Download Organelle motility and metabolism in axons vs dendrites of cultured

Journal of Cell Science 109, 971-980 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS7074
971
Organelle motility and metabolism in axons vs dendrites of cultured
hippocampal neurons
Caroline C. Overly, Heather I. Rieff and Peter J. Hollenbeck*
Department of Neurobiology and Program in Neuroscience, Harvard Medical School, 220 Longwood Avenue, Boston MA 02115,
USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
Regional regulation of organelle transport seems likely to
play an important role in establishing and maintaining
distinct axonal and dendritic domains in neurons, and in
managing differences in local metabolic demands. In
addition, known differences in microtubule polarity and
organization between axons and dendrites along with the
directional selectivity of microtubule-based motor proteins
suggest that patterns of organelle transport may differ in
these two process types. To test this hypothesis, we
compared the patterns of movement of different organelle
classes in axons and different dendritic regions of cultured
embryonic rat hippocampal neurons. We first examined
the net direction of organelle transport in axons, proximal
dendrites and distal dendrites by video-enhanced phasecontrast microscopy. We found significant regional
variation in the net transport of large phase-dense
vesicular organelles: they exhibited net retrograde
transport in axons and distal dendrites, whereas they
moved equally in both directions in proximal dendrites. No
significant regional variation was found in the net
transport of mitochondria or macropinosomes. Analysis of
individual organelle motility revealed three additional dif­
ferences in organelle transport between the two process
types. First, in addition to the difference in net transport
direction, the large phase-dense organelles exhibited more
persistent changes in direction in proximal dendrites where
microtubule polarity is mixed than in axons where micro­
tubule polarity is uniform. Second, while the net direction
of mitochondrial transport was similar in both processes,
twice as many mitochondria were motile in axons than in
dendrites. Third, the mean excursion length of moving
mitochondria was significantly longer in axons than in
dendrites. To determine whether there were regional dif­
ferences in metabolic activity that might account for these
motility differences, we labeled mitochondria with the vital
dye, JC-1, which reveals differences in mitochondrial
transmembrane potential. Staining of neurons with this dye
revealed a greater proportion of highly charged, more
metabolically active, mitochondria in dendrites than in
axons. Together, our data reveal differences in organelle
motility and metabolic properties in axons and dendrites of
cultured hippocampal neurons.
INTRODUCTION
(Kiss, 1977; Gorenstein et al., 1985; Gorenstein and Ribak,
1985; Parton et al., 1992). The first step toward an under­
standing of how selective organelle transport influences
neuronal polarity is the identification and characterization of
regional differences in organelle traffic and sub cellular metab­
olism in axonal and dendritic domains of neuronal cells.
Regional differences in cytoskeletal organization are likely
accompanied by organelle transport differences. The majority
of long-distance organelle transport is thought to be achieved
by the active movement of microtubule-associated motor
proteins, such as kinesins and cytoplasmic dyneins, along
microtubule tracks (Brady, 1991; Vallee and Bloom, 1991;
Skoufias and Scholey, 1993). Quantitative electron micro­
scopic studies of microtubules have shown that the organiz­
ation of these tracks is not uniform throughout all neuronal
processes. Axonal microtubules have greater maximum
lengths and closer spacing than those in dendrites (Wuerker
Neurons are highly polarized cells with distinct axonal and
dendritic domains whose integrity is critical to their proper
functioning. A fundamental question in cellular neurobiology
is how neurons establish and maintain these different domains,
specifically how they achieve the necessary cytoplasmic com­
partmentation and how they meet regional metabolic demands.
Selective and spatially regulated organelle transport seems
likely to play an important role in the establishment and main­
tenance of the known structural, functional and biochemical
differences between axons and dendrites. However, virtually
nothing is known about regional differences in organelle
traffic. Our knowledge of organelle transport in neurons stems
largely from a rapidly growing body of biochemical and bio­
physical data, in vitro motility analyses, and from studies of
axons. Only a few studies have addressed dendritic transport
Key words: Organelle motility, Microtubule polarity, Axon,
Dendrite, Mitochondrion
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C. C. Overly, H. I. Rieff and P. J. Hollenbeck
and Kirkpatrick, 1972; Bray and Bunge, 1981; Abelson, 1984;
Burton, 1987; Chen et al., 1992; Yu and Baas, 1994). In
addition, the polarity orientation of microtubules differs in
different process regions. In axons, these polar protein
polymers are uniformly oriented with their plus-ends directed
distally (Burton and Paige, 1981; Heidemann et al., 1981; Baas
et al., 1988). In dendrites, including those of the frog mitral
cells (Burton, 1988), cultured sympathetic neurons (Baas et al.,
1991), and cultured hippocampal neurons (Baas et al., 1988,
1989), microtubules are of mixed orientation and exhibit
changes in this mixture along their length (Baas et al., 1988).
Within the proximal half of the dendrite, microtubule orienta­
tion is mixed: half of the microtubules have their plus ends
directed distally, the other half their minus ends. With increas­
ing distance from the cell body, the relative abundance of
microtubules with their minus ends directed distally gradually
decreases, with the most distal 10% of dendritic length con­
taining only plus-end distal microtubules, like the axon. These
known differences in microtubule organization and the uni­
directional character of known microtubule-based motors
(Brady, 1991) have prompted speculation about possible dif­
ferences in axonal and dendritic organelle transport (Black and
Baas, 1989; Peters et al., 1991).
