of these fibers by 2-8 mV over the time course of these measurements (30-90 min). The reversibility of these effects was not tested. However, Acosta-Urquidi (1980) has demonstrated (for crayfish opener muscle fibers) a partial restoration of membrane potentials after DNP washout.

Synaptic fatigue observed during tetanic stimulation in DNP and azide cannot be attributed to changes in rin, and must be mainly presynaptic in origin. The CCCP-induced reduction in rin could account for part of the decrease in EPSP amplitude. To avoid difficulties in interpretation of results, CCCP was not used in further studies of synaptic fatigue.

PHASIC ABDOMINAL AXON 3. Synaptic depression was also assayed in phasic extensor axon 3 during acute exposure to DNP, azide, and IAA (Fig. 9). Average initial EPSP amplitude was larger in 0.8 mM DNP (27.3 ± 3.4 mV, n = 3) than in untreated controls (16.2 ± 5.0 mV, n = 5, P > 0.2, unpaired t-test comparison). Azide (20 mM) and IAA (0.1 mM) had no significant effect on mean initial EPSP sizes.


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FIG. 9. Inhibitors of oxidative ATP synthesis enhance synaptic fatigue in phasic extensor axon 3. DNP (0.8 mM, n = 3) and azide (20 mM, n = 3) both reduced time taken for EPSPs to decline to 50% of initial values during 5-Hz stimulation. In contrast, IAA (0.1 mM, n = 3) did not affect depression in axon 3 (compare with dashed curve for saline controls). Error bars are shown only for initial EPSP amplitudes; bars on final EPSP points are too small to be shown. Dashed curve is taken from data of Fig. 7B. Sample EPSP traces measured from 3 different preparations are shown to illustrate effects of the 3 drugs. Numbers: time (in min) of recording during 5-Hz stimulation. Scale bars: 10 mV, 5 ms.

Synaptic depression during 5-Hz stimulation of axon 3 was significantly accelerated by both DNP and azide (Fig. 9). The mean time taken for EPSPs to depress to 50% of their initial amplitudes (t0.5) was 1.3 ± 0.2 min for DNP(n = 3). This was significantly less than the t0.5 values for both untreated controls and DNP-treated tonic axon 6 (Table 2). The t0.5 value for azide-treated preparations was 3.0 ± 0.6 min (n = 3), which was significantly less than the values obtained for untreated controls and azide-treated tonic axon 6 (Table 2).

 
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TABLE 2. Synaptic fatigue in tonic flexor axon 6 and phasic extensor axon 3

In marked contrast, IAA had no significant effects on either initial EPSP amplitude or rate of synaptic fatigue(P > 0.1 for an unpaired t-test comparison with untreated saline controls, Fig. 9). Thus glycolysis per se does not appear to be critical for maintenance of synaptic transmission during activation of phasic extensor axon 3. In both phasic and tonic axons, the time course of synaptic depression is linked to oxidative metabolism.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Correlation between synaptic physiology and mitochondrial function

The present findings demonstrate a strong correlation between relative mitochondrial oxidative activity and capacity (as measured by confocal imaging of mitochondrial Rh123 fluorescence and relative numbers of mitochondria, respectively) and resistance to synaptic depression in crayfishmotor axons. Tonically active motor axons showed significantly higher mitochondrial fluorescence than phasically active axons, along with higher numbers of mitochondria in both axons and terminals. Correspondingly, synaptic stamina during high-frequency stimulation was greater in tonic than in phasic axons. Inhibitors of oxidative phosphorylation, but not inhibitors of nonoxidative metabolism, abolished the synaptic fatigue resistance normally seen in tonic axons, and accelerated synaptic depression in phasic extensor axons. Synaptic transmission in tonic axons assumed a more phasic pattern of transient augmentation and subsequent rapid depression after treatment with mitochondrial inhibitors. Thus, for abdominal motor axons (and also for the leg opener muscle's motor axon) (Lang and Atwood 1973), synaptic stamina requires metabolically intact mitochondria, and is correlated with different levels of oxidative activity in phasic and tonic neurons.

