Activity-dependent differences in the mitochondrial density of crayfish phasic and tonic motor axons

18 G.A. LNENICKA ET AL.THE JOURNAL OF EXPERIMENTAL ZOOLOGY 280:18–27 (1998)

© 1998 WILEY-LISS, INC.

JEZ 890

Activity-Dependent Differences in theMitochondrial Density of Crayfish Phasicand Tonic Motor Axons

G.A. LNENICKA,* C.J. CASE, AND J.L. TRAVISNeurobiology Research Center, Department of Biological Sciences, StateUniversity of New York, Albany, New York 12222

ABSTRACT We examined whether impulse activity influences the density of mitochondria inthe motor axons innervating the crayfish claw closer muscle. Mitochondria were compared in thephasic, tonic, and chronically stimulated phasic motor axons. Tonic axons had twice the mitochon-dria density (total mitochondria per cross-sectional area [CSA] per axon CSA) than did phasicaxons. Although individual mitochondria in phasic and tonic axons were cylindrical and similarlysized, the tonic axons had twice the number of mitochondrial per axon CSA of phasic axons. Dif-ferences in mitochondrial density included differences in transported mitochondria: the density ofmitochondrial transport (total length of motile mitochondria per volume axoplasm) was approxi-mately 50% greater in the tonic axon compared to the phasic axon. Motile mitochondria compriseda small proportion of the total axonal mitochondrial volume because the majority (60–70%) ofmitochondria were stationary, and these stationary mitochondria were considerably longer thanthe motile ones. The differences in axonal mitochondrial density were activity-dependent. We ob-served a 50% increase in the density of mitochondria in phasic axons that had been electricallystimulated for 7 days. The increase in mitochondrial density appears to result from an increase inboth mitochondrial length and number. These results show that tonic axons have a greater den-sity of both transported and stationary mitochondria, and the differences in mitochondrial densityare activity dependent. J. Exp. Zool. 280:18�27, 1998. © 1998 Wiley-Liss, Inc.

Impulse activity and mitochondrial density areclearly related in identified motor terminals ofarthropods. Tonically active motor terminals havea greater density of mitochondria than theirphasically active counterparts in crab (Atwood andJohnston, ’68), lobster (Hill and Govind, ’81), cray-fish (Lnenicka et al., ’86), locust (Titmus, ’81), andmoth (Rheuben, ’84). Given the important roleplayed by mitochondria in energy metabolism andcalcium buffering (Miller, ’91), these differencesare likely to be related to the different transmit-ter-releasing properties of phasic and tonic motorterminals (Lnenicka et al., ’86; Nguyen andAtwood, ’94). Mitochondrial density in these mo-tor terminals is activity dependent. When a pha-sic motoneuron was electrically stimulated for 2wk, the mitochondrial density in its synaptic ter-minals increased to levels similar to those of atonic motoneuron (Lnenicka et al., ’86).

It is not known whether these activity-depen-dent differences in mitochondria are specific to themotor terminals or occur throughout the motoneu-ron. Motor terminals represent only a small frac-tion of total motoneuron volume and are not

necessarily representative of the entire cell. Infact, mitochondrial distribution is different inaxons and terminals: mitochondria often appearconcentrated at the presynaptic terminal, presum-ably because of its high energy and calcium-buff-ering requirements.

Increased mitochondrial density in stimulatedmotor terminals could reflect an activity-depen-dent increase in mitochondrial density through-out the neuron. This seems reasonable becausemitochondrial content has been shown to respondto changes in metabolism in a variety of cell types(Smith and Ord, ’83). In the mammalian visualsystem, neuronal mitochondria undergo activity-dependent changes; blocking the impulse activityin monkey retinal cells for 2 wk results in a de-crease in the mitochondrial density in neuronalcell bodies of the striate cortex (Wong-Riley et al.,

Grant Sponsors: NSF grants IBN-9511558 (G.A.L.) and MCB-9505855 (J.L.T.).

*Correspondence to: Gregory Lnenicka, Neurobiology Research Cen-ter, Department of Biological Sciences, State University of New Yorkat Albany, Albany, NY 12222. E-mail: [email protected]

Received 15 May 1997; accepted 22 August 1997.