Differences in organelle transport in axons and dendrites are
also likely to be influenced by differences in local metabolic
demands within neuronal cells. It is known that regional
metabolic needs within cells influence organelle traffic, and
that changes in such local needs result in new patterns of
organelle transport and distribution (e.g. Fawcett, 1981; Morris
and Hollenbeck, 1993). But how the transport and localization
of a class of organelles is related to subcellular metabolic needs
remains poorly understood. In two studies organelle transport
has been shown to correspond to presumed subcellular
metabolic needs in neurons. Mitochondria are known to be
driven toward and accumulate in active axonal growth cones,
which are probable regions of high ATP consumption, and to
evacuate inactive growth cones (Morris and Hollenbeck,
1993). In addition, autophagic vacuoles exhibit an increased
rate of formation and upregulation of net retrograde axonal
transport when axon outgrowth is blocked (Hollenbeck and
Bray, 1987; Hollenbeck, 1993; Hollenbeck and Weld, 1994).
Before additional similar relationships can be identified, local
metabolism throughout neuronal cells must be further charac­
terized. Although the structural, biochemical, and functional
differences between axons and dendrites strongly suggest that
there must be metabolic differences between these two subcellular domains, such differences have not been demonstrated.
This report represents the first extensive comparative study
of organelle transport and metabolism in axons and dendrites
of neuronal cells. Our data clearly demonstrate differences in
both general organelle traffic and individual organelle motility
behaviors specific to process region as well as organelle type.
Large phase-dense organelles exhibit significant net retrograde
transport in axons and distal dendrites but not in proximal
dendrites. Also, these organelles exhibit more persistent
changes in direction in the proximal dendrites where micro­
tubule polarity is mixed than in axons where microtubules are
uniformly aligned. Although quantitative analysis of mito­
chondrial traffic did not reveal regional differences in their net
transport direction, our data do demonstrate regional variation
in individual mitochondrial motility patterns; axonal mito­
chondria move more frequently and move farther than
dendritic mitochondria. Furthermore, our data definitively
show that there are differences between axonal and dendritic
metabolism; dendritic mitochondria are more metabolically
active than those in axons.
MATERIALS AND METHODS
Materials
Minimum essential medium (MEM), horse serum, Hanks’ buffered
saline solution (HBSS) and trypsin were purchased from Gibco
(Grand Island, NY). All other cell culture reagents were purchased
from Sigma (St Louis, MO). Glass coverslips were purchased from
Carolina Biological Supply Co. (Burlington, NC). Rhodamine 123
was obtained from Molecular Probes, Inc. (Eugene, OR). JC-1 was a
gift from Dr Lan Bo Chen.
Cell culture
Embryonic rat hippocampal neurons were cultured on glass coverslips
using the method of Goslin and Banker (1991). Neurons were main­
tained in glial conditioned neuronal maintenance medium supple­
mented with 10 mM Hepes, pH 7.3, for all fluorescent labeling and
microscopy. Axons and dendrites were distinguished based on wellestablished morphologic characteristics and only spatially isolated
regions and unambiguous processes were selected for observation.
Observations were made at previously characterized developmental
stages (Dotti et al., 1988). Axons were observed in stage 4 cells (4-6
days in vitro). Dendrites were observed in stage 5 cells (8-10 days in
vitro), with some observations of mitochondria made in late stage 4
cells (6 days in vitro). These developmental stages were chosen for 2
reasons: (i) At these stages, the two process types are as mature as
possible without compromising spatial resolution of individual
processes; and (ii) the microtubule polarity orientations are in their
mature states (Baas et al., 1989). Data from different developmental
stages were pooled only when there was no significant difference in
results from the different stages.
Video microscopy
For all experiments, live neurons were observed using a Zeiss IM 35
inverted microscope equipped with a ×63 planapochromatic objective
and maintained at 37°C using an air curtain stage heater. Cultured
neurons were maintained in a sandwich of 2 coverslips separated by
coverslip fragments, filled with culture medium and sealed with a
mixture of Vaseline, lanolin and paraffin (1:1:1). Video-enhanced
phase-contrast images were obtained using a Hamamatsu Newvicon
camera mounted on a video port using relay lenses to obtain a field
width of 47 µm. Rhodamine 123 labeled mitochondria were imaged
using a Hamamatsu intensified CCD video camera mounted in the
ocular position on the microscope, giving a field width of 102 µm.
Images of JC-1 labeled mitochondria were collected with this same
camera mounted on the video port to obtain a field width of 47 µm.
All video sequences were recorded using a Panasonic AG-7300
Super-VHS video cassette recorder.
Motility analysis
Organelles were categorized based on morphological characteristics
seen by video-enhanced phase-contrast microscopy. An organelle was
identified as a mitochondrion if it was phase-dense and had a length
at least twice that of its width, a large phase-dense organelle if it was
phase dark with an apparent diameter of at least 0.4 µm, or a
macropinosome if it were phase bright and at least 0.4 µm in diameter
(Dailey and Bridgman, 1993). Organelles which did not meet any of
these criteria were categorized as ‘other organelles’. Organelle
movement was observed in three neuronal domains: axon shafts
greater than 100 µm from cell bodies or growth cones, the proximal
Axonal vs dendritic organelle motility
half of dendritic length, and the most distal 10% of dendritic length.
