H . Hiruma et al. Brain Research 883 2000 184 –191
185
genesis [19], and synaptic formation [7]. Thus, axonal labelling immunofluorescent image, the immunostained
transport is considered to be closely related to the develop- cells were examined with an inverted Zeiss Axiomat
ment and function of the sensory nervous system as well as microscope Carl Zeiss, Oberkochen, Germany equipped
other neuronal systems. In the present study, we attempted with exciting filters 546 nm for rhodamine, 450–490 nm
to clarify the roles of major sensory neuropeptides, sub- for FITC and emission filters 590 nm for rhodamine,
stance P and CGRP, in the regulation of axonal transport in 515–565 nm for FITC. The number of immunoreactive
isolated and cultured adult mouse DRG neurons. neurons was determined under microscopic observation
and also by examining microphotographs. DRG neurons used for the experiments on axonal transport were also
2. Materials and methods double immunostained for substance P and CGRP.
2.1. Cell isolation and culture 2.3. Video-enhanced microscopic recordings
Adult male c57BL 6 mice purchased from Japan SLC After a 48-h period of cell culture, the coverslip on
Hamamatsu, Japan were killed with ether, and dorsal root which cells were plated was attached with waterproof tape
ganglia were removed. The ganglia were incubated for 90 at the bottom of a 0.5-mm thick stainless steel chamber
min at 378C in Hams’ F-12 medium Gibco-BRL, Grand 50380 mm whose center was hollowed out in the shape
Island, NY, USA containing 2 mg ml collagenase Worth- of a lozenge 25335 mm. The top of the chamber was
ington Biochemical, Freehold, NJ, USA. Subsequently, covered with another coverslip, leaving small openings on
21
the ganglia were incubated for 15 min at 378C in Ca -
both sides to perfuse the solution. The culture medium was
21
and Mg -free Hanks’ balanced salt solution g l: KCl,
then replaced with Hepes-buffered saline 378C, pH 7.3 0.4; KH PO , 0.06; NaCl, 8; Na HPO 7H O, 0.09;
D
- containing 135 mM NaCl, 5 mM KCl, 1 mM CaCl , 1 mM
2 4
2 4
2 2
glucose, 1; phenol red, 0.01; Hepes 3.6; NaOH, 0.3 MgCl , 10 mM Hepes, and 5.5 mM
D
-glucose. The
2
containing 2.5 mg ml trypsin Sigma, St. Louis, MO, chamber was mounted onto the temperature-controlled
USA. Trypsin activity was then inhibited by addition of stage 378C of an inverted Zeiss Axiomat microscope,
trypsin inhibitor 0.125 mg ml, Sigma. Following a three- with an oil-immersed planapochromat 643 NA 1.40
times rinse with enzyme-free Hams’ F-12 medium, single objective Carl Zeiss. The drug-containing solution 3 ml
cells were obtained by triturating the ganglia through was injected into one opening using a Pasteur pipette and
fire-polished pipettes 0.2–0.5 mm inner diameter. The the solution spilled from another opening was removed
cells were plated onto polylysine Sigma-coated glass using a peristaltic pump.
coverslips 30340 mm, 50 mm thickness, and were Nomarski images of axon-like neurites length, 200
cultured in Ham’s F-12 medium containing 10 fetal mm; width, 1.0 mm obtained by the inverted microscope
bovine serum and penicillin 100 units ml–streptomycin were transformed into video images by an analogue video
100 mg ml at 378C under humidified conditions in 95 camera Harpicon, Hamamatsu Photonics, Hamamatsu,
air and 5 CO . Japan. The analogue signal was processed by a real-time
2
digital video
image enhancement
system DVS-20,
2.2. Immunocytochemistry Hamamatsu Photonics. Video images were displayed on a
video monitor C1846, Hamamatsu Photonics and were DRG neurons cultured for 48 h on a coverslip were
stored on a video recorder PVW-2800, Sony, Tokyo, fixed with 4 paraformaldehyde for 5 min at room
Japan. Such processing provided a final magnification of temperature. After fixation, they were washed with 0.025
approximately 310 000 on a video monitor. M phosphate-buffered saline PBS containing 0.3 Triton
X-100 PBST for 3 min, and were treated for 10 min with 2.4. Analysis of axonal transport
protein blocking agent Immunon, Pittsburgh PA, USA at room temperature to block nonspecific protein sites. Cells
The measurements of intraaxonal particle movement were incubated overnight at 48C with rat anti-substance P
were performed at the axon hillock and proximal parts of serum 1:1000, Chemicon International, Temecula, CA,
the parent axon distance from the cell body, ,100 mm. USA and rabbit anti-CGRP serum 1:1500, Cambridge
The number of particles diameter, 50 nm moving Research Biochemicals, Northwich, UK. The antisera
toward the axon terminal anterograde and back to the cell were diluted with 0.2 bovine serum albumin, 1 normal
body retrograde was counted for 2 min at 3-min intervals goat serum, and 0.1 sodium azide in PBST. After
during periods before and after the injection of neuro- washing with PBS, cells were incubated with rhodamine-
peptides. Data are expressed as mean percentage of control conjugated goat anti-rat IgG 1:100, Organon Teknika,
value that was obtained before the drug application. Durham, NC, USA and Fluorescein isothiocyanate FIT-
Analysis of variance ANOVA was used to evaluate the C-conjugated goat anti-rabbit IgG 1:100, Organon Tek-
statistical significance of fluctuations over time. The nika for 30 min at room temperature. For a double-
significances of differences between the control and the
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