It is pertinent to ask whether the more rapidly depressing EPSP of phasic neurons is simply the result of a disproportionately high release of transmitter per nerve terminal. This would generate both the larger initial EPSP and (because available stores of transmitter are being more rapidly used) the more rapid depression. This situation may occur in neuromuscular junctions of phasic and tonic leg motor neurons (Atwood 1967; Msghina et al. 1995). However, for the abdominal muscles, studies of quantal release made for both tonic flexor axon 6 (Gillary and Kennedy 1969; Lnenicka and Mellon 1983) and phasic extensor axon 3 (Mercier and Atwood 1989) do not coincide with this argument. The EPSP of tonic axon 6 is usually <1 mV at 1 Hz, and facilitates to 4-10 times that amplitude at 10 Hz. The quantal content ranges from ~0.2 to 0.7 in intermediate to large animals at 1 Hz (Lnenicka and Mellon 1983), and can be inferred to facilitate at 10 Hz to values of 1-7. The phasic axon, examined in a high-Mg2+ solution (as in the present study), exhibits a quantal content averaging 1-2 initially at typical focal recording sites (Mercier and Atwood 1989). This value rapidly declines with further stimulation. Thus typical endings of tonic axon 6, examined at 10 Hz, should be releasing more transmitter per impulse than those of the phasic axon soon after stimulation begins. Despite this, there is no depression at tonic synapses unless mitochondrial function is suppressed. We can conclude that in the abdominal muscles, the larger initial EPSP of the phasic axon arises in large measure from much more profuse innervation, rather than from a proportionately higher output of transmitter at each release site. In terms of transmitter release, the tonic terminals are "working harder" at 10 Hz than the phasic terminals at 5 Hz, and would be metabolically more active. Such a situation would require more mitochondrial ATP production in the tonic axon.

Effects of metabolic inhibitors on synaptic transmission

UNCOUPLERS OF OXIDATIVE PHOSPORYLATION. There is ample evidence to support a presynaptic site of action for some of these drugs. Both DNP and CCCP increase miniature endplate potential frequency at frog neuromuscular synapses (Moffatt and Miyamoto 1988; Molgo and PecotDechavassine 1988). Atwood et al. (1972) demonstrated that DNP interferes with synaptic vesicle recycling and mobilization in acutely stimulated crayfish motor nerve terminals. In the present study, DNP and CCCP both reduced mitochondrial Rh123 fluorescence in tonic flexor motor axons. Azide, like cyanide, inhibits the electron transport chain and depolarizes Vmt (Kauppinen and Nicholls 1986; Zubay 1983), thereby attenuating Rh123 fluorescence of mitochondria in living cells (Johnson et al. 1981; present study). Thus the available evidence supports a presynaptic locus for the induction of synaptic fatigue in tonic axons by DNP, azide, and CCCP (with the latter drug also reducing postsynaptic rin).

Maintenance of synaptic transmission during recurrent activity involves mobilization and recycling of synaptic vesicles (Heuser and Reese 1973); these processes require ATP for their continual operation (Atwood et al. 1972; Lang and Atwood 1973; Llinas et al. 1989). Our findings suggest that DNP and azide both promote synaptic fatigue by inhibiting oxidative ATP production, which might impede vesicle mobilization and recycling (Lang and Atwood 1973). However, other sites of action, not necessarily linked to ATP turnover, must be considered. Inhibitors of oxidative phosphorylation can elicit calcium efflux from mitochondrial (Akerman and Nicholls 1981; Heinonen et al. 1984; Scott et al. 1980) and nonmitochondrial (Jensen and Rehder 1991) stores. Elevated intracellular free calcium arising from intracellular and extracellular sources may, in turn, impair activity of the Na+/K+ pump (Fukuda and Prince 1992; Skou 1957), which would depolarize the axonal membrane potential and impede spike conduction into presynaptic terminals. Eventually, mitochondrial function would also be compromised by sustained buildup of intracellular Ca2+ (Atwood and Lnenicka 1992). In the tonic motor axon supplying the crayfish opener muscle, both ouabain (Wojtowicz and Atwood 1985) and DNP (Acosta-Urquidi 1980) decrease spike amplitude, blocking conduction. DNP also broadens the presynaptic action potential (Acosta-Urquidi 1980), leading to increased transmitter release and more rapid synaptic depression in the absence of vesicular recycling.

The smaller volume (and larger surface-to-volume ratio) of phasic terminals could promote more rapid intracellular Ca2+ concentration buildup during sustained stimulation, leading to calcium overload of mitochondria (Atwood and Lnenicka 1992) and subsequent synaptic depression. Calcium imaging of phasic and tonic terminals in the leg extensor muscle has directly demonstrated this differential rate of calcium uptake (Msghina et al. 1995).