MITOCHONDRIA IN CRAYFISH MOTOR AXONS 19

’89). Alternatively, more mitochondria could bepartitioned into stimulated motor terminals with-out an increase in the mitochondrial density ofthe axon. After all, axonal mitochondria move to-ward regions of greatest energy demand (Morrisand Hollenbeck, ’93). In fact, 3 days of electricalstimulation failed to produce a significant increasein the mitochondrial density of crayfish motoraxons (Nguyen and Atwood, ’94); however, this pe-riod of stimulation may have been too brief to pro-duce mitochondrial changes.

It is possible that impulse activity levels affectthe axonal transport of mitochondria. Crustaceanaxons include both stationary and transportedmitochondria (Forman et al., ’87). The transportedmitochondria are believed to supply the axon andterminals (Grafstein and Forman, ’80). Differencesin the mitochondrial density of the axon and/orterminals may be accompanied by differences inthe axoplasmic transport of mitochondria.

To address these questions, we have examinedthe distribution of motile and stationary mitochon-dria in the phasic and tonic axons innervating thecrayfish claw closer muscle. We also examined themitochondria in the phasic axon after chronicstimulation to determine the role of impulse ac-tivity in regulating mitochondrial density. Thesephasic and tonic axons are easily identified, andtheir well-documented differences in impulse ac-tivity levels (Pahapill et al., ’85) can be alteredfor prolonged periods (Lnenicka and Atwood, ’85).In addition, the terminals of these axons havewell-characterized, activity-dependent differencesin mitochondrial density. Because these axons con-tain most of the motoneuron volume, measure-ments of axonal mitochondrial density provide agood estimation of total cellular mitochondria.

We report that tonic axons have a greatermitochondrial density than do the phasic axons,including a greater density of transported mito-chondria. These differences in mitochondrial den-sity are activity dependent: chronic stimulationof the phasic axon for 1 wk results in an increasein the density of mitochondria in the axon.

MATERIALS AND METHODSIsolation of the phasic and tonic axons

Crayfish, Procambarus clarkii, were obtainedfrom Atchafalaya Biol. Supp. (Raceland, LA), andmaintained on a mixed diet. Crayfish with cara-pace lengths 2.5–3.0 cm and claw lengths 1.2–1.5cm were used in studies of the mitochondrial den-sity of fixed axons and in the studies of the effect

of chronic stimulation. Young crayfish were usedsince they show activity-dependent changes inmotor terminal mitochondria (Lnenicka et al., ’86).Studies of axoplasmic transport were performedupon larger crayfish (carapace lengths 4.3–5.8 cmand claw lengths 3.3–5.4 cm) because their clawnerves were longer, allowing isolation of a greaterlength of axon.

For examination of the closer axons, claws wereremoved from cooled animals. The dorsal exoskel-eton of the claw was removed before securing theclaw in a dissecting dish. For the examination offixed mitochondria, the axons were fixed in situwith 2.5% glutaraldehyde in 0.05 M PBS with 5%sucrose (pH 7.4) for 2 hr at room temperature.After fixation, the tissue was washed in 0.05MPBS with 5% sucrose and remained in this solu-tion for the remainder of the dissection. We lo-cated the phasic and tonic axons at the pointwhere they entered the closer muscle and thenremoved the adjoining sensory bundle. The fixedmotor bundle was then dissected from the claw.Living axons were isolated in a similar mannerexcept that the dissection was performed in VanHarreveld’s solution (Van Harreveld, ’36) bufferedwith 10 mM HEPES (pH 7.4).

In vivo stimulation of the phasic axonThe phasic axon was chronically stimulated in

vivo using implanted electrodes as previously de-scribed (Lnenicka et al., ’86). Stimulation was ap-plied at 5 Hz for 2 hr per day for 7 consecutivedays. One day after the final stimulation, clawswere removed for examination of the phasic andtonic axons.

Examination of mitochondria in fixed axonsIn order to examine the mitochondria, we used

both differential-interference-contrast-microscopy(DIC) and transmission electron microscopy(TEM). We used DIC to section optically alongthe length of the axon to determine the mitochon-drial length, whereas cross-section electron mi-crographs were used to determine mitochondrialdiameter and density.