Quantitative analysis was done by drawing a line across the video
screen perpendicular to and bisecting the process and counting the
number of organelles that crossed the line during a 4× time lapse
playback of the video sequence. Anterogradely and retrogradely trans­
ported organelles were counted from separate video playbacks, as
were organelles of different classes. Counts were often repeated 2 to
5 times then averaged to ensure accuracy.
Individual motility behaviors of the large phase-dark organelles
were analyzed to characterize changes in direction in axons and
proximal dendrites. To eliminate bias, all large phase-dark organelles
visible in each video sequence were followed for the duration of the
sequence, until they moved out of the plane of focus, or until they
moved out of the video field. An organelle was considered stopped if
it remained stationary for at least 1 second, and a movement was
counted only if the displacement was at least 0.5 µm. All observed
stops were scored, and the direction of resumed movement was
recorded as either the same or changed. A change in direction was
considered persistent if the organelle moved continuously in the new
direction for at least 3 µm or if it continued movement in this new
direction even after one or more subsequent stops. The data from all
observed organelles was pooled to get a total number of stops
observed, and numbers of stops followed: (i) by resumed movement
in the original direction; (ii) by any change in direction; and (iii) by
a persistent change in direction.
Observation of rhodamine 123 labeled mitochondria
To more clearly visualize mitochondria for analysis of individual
organelle motility, neurons were labeled with the mitochondrial vital
dye rhodamine 123 (0.2 µg/ml) for 30 minutes at 37°C, washed once
with HBSS, then returned to culture medium for immediate observa­
tion. For fluorescent images, a 50 W mercury lamp and a 1.0 neutral
density filter were used. For phase images, a tungsten lamp and 1.5
neutral density filter was used. Live sequences were recorded as
running averages of 8 video frames using Image1 image processing
software (Universal Imaging Corporation). A process was recorded
for as long as its fluorescent image remained in focus. Brief phase
images were also recorded so that the type of process and the direction
to the cell body could be determined.
To quantify individual mitochondrial motility behaviors, one
minute video sequences of axons and proximal dendrites were
analyzed. To eliminate bias, the first minute of each video sequence
throughout which all mitochondria remained in focus was used for
analysis, and every mitochondrion in the process was individually
tracked for the full minute. A fluorescent organelle was counted only
if more than one end was visible. For example, a cluster of mito­
chondria with three visible ends was counted as only one mitochon­
drion, and one with four ends as two mitochondria. For quantitative
analysis, three motility characteristics were recorded for each mito­
chondrion observed: (i) whether or not it moved; (ii) its net direction
of movement (anterograde or retrograde); and (iii) its excursion
length (net displacement). Net displacement was measured in µm
using a calibrated ruler taped to the video monitor.
JC-1 labeling and analysis
To label mitochondria for metabolic analysis, neurons were incubated
in medium containing freshly prepared JC-1 (2 µg/ml) for 3 minutes
at 37ºC, washed 2 times in HBSS for 1 minute each, then returned to
normal medium. Cells were observed immediately following labeling
and for no more than 45 minutes. Fluorescent mitochondria were visu­
alized using shuttered tungsten illumination to minimize bleaching.
The fluorescent green and red JC-1-loaded mitochondria were viewed
separately using standard fluorescein and Texas Red filter sets, respec­
tively. MetaMorph imaging software (Universal Imaging Corpor­
ation) was used for all digital image collection and image processing.
Separate images of fluorescent green and red mitochondria were
acquired as digital grey scale averages of 16 video frames. For each
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field, the red image was obtained immediately after the green image
to minimize mitochondrial movements between channels.
For analysis, green/red image pairs were colorized and overlaid,
yielding single images containing green and red mitochondria. Each
labeled mitochondrion was categorized as either green, red or both
green and red. If greater than 90% of a mitochondrion’s area was
green or red, it was counted as green or red, respectively. All other
mitochondria were categorized as both green and red.
Statistical analysis
Organelles observed by phase-contrast microscopy were evaluated for
net transport in one direction using a chi-square test. Data was
compared to an expected distribution of 50:50 for anterograde:retro­
grade movement, and statistically significant differences reflected net
transport in one direction. Comparisons of mean excursion lengths
and of mitochondrial metabolic categories were done using an
unpaired two-tailed t-test. For all other data, the proportions from
different data sets were compared assuming normal distributions and
a null hypothesis of equality. In all cases, differences were considered
statistically significant where P<0.05.
RESULTS
There are differences in overall organelle traffic in
axons vs dendrites
To begin to characterize organelle transport in different process
regions of neurons, we examined general organelle traffic
using quantitative video-enhanced phase contrast light
microscopy. Using this technique, we were able to distinguish
three distinct classes of organelles based on morphologic
criteria (Fig. 1). First, elongated phase dense organelles were
identified as mitochondria. Second, large phase-dense
vesicular organelles were a distinct class and probably
represent autophagic vacuoles since their light microscopic
characteristics and axonal transport properties are similar to
those previously described for neuronal autophagic vacuoles
(Hollenbeck and Bray, 1987; Hollenbeck, 1993). Third,
A
C
B
D
Fig. 1. Four morphologic classes of organelles can be distinguished
by video-enhanced phase-contrast microscopy. Large phase-dense
vesicular organelles (arrow in A), large phase-clear macropinosomes
(arrow in B), and mitochondria (brackets in C) could be
distinguished in video-enhanced phase-contrast images of
hippocampal neuronal processes. A variety of other smaller vesicular
organelles, many of which could only be detected when moving, did
not fall into any of these categories (arrow in D). Bar, 2 µm.