For IAA, Acosta-Urquidi and Atwood (1977) have shown that acute application of DNP and IAA, at the same concentrations used in the present study, produces greater synaptic fatigue in crayfish tonic motor axons than DNP per se. This finding suggests that IAA can enter the terminal and inhibit glycolysis, but will induce synaptic depression only when additional inhibition of oxidative metabolism (by DNP) has been effected. Also, even after 40 min of exposure to IAA, mitochondrial oxidative activity (as measured with Rh123 fluorescence) was not significantly attenuated in tonic flexor axons (Fig. 2E). Thus neither of these drugs had any demonstrable effects on either synaptic stamina or mitochondrial fluorescence in tonic axons. This is consistent with the hypothesis that mitochondrial oxidative phosphorylation (but not nonoxidative metabolism) is essential for sustainingsynaptic transmission during stimulation.

Assessment of Rh123 as a supravital probe for oxidative activity in living neurons

Electron transport contributes to Vmt (approximately -150 mV, inside negative), which drives ATP synthesis (Mitchell 1966; Racker and Stoeckenius 1974) and is linearly related to Rh123 fluorescence (Emaus et al. 1986). Protonophores that dissipate and depolarize Vmt (e.g., DNP and CCCP) attenuate the normal punctate fluorescence pattern of prestained mitochondria in living cells, and induce release of Rh123 into the cytosol (Chen et al. 1981; Johnson et al. 1981). The present findings (Fig. 2) are consistent with these observations: DNP, CCCP, and azide (an electron transport inhibitor) all reversibly released Rh123 from mitochondria, thereby attenuating the punctate pattern of mitochondrial fluorescence.

In various cell types, mitochondrial Rh123 fluorescence is graded according to cellular activity levels. For cultured mouse bladder epithelial cells, those cells present at the migratory leading edge show higher Rh123 fluorescence than neighboring stationary cells (Johnson et al. 1981). Human fibroblasts show higher Rh123 fluorescence during growth; a decrease in oxygen consumption during dormancy is accompanied by a reduction in such fluorescence (Goldstein and Korczack 1981). Contracting cardiac muscle cells fluoresce more intensely than noncontracting cells in the same culture dish (Johnson et al. 1981). Our results are consistent with these and other studies, because tonically active motor axons, which are known to experience prolific amounts of impulse activity (Atwood and Wojtowicz 1986; Atwood et al. 1991; Hill and Govind 1983), showed higher mitochondrial Rh123 fluorescence than the less active phasic motor axons.

Possible links between neuronal impulse activity, mitochondrial metabolism, and mitochondrial content

Imposed alterations in impulse activity can modify oxidative activity in the brain (Hevner and Wong-Riley 1990). Tonically active motor neurons may possess higher basal levels of oxidative activity in the soma (Sickles and Oblak 1984), and thus higher rates of electron transport and larger Vmt values (Chen 1988, 1989).

Tonically active neurons would require more energy to maintain both ionic gradients and the operation of numerous homeostatic processes (Erecinska and Silver 1989). The higher mitochondrial content in tonic axons is thus consistent with an activity-dependent coupling between energy demand and supply, and mirrors parallel results obtained from the crayfish claw closer's tonic and phasic motor axons (Case and Lnenicka 1992). The fact that tonically stimulated phasic axons acquire a higher level of mitochondrial fluorescence (Nguyen and Atwood 1994) indicates that adjustments occur in individual mitochondria that adapt them for more active metabolism, a necessary component of the adaptive changes in synaptic physiology.

    ACKNOWLEDGEMENTS

  We thank M. Hegström-Wojtowicz for technical assistance and help in preparing the figures. Critical input by Drs. U. De Boni, A. Roach, and P. Pennefather is gratefully acknowledged. We thank Drs. R. Robitaille and R. Cooper for advice and assistance on confocal microscopy and A. Shayan for help in obtaining and analyzing data on mitochondria in axons. We also thank a reviewer for constructive comments that helped to improve this paper.

  This work was supported by a Medical Research Council (MRC, Canada) grant to H. L. Atwood and by National Sciences and Engineering Council (Canada) and MRC (Canada) Predoctoral Fellowships to P. V. Nguyen.

    FOOTNOTES

   Present address of P. V. Nguyen: Centre for Neuroscience Research, Montreal General Hospital Research Institute, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada.

  Address for reprint requests: H. L. Atwood, Dept. of Physiology, University of Toronto, Faculty of Medicine, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

  Received 25 April 1996; accepted in final form 19 March 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society