After fixation, the axons were placed upon a slideand viewed with an Olympus BH-2 microscope us-ing DIC and an Olympus 100× (N.A. 1.25) objec-tive. As previously described (Smith, ’71), axonalmitochondria can be visualized by DIC where theyappear as cylindrical structures. A region of the axonwas randomly selected, and the mitochondria in afocal plane centered in the vertical axis were ex-amined in adjacent regions of the phasic and tonic

20 G.A. LNENICKA ET AL.

axons. Camera lucida drawings were made of allelongated structures that crossed this focal plane.The total length of individual mitochondria wasdetermined by including the regions that extendedabove or below the central plane of focus.

For electron microscopy, the axons were fixedas described above, then postfixed in 2% osmiumtetroxide, dehydrated, and embedded in an Epon-Araldite mixture. Ultrathin sections were mountedon single-slot Formvar-coated grids, poststainedwith uranyl acetate and lead citrate, and viewedeither on a Philips 201 or Zeiss EM 902 transmis-sion electron microscope operated at 80 Kv.

Examination of mitochondriain living axons

As in previous studies (Forman et al., ’87), mi-tochondria were visualized in living axons usingAllen video-enhanced contrast-DIC (AVEC-DIC,Allen et al., ’81). After isolation, the tonic and pha-sic axons were positioned on a coverslip cationizedwith alcian blue to improve adhesion (Sommer,’77). The coverslip was attached to a gravity flowperfusion chamber (Travis and Allen, ’81), and VanHarreveld’s solution (Van Harreveld, ’36) contain-ing 1 mM glucose and 10 mM HEPES (pH 7.4)was continuously perfused at 20°C.

The axons were viewed with a Zeiss IM-35 in-verted microscope equipped with high-resolutionDIC optics and a Zeiss 63× (1.4 N.A.) lens. Thelight from a 50-W mercury arc lamp was passedthrough serial u.v. and heat cut filters and a 546-nm filter to minimize specimen irradiation dam-age. The image was enhanced with a HamamtsuDVS 3000 digital image processor using back-ground subtraction, contrast-maximization, and arolling four-frame average to reduce noise. Imageswere recorded on ¾-inch videotape and subse-quently analyzed.

A region of the axon equidistant between thetwo cut ends was selected for analysis. Becausethe axons were approximately 3 cm long, mito-chondrial transport was less than 4 mm per hr,and all measurements were performed within 1hr of the dissection, the cut ends were unlikely todirectly influence the measurements of transport.Mitochondria were viewed in the central plane offocus in adjacent regions of the phasic and tonicaxons. Mitochondrial movements were taped forat least 10 min in each axon.

The mitochondrial flux was measured by count-ing the number of mitochondria that moved acrossa line transecting the axon. As each mitochondriacrossed the line, its contour length was measured

directly from the video screen. To compare mito-chondrial velocity in phasic and tonic axons, wedetermined the time required for mitochondria tomove a distance of 10 µm along the longitudinalaxis of the axon. All measurements were per-formed by an individual unaware of the identityof the axon.

Statistical comparisons were performed usingindependent or paired t-tests. All values representthe mean ± SE.

RESULTSThe motor bundle that innervates the crayfish

claw closer muscle contains three axons (Fig. 1):the largest of these axons is the phasic excitor;the intermediate-sized axon is the tonic excitor;and the smallest one is the inhibitor (Van Harre-veld and Wiersma, ’36). As shown in Table 1 the

Fig. 1. Isolated motor bundle containing the motor axonsthat innervate the crayfish claw closer muscle. The motorbundle was dissected free from the claw nerve and viewedwith DIC. Normally, the tonic (T) and phasic (P) axons canbe clearly identified based upon their characteristic differ-ences in size. The smallest axon positioned on the left is theinhibitor (I). Preparation was viewed with an Olympus 10×(N.A. 0.3) objective. Calibration: 30 µm.


diameter of the larger phasic axon was approxi-mately 30% greater than that of the tonic axon.

Mitochondria in phasic and tonicmotor axons

Using DIC microscopy, mitochondria in crayfishmotoneurons appear as dense, elongated rods (Fig.2) similar to those of lobster motoneurons (Formanet al., ’87). We examined a total of 3,246 mito-chondria in 17 pairs of phasic and tonic axons.The length of individual mitochondria ranged fromless than 5 µm to more than 100 µm, and nonewere branched. The mean mitochondrial lengthwas similar in phasic and tonic axons (Table 1).The apparent width of the mitochondria was tooclose to the resolution limit of the light microscopefor reliable measurements; therefore, mitochondrialdiameter was measured in electron micrographs ofpaired phasic and tonic axon cross-sections (Fig. 3).The diameters were similar in the phasic and tonicaxons (Table 1) and consistent with those previ-ously reported for lobster motor axons (Formanet al., ’87). Thus, the size of individual mitochon-dria in phasic and tonic axons did not differ.

