Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol883.Issue2.2000:

(1)

www.elsevier.com / locate / bres

Research report

Effects of substance P and calcitonin gene-related peptide on axonal

transport in isolated and cultured adult mouse dorsal root ganglion

neurons

a ,

_*

a b c d

Hiromi Hiruma

, Ayako Saito , Takafumi Ichikawa , Yoriko Kiriyama , Sumio Hoka ,

e c a

Tatsumi Kusakabe , Hirosuke Kobayashi , Tadashi Kawakami

Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan

Department of Biochemistry, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan

Department of Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan

Department of Anesthesiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan

Department of Sport and Medical Science, Kokushikan University, 7-3-1 Nagayama, Tokyo 206-8515, Japan Accepted 22 August 2000

Abstract

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (.98%) small (diameter

,25mm) and medium (diameter, 25–40mm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66.0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n55). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast,a-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n55), and also increased the instantaneous velocities of particles transported bidirectionally. Application ofb-CGRP (100–1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P anda-CGRP, both of which can be derived from its own and adjacent sensory neurons.  2000 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Axon guidance mechanisms and pathways

Keywords: Axonal transport; Dorsal root ganglion neuron; Substance P; Calcitonin gene-related peptide; Video-enhanced microscopy; Immuno-cytochemistry

1. Introduction dorsal root ganglion (DRG) [24–26,31,36,42] and axonally transported to the central and peripheral endings of sensory Neuropeptides such as substance P and calcitonin gene- nerve fibers [5,12,23,34,35]. They are released from cen-related peptide (CGRP) play a critical role in regulating tral terminals into the dorsal horn of the spinal cord to the inflammatory response in peripheral sensory neurons. convey noxious information as synaptic transmitters [53]. These neuropeptides are synthesized in cell bodies of the These neuropeptides are also released from peripheral terminals to induce extravasation, vasodilatation, inflam-mation, and nociception [29,40,45].

*Corresponding author. Tel.: 181-42-778-9159; fax: 1

81-42-778-Axonal transport is recognized as a cellular function 9841.

(2)

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

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;₂ ₄ ₂ ₄ ₂ D- containing 135 mM NaCl, 5 mM KCl, 1 mM CaCl , 1 mM₂ glucose, 1; phenol red, 0.01; Hepes 3.6; NaOH, 0.3) MgCl , 10 mM Hepes, and 5.5 mM₂ D-glucose. The 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,>1.0mm) 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 .2 Japan). The analogue signal was processed by a real-time

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 approximately310 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

(3)

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 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,$200mm; width,$1.0mm) 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,,25mm) and medium

(diameter, 25–40mm) 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: 20mm.

(4)

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 ofb-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 nMb-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 ofa-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 nMa-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 byb-CGRP (Fig. 4B).

(5)

Fig. 3. Effects ofa-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 nMa-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 ofa-CGRP.

4. Discussion CGRP is modified by culture [8,54], influences of culture are also considerable factors.

In the present study, we clarified the effects of sensory We applied video-enhanced microscopy to investigate neuropeptides, substance P and CGRP, on axonal transport the effects of neuropeptides on axonal transport in such in isolated and cultured adult mouse DRG neurons. These small- and medium-sized DRG neurons. Application of neurons were small and medium in size, and 89.5% of the substance P decreased the number and instantaneous DRG neurons were immunoreactive for substance P and velocity of particles transported in anterograde and retro-66.0% of neurons expressed immunoreactivity for CGRP. grade directions. In contrast,a-CGRP increased the num-In the intact DRG, it has been reported that 10–25% of ber and velocity of particles transported bidirectionally. neurons are immunoreactive for substance P Application ofb-CGRP had no effect on axonal transport. [2,6,21,25,27,48] and 25–100% of neurons are immuno- These responses occurred in all the DRG neurons tested. reactive for CGRP [6,22,43,48,62]. Thus, the expression The immunocytochemical staining revealed that the neu-ratios of these neuropeptides in isolated and cultured DRG rons examined were positive as well as negative to neurons are different from those in DRG neurons in situ. It substance P and CGRP, although positive neurons were has been described that immunoreactivity for substance P predominant in accord with the proportion of positive is present mainly in small- and medium-sized DRG cells. From these findings, it is suggested that substance P neurons [25], whereas CGRP immunoreactivity distributes and a-CGRP can modulate axonal transport in most of in small to large-sized cells [15,30,32,37,39]. Therefore, isolated and cultured DRG neurons, regardless of whether the difference in expression ratios may be due to the size or not the cells contain these neuropeptides. Supporting distribution of cells. In addition, because the percentage of our findings, several experimental methods have demon-adult DRG neurons immunostaining for substance P and strated that substance P and CGRP receptors are expressed