974
C. C. Overly, H. I. Rieff and P. J. Hollenbeck
macropinosomes were identified by their relatively large size
and their characteristic phase-bright properties. All remaining
organelles were too small to resolve clearly and were thus cat­
egorized as other organelles.
To identify regional differences in organelle traffic, the
relative frequencies of anterogradely and retrogradely moving
organelles were determined for each organelle class in different
axonal and dendritic domains. To test the hypothesis that the
organization of the microtubule tracks might influence
organelle transport, this motility was quantified in three
process regions with characteristic microtubule arrangements:
(i) axon shafts; (ii) the distal 10% of dendrites where microtubules are uniformly aligned with all plus ends directed
distally; and (iii) the proximal half of dendrites where micro­
tubule polarity orientation is evenly mixed (Baas et al., 1988).
Our data suggest that the traffic patterns of most organelle
classes are similar in the three process regions (Fig. 2). There
was neither a detectable net anterograde nor net retrograde flow
of the total organelle population in any of the observed
domains. Observations of individual organelle classes revealed
that mitochondria were transported equally in both directions
and macropinosomes exhibited retrograde flow in every
process region examined.
In contrast, other organelle classes did exhibit regional dif­
ferences in trafficking patterns. In axons, the large phase-dense
vesicular organelles exhibit significant net retrograde transport
as shown previously for autophagic vacuoles in axons of sym­
pathetic neurons, with more than twice as many organelles
moving retrogradely as moved anterogradely. A similar pattern
was observed in distal dendrites, where the microtubule polarity
orientation is uniform as it is in axons. In contrast, a different
pattern was observed in proximal dendrites, where the micro­
tubule polarity orientation is mixed. In this process region, the
large phase-dense vesicular organelles were transported equally
in both directions, with no significant difference in the propor­
tions of anterogradely and retrogradely moving organelles.
Another regional transport difference was observed for
organelles categorized as other. These organelles were trans­
ported equally in both directions in both dendritic regions
observed, but exhibited significant net anterograde transport in
axons. Although the precise identity of these organelles cannot
be determined using this technique, it is likely that they represent
small carrier vesicles conveying membrane and protein to and
from the distal ends of the processes, such as endosomes and
synaptic vesicle precursors. In summary, these data demonstrate
that there are indeed regional differences in organelle transport
that are specific to process region and organelle class.
Dendritic organelles exhibit more persistent
changes in direction than axonal organelles
To determine whether or not there were any regional differ­
ences in individual organelle motility behaviors, we first quan­
tified the patterns of transport of the large phase-dense
organelles in axons and proximal dendrites, where we had
observed differences in gross traffic patterns for this organelle.
Because the mixed polarity orientation of the dendritic microtubules might facilitate directional changes, and because
dendritic organelles exhibited no net anterograde or retrograde
flow in contrast to the net retrograde transport in axons, we
specifically addressed the following question: do organelles
change direction more frequently in dendrites than in axons?
To address this question, we tracked every large phase dense
organelle in our axonal and dendritic video sequences. Two
distinct classes of directional changes were observed (Fig. 3).
First, there were temporary changes, in which an organelle
traversed less than 3 µm in the new direction after a stop before
AXONS
all organelles
n = 350
large dense
n = 100
mitochondria
n = 25
macropinosomes
n=7
other
n = 218
100 80 60 40 20
% RETROGRADE
20 40 60 80 100
% ANTEROGRADE
PROXIMAL DENDRITES
all organelles
n = 347
large dense
n = 69
mitochondria
n = 31
macropinosomes
n=7
other
n = 240
100 80 60 40 20
% RETROGRADE
20 40 60 80 100
% ANTEROGRADE
DISTAL DENDRITES
all organelles
n = 141
large dense
n = 46
mitochondria
n = 1*
macropinosomes
n = 2*
other
n = 92
100 80 60 40 20
% RETROGRADE
20 40 60 80 100
% ANTEROGRADE
Fig. 2. The net direction of transport of some organelles differs in
axons vs dendrites. Organelle transport was visualized using videoenhanced phase-contrast light microscopy. The data indicate the
percentage of organelles in each class moving in each direction past
a fixed point within the process. Axonal transport data was obtained
from central regions of axon shafts (n=7), at least 100 µm from
either cell bodies or growth cones. Dendritic transport was
characterized in two dendritic regions: within the proximal 50% of
dendritic length (n=7), where microtubules are mixed in their
polarity orientation, and in the most distal 10% of dendritic length
(n=5), where microtubules are aligned uniformly with their plus ends
distal. The large phase-dense vesicular organelles exhibit regional
differences in traffic patterns: net transport is retrograde in axons
(P=0.0001) and distal dendrites (P=0.0390), but not in proximal
dendrites (P=0.7180). The other small vesicular organelles exhibit
significant net anterograde transport in axons (P=0.0012), but not in
proximal (P=0.1156) or distal (P=1.0000) dendrites.