The mitochondrial density was examined inelectron micrographs from axon cross-sections(Fig. 3). For each of the four axon pairs exam-ined, the tonic axon had more mitochondrial pro-files per CSA of axon than the phasic axon (Fig.4). Overall, the mean value for the tonic axon wasapproximately twice that of the phasic axon (Table1). Because the mitochondrial lengths are thesame for the two axon types, the tonic axons musthave twice the number of mitochondria per vol-ume of axoplasm as do the phasic axons. Thesedifferences in mitochondria number resulted in a

TABLE 1. Axon diameters and mitochondrial measurements in fixed axons using DIC and TEM1

Mitochondria perIndividual mitochondria 100 µm2 axon CSA

Axon Length Diameter Total CSAdiameter (µm) (µm) (µm) Number (µm2)

Control clawPhasic axon 40.5 ± 2.3 14.0 ± 0.6 0.19 ± 0.01 8.8 ± 0.7* 0.25 ± 0.02*Tonic axon 31.5 ± 1.6 15.4 ± 0.6 0.20 ± 0.01 16.7 ± 2.0 0.53 ± 0.08

n = 21 n = 17 n = 4 n = 4 n = 4Stimulated clawPhasic axon 41.1 ± 4.1 20.4 ± 1.6* 0.18 ± 0.01 15.2 ± 2.1 0.38 ± 0.06Tonix axon 30.6 ± 2.9 16.4 ± 1.3 0.18 ± 0.01 20.7 ± 4.3 0.47 ± 0.02

n = 11 n = 7 n = 4 n = 4 n = 41All measurements were performed in pairs of phasic and tonic axons. Values for n represent number of animals. The mitochondrial mea-surements were compared for the phasic and tonic axons using a paired t-test. For the control claw diameters were measured in a total of 690mitochondria and lengths were measured in a total of 3,246 mitochondria. For the stimulated claw diameters were measured in a total of 632mitochondria and lengths were measured in a total of 569 mitochondria.*Significantly different than the accompanying tonic axon; P < 0.05, paired t-test.

Fig. 2. Mitochondria visualized in a tonic axon after fixa-tion. The elongated structures (mitochondria) can be resolvedin the fixed tissue with DIC. In order to quantify the lengthof the mitochondria in the two axons, mitochondria withinan optical section equidistant between the axon top and bot-tom were drawn using camera lucida. The mitochondria inthe phasic and tonic axons were similar in length. Prepara-tion was viewed with an Olympus 100× (N.A. 1.25) objective.Calibration: 6 µm.


twofold greater total CSA of mitochondria per unitaxon CSA in the tonic axon compared to the pha-sic axon (Table 1).

Differences in mitochondrial transport inphasic and tonic axons

AVEC-DIC allowed us to visualize and recordaxoplasmic transport of mitochondria (Fig. 5). Be-cause of the uniform diameters of axonal mito-chondria, we determined mitochondrial flux as thetotal contour length of mitochondria traveling ineither anterograde or retrograde directions. Allvalues were standardized to express mitochondrialflux occurring across a 10 µm width of axon.

As shown in Figure 6, the mitochondrial flux inthe tonic axon (4.0 ± 0.5 µm mitochondrial length/min) is significantly greater than in the phasicaxon (2.7 ± 0.6 µm mitochondrial length/min, n =7 axon pairs; P < 0.02, paired t-test). For bothphasic and tonic axons, mitochondrial flux wasgreatest in the anterograde direction. The meanlength of the individual transported mitochondriawas similar in the tonic (1.7 ± 0.1 µm) and phasic

axons (1.5 ± 0.2 µm, n = 7 axon pairs; P > 0.10,paired t-test). Thus, the observed differences inmitochondrial flux were likely due to differencesin the number of mitochondria being transportedin tonic (2.4 ± 0.2 mitochondria/min) and phasicaxons (1.8 ± 0.3 mitochondria/min, n = 7 axonpairs, P > .05, paired t-test), although the differ-ence was not significant.