(6)

Fig. 4. Effects ofb-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 nMb-CGRP. Each point indicates the mean (6S.D.). (B) Histograms of the instantaneous velocities of individual particles moving in anterograde and retrograde directions before (control) and during application ofb-CGRP.

in DRG neurons. Substance P receptor (neurokinin-1 andb-CGRP can both couple all CGRP receptor subtypes receptor) immunoreactivities and binding sites have been [60]. Therefore, one possibility for explaining our results is found in isolated and cultured rat DRG neurons [55,59] as that mouse DRG neurons may express CGRP receptor well as in the intact rat DRG [58]. Neurokinin-1 receptor subtypes exclusively selective to a-CGRP.

mRNA has also been identified in the mouse DRG by It has been well documented that substance P and using reverse transcription-polymerase chain reaction (RT- mRNA encoding the precursor for substance P are present PCR) [1]. The neurokinin-1 receptor seems to be present in the rat DRG [3,25,46]. Botha-CGRP andb-CGRP and mostly in small- and medium-sized DRG cells [41,55]. their mRNAs are also expressed in rat DRG neurons Several electrophysiological studies have revealed that the [16,44,47,57], but a-CGRP is predominant [16,44,57]. majority (90%) of acutely isolated rat DRG neurons are Thus, axonal transport in sensory neurons may be con-sensitive to exogenously applied substance P [10,28], trolled by substance P anda-CGRP which can be released suggesting that substance P receptor is expressed on the from its own and adjacent sensory neurons, through cell membrane of most isolated DRG cells. Although autocrine and paracrine mechanisms. In addition, recent CGRP receptors have not yet been identified in DRG evidence has shown that substance P and CGRP are neurons, electrophysiological studies have shown that synthesized and released by lymphocytes [38,61]. Thus, CGRP elicits excitatory responses in rat DRG neurons in these neuropeptides derived from lymphocytes may also be situ [51,52], suggesting that DRG neurons functionally involved in the modification of axonal transport.

express CGRP receptors. As described above, we found Substance P and CGRP are released from sensory that a-CGRP but not b-CGRP was effective to modify neurons simultaneously in response to inflammation [13]. axonal transport. In this regard, it is described that there These neuropeptides cooperatively induce vasodilatation may be some subtypes of CGRP receptors with different and extravasation in peripheral tissue [4,14,20]. In a selectivity to a-CGRP and b-CGRP, although a-CGRP similar way, nociceptive transmission is modulated by

(7)

[10] A. Dray, R.D. Pinnock, Effects of substance P on adult rat sensory synergic action of these neuropeptides [33,49]. However,

ganglion neurones in vitro, Neurosci. Lett. 33 (1982) 61–66. the effects of substance P and a-CGRP observed in this

[11] H.A. Fernandez, J.A. Donoso, Axonal transport involvement in study were opposite, the former was inhibitory and the _{long-lasting synaptic modifications in Blatta orientalis, J. Neurobiol.} latter was stimulatory. In addition, the duration of the 14 (1983) 61–75.

effect of CGRP was transient whereas the effect of [12] H.L. Fernandez, C.A. Hodges-Savola, Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as a function substance P was prolonged. Thus, sensory neuropeptides

of age, Neurochem. Res. 19 (1994) 1369–1377. may contribute to the fine and balanced regulation of

[13] M.T. Galeazza, M.G. Garry, H.J. Yost, K.A. Strait, K.M. Har-axonal transport in sensory neurons. _{greaves, V.S. Seybold, Plasticity in the synthesis and storage of} In the present study, we demonstrated that substance P _{substance P and calcitonin gene-related peptide in primary afferent} inhibited and a-CGRP stimulated axonal transport in neurons during peripheral inflammation, Neuroscience 66 (1995)

443–458. sensory neurons. Axonal transport is fundamental to

[14] R. Gamse, A. Saria, Potentiation of tachykinin-induced plasma neuronal formation and synaptic transmission, therefore,

protein extravasation by calcitonin gene-related peptide, Eur. J. these neuropeptides can modify axonal transport to affect _{Pharmacol. 114 (1985) 61–66.}

development and function of the sensory nervous system. _{[15] S.J. Gibson, J.M. Polak, S.R. Bloom, I.M. Sabate, P.M. Mulderry,} M.A. Ghatei, G.P. McGregor, J.F. Morrison, J.S. Kelly, R.M. Evans, M.G. Rosenfeld, Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci. 4 Acknowledgements

(1984) 3101–3111.