Macropinosomes exhibit retrograde flow in all three process regions,
though they are observed so rarely that these data yield strong
statistical significance only in axons (P=0.0082 in axons, P=0.0588
in proximal dendrites, P=0.1573 in distal dendrites). Neither
mitochondria nor the entire population of moving organelles show a
net directional transport in any of the three process regions
examined. The total number of organelles counted in each category
is indicated on the right. Asterisks indicate a sample size too small to
have statistical significance.
Axonal vs dendritic organelle motility
stopping and changing direction again, thus finally continuing
movement in the original direction (Fig. 3A). The other class
of directional changes was that of persistent changes, in which
the organelle exhibited sustained movement in the new
direction (Fig. 3B). These organelles were either carried a long
distance, further than 3 µm, in the new direction after a stop,
or stopped one or more times, each time resuming movement
in the same new direction.
For quantitative analysis, each time an organelle stopped, the
direction of its resumed movement was recorded as either the
same or changed, and persistent changes of direction were
noted. When the two classes of directional changes were con­
sidered together, there was no significant difference in the
frequency of directional changes between the axonal and
proximal dendritic domains (Fig. 4A). However, separation of
the two classes and comparison of the relative abundance of
(A)
Fig. 3. Two types of direction
changes are exhibited by large phasedense vesicular organelles.
Individual motility behaviors of large
phase-dense vesicular organelles
were observed by video-enhanced
phase-contrast microscopy. These
two sequences of video frames
illustrate the two different types of
directional changes exhibited by
these organelles (arrows) in neuronal
processes. Transient changes of
direction (A) were characterized by a
brief, short excursion in the new
direction followed by a second
reversal of direction yielding
continued movement in the first,
original direction. In contrast,
persistent changes of direction (B)
resulted in continued transport in the
new direction. Elapsed time is
indicated in minutes:seconds in
lower right of each video frame. Bar,
5 µm.
975
(B)
0:00
0:00
0:04
1:31
0:12�
2:15�
0:15�
2:21�
0:19�
2:28�
0:26
2:32
0:29
2:34
C. C. Overly, H. I. Rieff and P. J. Hollenbeck
each type of directional change in axons and proximal
dendrites did reveal a striking difference in motility between
these two neuronal domains (Fig. 4B). Nearly 20% of the
observed changes in direction in dendrites were persistent,
whereas none of those in axons were. In summary, these data
demonstrate that while large phase dense vesicular organelles
change direction with similar frequency when they stop and
resume movement in axons and dendrites, the proportion of
these changes that result in sustained or persistent transport in
the new direction is significantly greater in dendrites where
microtubule polarity orientation is mixed than in axons where
it is uniform.
Percent of mitochondria that move
976
30
20
10
0
AXONS
Mean excursion length (µm ± s.e.m.)
8.0
Axonal mitochondria move more of the time and
greater distances than those in dendrites
Individual motility behaviors of axonal and dendritic mito­
chondria were next characterized to see whether or not there
were any regional differences despite the similarity in general
trafficking. To facilitate observation of mitochondrial motility,
mitochondria were specifically labeled with the fluorescent
lipophilic cationic vital dye, rhodamine 123 (Johnson et al.,
1981). Epifluorescence images of mitochondria in axons and
(A)
7.0
6.0
(B)
DENDRITES
anterograde
retrograde
overall
5.0
4.0
3.0
2.0
1.0
0.0
(A)
AXONS
70/192
PROXIMAL DENDRITES
32/106
0.0
0.1
0.2
0.3
0.4
Probability of change of direction
0.5
(B)
AXONS
0/70
PROXIMAL DENDRITES
6/32
0.00
0.05
0.10
0.15
0.20
0.25
Probability that change of direction is persistent
Fig. 4. Relative frequency of persistent changes of direction differ in
axons vs dendrites. Large phase-dense vesicular organelles were
individually tracked throughout video sequences of axons and
proximal dendrites, and the total number of stops and the number of
stops followed by a change in direction were counted. (A) The
proportion of stops that resulted in any change of direction was
calculated for axons and for proximal dendrites. There was no
significant difference in the frequencies of direction changes in axons
vs dendrites (P=0.2758). (B) The proportion of reversals of organelle
transport direction that resulted in sustained movement in the new
direction was calculated for axons and proximal dendrites. Dendritic
organelles exhibited a significantly higher frequency of persistent
changes of direction than did axonal organelles (P=0.0005).
AXONS
DENDRITES
Fig. 5. Mitochondrial transport in axons vs dendrites differs in its
duty cycle and excursion length. Mitochondrial movements were
quantified from one-minute video sequences of rhodamine 123­
labeled hippocampal neurons. (A) The population duty cycles were
calculated by counting the mitochondria which exhibit movement
and the total number of mitochondria observed in axons (n=197) and
dendrites (n=96). The percentage of mitochondria that exhibit
movement is significantly greater in axons than in proximal dendrites
(P=0.0014). (B) The excursion lengths of moving mitochondria in
axons (n=41) and proximal dendrites (n=24) were measured then
averaged for anterogradely, retrogradely, and all moving
mitochondria. There was no significant difference between
anterograde and retrograde movements in axons (P=0.7759) or in
proximal dendrites (P=0.2105). However, axonal mitochondria
traverse significantly greater distances than dendritic mitochondria,
whether moving anterogradely (P=0.0262), retrogradely (P=0.0186),
or either direction (P=0.0004). Error bars = 1 s.e.m.
proximal dendrites of live neurons were captured on video tape
for motility analysis.