The greater flux of mitochondria in tonic axonsresults from the presence of more motile mito-chondria per volume of axoplasm rather thanmore rapid movement of mitochondria along theaxon. Mitochondrial velocity was similar in thephasic (0.67 ± 0.04 µm/sec) and tonic axons (0.65± 0.05 µm/sec, n = 7 axon pairs; P > 0.10, pairedt-test). In general, these velocities are in agree-ment with earlier studies performed in lobsteraxons (Forman, ’87; Forman et al., ’87).

The mean length of transported mitochondria(1.6 ± 0.1 µm, n = 14 axons) was considerably lessthan the length of mitochondria measured in fixedtissue (14.7 ± 0.4 µm, n = 34 axons; P < 0.0001, t-test), indicating that transported mitochondria are

Fig. 3. Representative electron micrographs of mitochon-drial cross-sections from the periphery of a phasic and tonicaxon. Mitochondria were measured in montages of paired pha-sic and tonic axons from four animals. Although there was

no difference in mitochondrial diameter, the density of mito-chondria was significantly greater in the tonic axon. Arrowspoint to 1 of 11 mitochondrial profiles in the tonic axon and 1of 5 profiles in the phasic axon. Calibration: 1 µm.


significantly shorter than stationary mitochondria.This was directly examined in living axons bymeasuring the length of the mitochondria that re-mained stationary during a 10-min period. Themajority of mitochondria in the tonic (64.9% SD= 19.5, n = 7 axons) and phasic axons (67.1% SD= 16.6, n = 7 axons) remained stationary duringthis period. These stationary mitochondria (7.1 ±1.0 µm, n = 14 axons) were significantly longerthan the transported mitochondria reported above(P < 0.001, paired t-test). The stationary mitochon-drial length likely was an underestimation: 20%of the stationary mitochondria extended beyondthe video screen and their total length was notmeasured.

Activity-dependent increase in themitochondrial density of the phasic axonTo determine whether the differences in the mi-

tochondria in the phasic and tonic axons were ac-tivity dependent, the phasic axon was electricallystimulated in vivo at 5 Hz for 2 hr per day for 7days. This stimulation procedure produced a dra-matic increase in its normal impulse activity(Pahapill et al., ’85). After 1 wk of stimulation,both phasic and tonic axons from the stimulatedclaw were examined using DIC and TEM.

Mitochondrial values for the tonic and phasicaxons in the stimulated claw are presented inTable 1. In general, stimulation increased the den-sity of mitochondria in the phasic axon to levelssimilar to those of the tonic axon. After stimula-tion, the mitochondrial diameter in the phasic didnot change, although mitochondria became sig-nificantly longer in phasic axons compared totonic axons.

Data from the control and stimulated claws arecombined in Figure 7 in order to compare directlymitochondrial measurements from stimulatedphasic axons, tonic axons, and control phasicaxons. The aggregate mitochondrial CSA per 100µm2 axon CSA was significantly greater in thetonic axon (0.50 ± 0.04, n = 8; P < 0.002, t-test)and stimulated phasic axon (0.38 ± 0.06, n = 4, P< 0.05, t-test) than the control phasic axon (0.25± 0.05, n = 4). The number of mitochondria per100 µm2 axon CSA was significantly greater inthe tonic axon (18.7 ± 2.3, n = 8; P < 0.02, t-test)and stimulated phasic axon (15.2 ± 2.1, n = 4; P <0.02, t-test) than the control phasic axon (8.8 ±0.6, n = 4). This increase in mitochondrial den-sity produced by stimulation of the phasic axonappeared to be due in part to an increase in mito-chondrial length (Fig. 7). The length of mitochon-

Fig. 4. Mitochondria were examined with TEM in axonpairs from four animals. Measurements were made from mon-tages of phasic and tonic axon cross-sections. The numberand total CSA of mitochondria per axon was determined andexpressed per 100 µm2 of axon CSA. Top: In all four axonpairs, the tonic axon had a greater number of mitochondriaper unit axon CSA. Bottom: In all four axon pairs, the totalmitochondrial CSA per unit axon CSA was greater for thetonic axon.


