186 H
values obtained during application of neuropeptides were Immunocytochemical staining revealed that a large
determined by Student’s paired t-test. The instantaneous portion of DRG neurons were immunoreactive for sub-
velocity of the individual moving particles was also stance P and CGRP. Of total 674 neurons, 579 85.9
analysed. Serial video images were transferred at 5–20 ms were immunoreactive for substance P, and 420 72.5 of
intervals to a Macintosh computer Power Macintosh, substance P-immunoreactive neurons contained CGRP.
7600 200 equipped LG-3 video capture board Scion, Immunoreactivity for CGRP was found in 445 66.0 of
Frederick, MD by using Scion image software Scion. total 674 DRG neurons, and 420 94.4 of CGRP-
Then, the movement distance time ratio was analysed and immunoreactive neurons contained substance P. The re-
defined as the instantaneous velocity. maining 69 10.2 of total neurons exhibited no immuno-
reactivity for substance P or CGRP. The immuno- 2.5. Drugs
reactivities for substance P and CGRP were found in both the cell bodies and neurites Fig. 1.
Substance P Sigma, rat a-CGRP Peptide Institude, Axonal transport of particles within the long axon-like
Osaka, Japan, and rat b-CGRP Peninsura Laboratories, neurite length, 200 mm; width, 1.0 mm was observed
Belmont, CA, USA were dissolved in Hepes-buffered under video-enhanced microscopy, and the particle move-
saline at concentrations indicated in the text. ment measurements were performed at the axon hillock
and proximal axon distance from the cell body, ,100 mm. In control extracellular medium Hepes-buffered
3. Results saline, pH 7.3, 378C, mean numbers of particles per min
transported in anterograde and retrograde directions were 3.1. Morphological and immunocytochemical properties
63.3616.3 mean6S.D., n519 and 63.1625.3 n519, respectively. The instantaneous velocity of moving par-
Most .98 of isolated and cultured adult mouse ticles varied from 0.03 to 2.48 mm s in control medium.
DRG neurons were small diameter, ,25 mm and medium diameter, 25–40 mm in size. This may be associated with
3.2. Effects of substance P on axonal transport loss of large cells during the process of cell isolation and
culture, because large cells are likely to be more abundant Application of 100 nM substance P decreased the
in the intact mouse DRG [56]. In general, small- and number of particles transported in both the anterograde and
medium-sized DRG neurons are chemosensitive whereas retrograde directions in each of five neurons tested Fig.
large neurons are not [9,17]. Therefore, small- and 2A. The maximum inhibition reached approximately 60
medium-sized DRG neurons were subjected to the follow- of control after 11 min of the application, and this value
ing experiments where the effects of sensory neuropeptides was sustained throughout the application. Expressions of
were investigated. substance P and CGRP in the five neurons used for the
Fig. 1. Double immunofluorescence of substance P and CGRP in the cultured DRG neuron. A Immunofluorescence using antibody for substance P visualized by rhodamine. B Immunofluorescence using antibody for CGRP visualized by FITC. Scale bar: 20 mm.
H . Hiruma et al. Brain Research 883 2000 184 –191
187
Fig. 2. Effects of substance P on axonal transport in DRG neurons. A Percent changes in the number of transported particles moving in anterograde and retrograde directions induced by application of 100 nM substance P. Each point indicates the mean 6S.D.. P,0.05, P,0.005, P,0.0005
compared to the value before the application. B Histograms of the instantaneous velocities of individual particles moving in anterograde and retrograde directions before control and during application of substance P.
experiments were investigated by immunocytochemical CGRP but one was not immunoreactive for CGRP. One
staining. Of the five neurons, four were immunoreactive neuron was not immunoreactive for either substance P or
for substance P. Of these four substance P-immunoreactive CGRP. The instantaneous velocities of individual particles
neurons, three were immunoreactive but one was not transported in anterograde and retrograde directions were
immunoreactive for CGRP. One neuron was immuno- increased by a-CGRP Fig. 3B.
reactive for neither substance P nor CGRP. When the instantaneous velocity of particles was analyzed in one of
3.4. Effects of b-CGRP on axonal transport the tested neurons, substance P was found to reduce the
instantaneous velocities of particles moving in both antero- Application of 100 nM b-CGRP did not elicit any effect
grade and retrograde directions Fig. 2B. on the number of particles transported in either anterograde
or retrograde direction n55 Fig. 4A. b-CGRP at higher 3.3. Effects of a-CGRP on axonal transport
concentration 1000 nM was also without effect data not shown; n54. In the five neurons tested with 100 nM
Application of 100 nM a-CGRP increased the number b-CGRP, three were immunoreactive but two were not
of particles transported in anterograde and retrograde immunoreactive for substance P. In three substance P-
directions in each of five neurons tested Fig. 3A. The positive neurons, two were immunoreactive for CGRP but
value peaked at 8 min after the start of the application, and the remaining one was not immunoreactive for CGRP. In
thereafter gradually declined. The peak value was 150– the two substance P-negative neurons, one was immuno-
160 of control. Of the five neurons examined, four were reactive but another was not immunoreactive for CGRP.
immunoreactive for substance P. In these substance P- The instantaneous velocity of individual particles was also
immunoreactive neurons, three were immunoreactive for not affected by b-CGRP Fig. 4B.
188 H
Fig. 3. Effects of a-CGRP on axonal transport in DRG neurons. A Percent changes in the number of transported particles moving in anterograde and retrograde directions induced by application of 100 nM a-CGRP. Each point indicates the mean 6S.D.. P,0.05, P,0.005, P,0.0005 compared
to the value before the application. B Histograms of the instantaneous velocities of individual particles moving in anterograde and retrograde directions before control and during application of a-CGRP.
4. Discussion CGRP is modified by culture [8,54], influences of culture