To characterize mitochondrial transport in the different
neuronal domains, three parameters of motility were quanti­
fied: duty cycle, net transport direction, and mean excursion
length. For each mitochondrion observed in all video
sequences analyzed, we recorded whether or not it moved, its
direction of net movement, and its excursion length (net dis­
placement) during the one-minute observation period. In
agreement with the video-enhanced phase-contrast analysis
(above), the observed mitochondria, although motile, exhibited
no significant net anterograde or retrograde transport in axons
or dendrites (not shown). Comparison of the proportions of
mitochondria that moved in axons and proximal dendrites
revealed significant regional differences in the mitochondrial
duty cycle; more axonal than dendritic mitochondria moved in
a defined period of time (Fig. 5A). In addition, the mean
excursion length of mitochondria moving in either direction
Axonal vs dendritic organelle motility
was significantly longer in axons than in dendrites (Fig. 5B).
Furthermore, we observed a nearly four-fold difference in the
maximum excursion lengths of 25 µm in axons and 7 µm in
dendrites. These data demonstrate that although the net
direction of flow is similar in axons and dendrites, mitochon­
dria do exhibit significant regional motility differences.
Dendritic mitochondria are more likely to remain stationary,
and when they move they are transported shorter distances than
axonal mitochondria.
Dendritic mitochondria are more metabolically
active than axonal mitochondria
To experimentally demonstrate regional metabolic differences
and to explore the possibility that they might account for at
least some of the observed organelle transport properties in
different subcellular domains, mitochondrial activity was
examined. Mitochondrial energy states in axons and dendrites
977
were examined and compared by incubating neurons in the flu­
orescent mitochondrial vital dye, JC-1, an indicator of relative
mitochondrial transmembrane potentials and activity (Smiley
et al., 1991; Reers et al., 1991). This molecule, like rhodamine
123, is a lipophilic cationic dye that becomes concentrated
inside mitochondria in proportion to their transmembrane
potentials; more dye accumulates in mitochondria with greater
transmembrane potentials, thus in those with greater ATP gen­
erative capacities. At lower concentrations, JC-1 emits a green
fluorescence (527 nm), whereas at higher concentrations this
molecule forms J-aggregates and shifts to a red fluorescence
with an emission peak of 590 nm (Smiley et al., 1991). Thus,
the fluorescence of JC-1 can be used as an indicator of relative
mitochondrial energy state. The least active mitochondria have
relatively low transmembrane potentials, thus accumulate less
dye and appear green. In contrast, the more active mitochon­
dria have higher transmembrane potentials, leading to higher
intramitochondrial dye concentrations and red fluorescence.
Fluorescence images of the labeled cells reveal hetero­
geneous populations of mitochondria in all neuronal domains,
with a greater number of highly charged red mitochondria in
dendrites than in axons (Fig. 6). To quantify this observation,
the proportions of relatively inactive (green), active (red), and
intermediate (red and green) mitochondria in axons and
dendrites were determined (Fig. 7). The data show that
dendrites contain nearly four times as many highly active mito­
chondria and approximately half as many relatively inactive
mitochondria as axons. These results demonstrate that the
dendritic mitochondrial population is more metabolically
active than the axonal population.
RELATIVE FREQUENCY
0.8
0.6
0.4
0.2
0.0
Fig. 6. Mitochondria in cultured hippocampal neurons labeled with
JC-1. Cultured hippocampal neurons were labeled with the
mitochondrial vital dye JC-1, which reveals relative differences in
mitochondrial energy state. Low-energy green (A), high-energy red
(B) and mixed red and green (arrow in C) mitochondria were
observed. Both axons (D) and dendrites (E) exhibited mixed
mitochondrial populations, but in different proportions. (D) Axons
contained more low-energy green mitochondria, and often contained
red mitochondria in the growth cone region (arrow). (E) Dendrites
exhibited more high-energy red mitochondria. Bars: 5 µm (A-C); 20
µm (D,E).
AXONS
DENDRITES
GREEN
BOTH
RED
Fig. 7. Relative frequency of mitochondria in different energy states
in axons vs dendrites. Cultured hippocampal neurons were labeled
with JC-1 and the numbers of low-energy (green), high energy (red)
and mixed (both green and red) mitochondria in axons and dendrites
were counted then converted to proportions of the total
mitochondrial population. Totals of 263 mitochondria in 55 axons
and 482 mitochondria in 43 dendrites were observed. Data from all
processes observed on single coverslips were pooled. The relative
frequencies calculated for the three mitochondrial populations from 3
axonal and 4 dendritic coverslips were averaged. Dendritic
mitochondria were more active than axonal mitochondria. Dendrites
contained significantly fewer green (P=0.0031), and more red
(P=0.0057) mitochondria than axons. Although there were
consistently more mixed red and green mitochondria in dendrites
than in axons, the data were not significantly different (P=0.1908).
Error bars = 1 s.e.m.