Fig. 5. Visualization of axoplasmic transport of organellesin a tonic axon using AVEC-DIC. Four organelles travelingin the anterograde direction are viewed in the central regionof the axon. Left: Three elongated organelles (arrows), whichwere classified as mitochondria, and one spherical organelle

(asterisk) are shown. Right: Subsequently (7 sec later), themitochondria and the vesicle have moved, whereas the twolong mitochondria that extend beyond the bottom of the screenremain stationary. Preparation was viewed with a Zeiss 63×(N.A. 1.4) objective. Calibration: 2 µm.

Fig. 6. The mitochondrial flux was compared in the pha-sic and tonic axons. The mitochondrial flux, total contourlength of mitochondria traveling across a line transecting theaxon, was determined for paired phasic and tonic axons. Val-ues were expressed as total mitochondrial length transportedper 10 µm axon width per minute. In six of seven animals,there was a greater total length of mitochondria transportedin the tonic axon compared to the phasic axon.

dria in the stimulated phasic axon (20.4 ± 1.6, n= 7; P < 0.002, t-test) was significantly greaterthan in the control phasic axon (14.0 ± 0.6, n =17). Because the magnitude of the increase inlength is not sufficient to account for the increasein the number of mitochondrial profiles, it appearslikely that there is also an increase in the num-ber of mitochondria per volume axoplasm as a re-sult of stimulation.

DISCUSSIONMitochondrial density of phasic

and tonic axonsTonic axons have approximately twice the total

mitochondrial volume per volume axoplasm thatphasic axons do because tonic axons have agreater number of mitochondria. This differencein axonal mitochondrial density affects a largepart of the neuron as these motor axons containapproximately 98% of total cell volume based uponestimates of soma diameter, axon length and CSA,and terminal length and CSA (Lnenicka et al., ’86;Lnenicka et al., ’91). The somata of crustaceanneurons also show differences in mitochondrialdensity; e.g., the somata of lobster tonic sensoryneurons have a greater volume of mitochondriathan their phasic counterparts. Similarly, the so-mata of tonically active mammalian motoneuronshave a greater level of mitochondrial enzyme ac-


tivity than do their phasic counterparts (Sicklesand Oblak, ’84).

In the crayfish, as in the lobster (Forman et al.,’87), the majority of the mitochondria (60–70%)are stationary. Stationary mitochondria appear tobe anchored in the axon’s cortical cytoskeleton(Forman et al., ’87; Lin et al., ’90). The stationarymitochondria are considerably longer than thetransported ones; therefore, differences observedin the mitochondrial density of phasic and tonicaxons can be largely attributed to the number ofstationary mitochondria.

The greater density of stationary mitochondriamay reflect greater energy demands of the toni-cally active axon. Most of the energy productionin neurons is used to operate the Na+/K+ pump(Di Rocco et al., ’89) and the energy demands ofthe tonic axon, which fires about 6,000 impulses/hr, should be larger than that of the phasic axon,which fires only about 1 impulse/hr (Pahapill etal., ’85). This difference is accentuated by the 25%smaller diameter of the tonic axon, which providesit with a greater surface to volume ratio than thephasic axon. Reduced energy metabolism in-creases susceptibility to excitotoxicity (Beal, ’92),suggesting that the greater resistance of tonicaxons to calcium neurotoxicity (Atwood andLnenicka, ’92; Arcaro and Lnenicka, ’97) reflectstheir higher mitochondrial activity.

Mitochondrial measurements in the claw closeraxons can be directly compared to those made pre-viously in the motor terminals (Lnenicka et al.,’86). The percentage of axoplasm occupied by mi-tochondria is greater in tonic (6.9%) than phasic(4.5%) terminals. The percent of axoplasm occu-pied by mitochondria for the tonic (0.53%) andphasic axons (0.26%) is much lower than that ofthe terminals; however, the percent difference be-tween phasic and tonic axons are similar to thatobserved for the terminals. Thus, the degree to

Fig. 7. A comparison of mitochondria in the tonic, stimu-lated phasic and control phasic axons. The data from Table 1were plotted to compare directly the tonic and stimulatedphasic axons to the control phasic axon. Values for the tonicaxons from control and stimulated claws were combined. Top:

The mitochondrial size was compared in the three classes ofaxons. Upper: The mitochondrial length in stimulated pha-sic and tonic axons was significantly greater than in the con-trol phasic axon. Lower: The mitochondrial diameter wassimilar for all three axons. Bottom: The mitochondria perunit axon CSA were compared in the three axons. Upper:Both the stimulated phasic and tonic axons had a signifi-cantly greater numerical density of mitochondria than thecontrol phasic axon. Lower: Both the stimulated phasic andtonic axons had a significantly greater areal density of mito-chondria than the control phasic axon. Numbers in paren-theses represent number of axons. Values from the stimulatedphasic and tonic axons were compared to the control phasicaxon using a t-test: *P < 0.05; **P < 0.02; ***P < 0.002.


which mitochondria are selectively partitioned intothe terminals is similar for phasic and tonic axons;differences in mitochondrial density in tonic andphasic motor terminals simply reflect the differ-ences observed throughout the neuron.

The characteristics of the individual mitochon-dria differed in the synaptic terminals, but notthe axons of the phasic and tonic motoneurons.The CSA of individual mitochondria was previ-ously shown to be greater in the tonic terminals(0.0672 µm2) than in the phasic terminals (0.0310µm2). In contrast, the mitochondria in phasic andtonic axons were similar in CSA (0.0284 µm2) aswell as length. Axonal and terminal mitochondriaare shaped differently. Branched mitochondriawere observed in the motor terminals, but not inthe axon.

Mitochondrial transport in phasic andtonic axons

The total length of mitochondria undergoingtransport per volume of axoplasm is greater inthe tonic axon than the phasic axon because of dif-ferences in the number of transported mitochon-dria. It is likely that mitochondria transported fromthe cell body play a role in supplying the remain-der of the neuron because mitochondria dependupon cytoplasmic synthesis for most of their pro-teins and lipids (Schatz, ’79). The transport of newlysynthesized, nuclear-encoded mitochondrial proteinsto axons and terminals occurs within mitochondria(Liu and Wong-Riley, ’94).

Transported mitochondria could replace exist-ing mitochondria or fuse with stationary mito-chondria to resupply them with necessarycomponents (Grafstein and Forman, ’80). Mito-chondria undergoing axoplasmic transport areshorter than most of the mitochondria in theaxon or terminals. This suggests that mitochon-dria transported to the axon or terminals fuse toform stationary mitochondria, and stationary mi-tochondria divide or fragment before being trans-ported back to the cell body. The greater densityof transported mitochondrial in the tonic axonwould be consistent with its greater requirementfor mitochondrial renewal.

Activity-dependent changes inmitochondrial density

Axonal mitochondrial density is activity depen-dent. In vivo stimulation of the phasic motoneu-ron over a period of 7 days resulted in an increasein the density of mitochondria in the axon. Theincreased mitochondrial density appears to have

resulted from an increase in both mitochondriallength and number.

Our findings are consistent with those of Wong-Riley et al. (’89), which showed a decrease in themitochondrial density in the soma of monkey stri-ate cortex neurons after chronic application of TTXto the retinal cells. This decrease in mitochondrialdensity presumably results from a decrease in theimpulse and/or synaptic activity of these corticalcells. Nguyen and Atwood (’94) did not observean increase in axonal mitochondrial density whencrayfish abdominal phasic extensor motoneuronswere stimulated for 3 days; however, there ap-peared to be an increase in the oxidative compe-tence of existing mitochondria. It may be that thisstimulation period was not long enough to pro-duce an increase in mitochondrial biogenesis. Theresponse to elevated impulse activity could includean initial enhancement of oxidative activity fol-lowed by an increase in mitochondrial volume.

It may seem surprising that the mitochondriallength in stimulated phasic axons is greater thanthat of both tonic and phasic axons; however, thisincrease appears consistent with the observationthat stimulation of the phasic axon increases thenumber of branches per mitochondrion in the mo-tor terminals (Lnenicka et al., ’86). It may be thatthe activity-induced increases in length and thenumber of branches both result from increasedfusion of transported mitochondria to stationarymitochondria as discussed above. These activity-induced mitochondrial changes may representtransient structures that develop initially duringa period of increased mitochondrial transport. Fur-ther studies will be necessary to determinewhether increased impulse activity causes an in-crease in the number of transported mitochondria.However, this seems likely given that the greatermitochondrial density of the tonic axon includesmore transported mitochondria.

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Activity-dependent differences in the mitochondrial density of crayfish phasic and tonic motor axons