978
C. C. Overly, H. I. Rieff and P. J. Hollenbeck
DISCUSSION
The morphological and cytoplasmic asymmetry of neuronal
cells critically underlies their functions and must be achieved
at least in part by an elaborate and tightly controlled system
for efficiently transporting and positioning organelles within
the cell. The different metabolic demands and requirements for
macromolecules likely necessitate different organelle traffick­
ing patterns in axons and dendrites. Furthermore, the differ­
ences in the organization of the microtubule tracks in these two
process types (Baas et al., 1988), in conjunction with the direc­
tional selectivity of microtubule based motor proteins (Brady,
1991), have prompted speculation about their role in selective
organelle transport in the different neuronal domains (Black
and Baas, 1989; Peters et al., 1991). Despite the extensive and
rapidly growing literature characterizing axonal transport,
dendritic transport has only briefly been examined (Kiss, 1977;
Gorenstein et al., 1985; Gorenstein and Ribak, 1985; Parton et
al., 1992). The data presented here represent the first direct and
quantitative comparative study of organelle traffic and
metabolic properties in different process regions of live
neurons. In this report, we demonstrate quantitative differences
in overall organelle traffic, individual organelle motility char­
acteristics and metabolic state between axons and dendrites of
developmental stage 4 and stage 5 (Dotti et al., 1988) cultured
embryonic rat hippocampal neurons, respectively.
Influence of microtubule organization on general
organelle trafficking
The discovery of the regional differences in microtubule
polarity orientation in neuronal cells has prompted speculation
about a mechanistic role for microtubule polarity organization
in selective organelle transport and cytoplasmic compartmen­
tation in neurons (Black and Baas, 1989; Peters et al., 1991).
The combination of unidirectional transport along microtubules and microtubule organization is known to play an
important role in directed motility and positioning of specific
organelles within cells. For example, there is evidence for
minus-end specific transport of the Golgi apparatus in various
cell types (Wehland et al., 1983; Rogalski and Singer, 1984;
Ho et al., 1989; Corthésy-Theulaz et al., 1992), and of
ribosomes in insect ovarian nutritive tubes (Stebbings et al.,
1991). There is also evidence for plus end directed movement
of Golgi elements (Lippincott-Schwartz et al., 1995) and bidi­
rectional movement of endoplasmic reticulum (Terasaki et al.,
1984, 1986; Lee et al., 1989), lysosomes (Herman and
Albertini, 1984; Matteoni and Kreis, 1987; Hollenbeck and
Swanson, 1990; Swanson et al., 1992), pigment granules
(Rozdzial and Haimo, 1986a,b; Lynch et al., 1986a,b), mito­
chondria (Morris and Hollenbeck, 1993), and autophagic
vacuoles (Hollenbeck and Bray, 1987; Hollenbeck, 1993).
Although the regulation of the motors and their association
with organelles is not clearly understood, there is also some
evidence for the association of specific motors with specific
organelles (e.g. synaptic vesicles; Leopold et al., 1992; Okada
et al., 1995); mitochondria (Leopold et al., 1992; Nangaku et
al., 1994); lysosomes (Hollenbeck and Swanson, 1990;
Swanson et al., 1992); Golgi elements (Schmitz et al., 1994;
Marks et al., 1994; Lippincott-Schwartz et al., 1995); pigment
granules (Rodionov et al., 1991).
Our quantitative analysis of the net transport direction of
four classes of organelles in three neuronal regions, axon
shafts, proximal dendrites and distal dendrites, revealed one
organelle type, the large phase-dense organelles, which
exhibited regional differences in trafficking consistent with
regional differences in microtubule organization. The size,
morphology, and net retrograde axonal transport properties of
these organelles strongly suggest that they are autophagic
vacuoles, a predegradative compartment rigorously character­
ized in cultured sympathetic neurons (Hollenbeck and Bray,
1987; Hollenbeck, 1993). While these organelles exhibit net
retrograde transport in both axons and the most distal 10% of
dendritic length, where microtubules are uniformly aligned
with their plus ends directed distally, they did not exhibit sig­
nificant net transport in either the anterograde or the retrograde
direction in proximal dendrites where the microtubule polarity
orientation is mixed. This difference might be driven merely
by regional differences in subcellular needs. However, because
the similarities and differences correlate with cytoskeletal
architecture rather than with process type, it is also likely that
these regional variations in organelle traffic are related to the
known differences in the organization of the microtubule
tracks.
Individual organelle motility characteristics and
microtubule polarity orientation
In addition to influencing general organelle traffic patterns, the
differences in microtubule polarity orientation in axons and
proximal dendrites likely affect the motility characteristics of
individual organelles. It is well known that many organelles of
many types move along microtubules in a saltatory manner and
often change their direction of movement, as discussed above.
We hypothesized that organelles in proximal dendrites where
microtubule polarity is mixed might change direction more fre­
quently than axonal organelles. Because of the mixed micro­
tubule polarity orientation in dendrites, reversal of direction
could be achieved by changing motor activation or association,
as is most likely in the axon, or could merely require the
organelle to switch to another microtubule, a phenomenon that
has been observed in vitro (Smith and Forman, 1988; Brady et
al., 1982). Our data demonstrate that large phase-dense
organelles exhibit significantly more frequent persistent
changes in direction in proximal dendrites than in axons. This
increased bidirectional transport of individual large phasedense organelles in proximal dendrites also might account for
the observed regional differences in net transport direction and
is consistent with the hypothesis that microtubule organization
influences both general organelle trafficking patterns and indi­
vidual organelle motility behaviors. Furthermore, in contrast to
suggestions that microtubules in only one orientation can
support organelle transport (Sheetz et al., 1989; Lopez and
Sheetz, 1993), our data are consistent with dendritic microtubules in both polarity orientations supporting organelle
transport.
Mitochondrial motility and subcellular metabolism
Although no significant regional variation in net transport
direction of mitochondria was detected, they, like the large
phase-dense organelles, exhibited significant regional differ­
ences at the level of individual organelle motility behaviors.
Our data demonstrate that mitochondrial movement is more
extensive in axons than in proximal dendrites. At any point in
Axonal vs dendritic organelle motility
time, a greater proportion of mitochondria in axons are
moving, and when they move, they move significantly farther
that they do in dendrites. The observed difference in excursion
lengths could reflect either differences in the microtubule
arrays or metabolic demands in axons and dendrites. Serial
reconstructions of microtubules from electron micrographs of
axons (Bray and Bunge, 1981) and dendrites (Fletcher et al.,
1994) revealed that axonal microtubules are significantly
longer than dendritic microtubules. The average length of
axonal microtubules was calculated to be 108 µm whereas the
longest observed dendritic microtubule was only 11 µm. Thus,
the longer mean excursion lengths in axons could merely be
related to the longer microtubules. Alternatively, the shorter
dendritic excursion lengths could result from more closely
spaced regions requiring high ATP consumption; while
energy-dependent processes are concentrated in growth cones
and synaptic terminals of axons, they are more evenly distrib­
uted along the lengths of dendrites.
The observed regional variation in mitochondrial duty cycle
is most likely related to regional differences in subcellular
metabolism. Our data demonstrate that dendritic mitochondria
remain stationary more of the time than axonal mitochondria.
The differential transport and targeting of mitochondria to
regions of high ATP consumption is well known in both
neuronal and non-neuronal cells (Fawcett, 1981; Morris and
Hollenbeck, 1993). The decreased mitochondrial motility in
dendrites could reflect greater ATP demands along their length
than along axon shafts. Characterization of relative differences
in mitochondrial transmembrane potentials in axons and
dendrites using the lipophilic cationic mitochondrial vital dye,
JC-1, yielded data consistent with this hypothesis. Dendritic
mitochondria exhibited higher transmembrane potentials, thus
were more metabolically active, than axonal mitochondria.
Although we cannot rule out developmental influences on
organelle motility, such influences were minimized by
comparing developmental stage 4 axons with stage 5 dendrites.
Our experimental observations were made in axons and
dendrites at these different stages of neuronal development for
several reasons. First, it is known that in this and other primary
neuronal culture systems, axons grow and mature earlier in
neuronal development than dendrites. They initiate outgrowth,
achieve their characteristic morphology (Dotti et al., 1988),
and become competent to form synapses (Fletcher et al., 1994)
before dendrites. Second, the chosen stages represent similar
stages in the growth of these two process types, after initial
outgrowth when growth is continuing slowly (Dotti et al.,
1988). Third, at these stages, the axons and dendrites have
matured as much as possible without sacrificing their spatial
resolution for these microscopy studies. With additional
growth, the axons and dendrites become a tangled meshwork
and cannot be traced to either end. At the stages observed, the
processes have the characteristics of older, mature processes.
Stage 4 axons have achieved their mature morphology (Dotti
et al., 1988), lost characteristic somatodendritic markers such
as MAP2 (Matus et al., 1981) and high levels of mRNA
(Kleiman et al., 1994), and are capable of making synapses
(Fletcher et al., 1994). Stage 5 dendrites exhibit the mature
tapered and branched morphology (Dotti et al., 1988) and are
competent to form synapses (Fletcher et al., 1994). Perhaps
most important for this study, the mature microtubule organ­
ization is established in dendrites of stage 5 cells (Baas et al.,
979
1989). Specific cases of organelle motility have been shown to
change with process growth state, but these changes are most
pronounced near the site of active elongation (Hollenbeck and
Bray, 1987; Hollenbeck, 1993; Hollenbeck and Weld, 1994;
Morris and Hollenbeck, 1993). By matching the growth states
of axons and dendrites, maximizing process maturity, and
examining organelle motility primarily in more proximal
process regions, we minimized the influence of specific devel­
opmental demands on our comparisons. Furthermore, by char­
acterizing organelle transport in axons and dendrites of stage
4 and stage 5 neurons, respectively, we can be certain of the
microtubule organization and thus can consider transport dif­
ferences in terms of differences in the organization of the
tracks.
The data in this report represent a first step toward an under­
standing of the role of organelle traffic in the establishment and
maintenance of neuronal polarity and specialized subcellular
domains. Our data definitively show that there are indeed
regional differences in both organelle motility and metabolism.
These observations are entirely consistent with the hypothesis
that organelle transport properties within living cells are influ­
enced both by the organization of the microtubule tracks and
by local metabolic demands.
The authors thank Dr Robert L. Morris for helpful suggestions and
Dr Marguerite Olink-Coux for comments on the manuscript. We are
also grateful to Rose Weld and Myrta Otero for valuable technical
assistance. This work was supported by grants from the National Insti­
tutes of Health and the March of Dimes Birth Defects Foundation,
and by an Albert J. Ryan Fellowship (C.C.O) and National Eye
Institute training grant #T32EY07110-05 (H.I.R).
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(Received 5 December 1995 - Accepted 7 February 1996)