[16] S.J. Gibson, J.M. Polak, A. Giaid, Q.A. Hamid, S. Kar, P.M. Jones, This work was partly supported by the Academic _{P. Denny, S. Legon, S.G. Amara, R.K. Craig, S.R. Bloom, R.J.S.} Frontier Project from the Ministry of Education, Science, Penketh, C. Rodek, N.B.N. Ibrahim, A. Dawson, Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of Sports and Culture, Japan; a Grand-in-Aid for Scientific

the dorsal root ganglia and also in spinal motoneurones in man and Research (No. 11670638) from the Ministry of Education,

rat, Neurosci. Lett. 91 (1988) 283–288. Science, Sports and Culture, Japan; and Comprehensive

[17] M.S. Gold, S. Dastmalchi, J.D. Levine, Co-expression of nociceptor Research on Aging and Health (H10-Aging and Health- _{properties in dorsal root ganglion neurons from the adult rat in vitro,} 008), Health Science Research Grants, from the Ministry Neuroscience 71 (1996) 265–275.

of Health and Welfare, Japan. [18] A. Gorio, The role of axoplasmic transport in the restoration of synaptic transmission and in the process of sprouting during nerve regeneration, Adv. Exp. Med. Biol. 209 (1987) 41–49.

[19] B. Grafstein, D.W. Burmeister, C.M. McGuinness, G.W. Perry, J.R. References _{Sparrow, Role of fast axonal transport in regeneration of goldfish} optic axons, in: Neural Regeneration, F.J. Seil, E. Herbert, B.M. [1] T. Andoh, T. Nagasawa, Y. Kuraishi, Expression of tachykinin NK1 Carlson (Eds.), Progress in Brain Research, Vol. 71, Elsevier,

receptor mRNA in dorsal root ganglia of the mouse, Brain Res. Mol. Amsterdam, 1987, pp. 113–120.

Brain Res. 35 (1996) 329–332. [20] P.G. Green, A.I. Basbaum, J.D. Levine, Sensory neuropeptide [2] G. Battaglia, A. Rustioni, Coexistence of glutamate and substance P interactions in the production of plasma extravasation in the rat,

in dorsal root ganglion neurons of the rat and monkey, J. Comp. Neuroscience 50 (1992) 745–749.

Neurol. 277 (1988) 302–312. [21] U. Hanesch, B. Heppelmann, R.F. Schmidt, Somatostatin-like [3] C.G. Boehmer, J. Norman, M. Catton, L.G. Fine, P.W. Mantyh, High immunoreactivity in primary afferents of the medial articular nerve levels of mRNA coding for substance P, somatostatin anda-tubulin and colocalization with substance P in the cat, J. Comp. Neurol. 354 are expressed by rat and rabbit dorsal root ganglia neurons, Peptides (1995) 345–352.

10 (1989) 1179–1194. [22] U. Hanesch, U. Pfrommer, B.D. Grubb, H.G. Schaible, Acute and [4] S.D. Brain, T.J. Williams, Inflammatory oedema induced by syner- chronic phases of unilateral inflammation in rat’s ankle are associ-gism between calcitonin gene-related peptide (CGRP) and mediators ated with an increase in the proportion of calcitonin gene-related of increased vascular permeability, Br. J. Pharmacol. 86 (1985) peptide-immunoreactive dorsal root ganglion cells, Eur. J. Neurosci.

855–860. 5 (1993) 154–161.

[5] S. Brimijoin, J.M. Lundberg, E. Brodin, T. Hokfelt, G. Nilsson, [23] A. Harmar, P. Keen, Synthesis, and central and peripheral axonal Axonal transport of substance P in the vagus and sciatic nerves of transport of substance P in a dorsal root ganglion-nerve preparation the guinea pig, Brain Res. 191 (1980) 443–457. in vitro, Brain Res. 231 (1982) 379–385.

[6] M. Burg, D.S. Zahm, M.M. Knuepfer, Immunocytochemical co- [24] T. Hokfelt, Neuropeptides in perspective: the last ten years, Neuron localization of substance P and calcitonin gene-related peptide in 7 (1991) 867–879.

afferent renal nerve soma of the rat, Neurosci. Lett. 173 (1994) [25] T. Hokfelt, R. Elde, O. Johansson, R. Luft, G. Nilsson, A. Arimura, 87–93. Immunohistochemical evidence for separate populations of

somatos-ˆ

[7] R.E. Cull, Role of axonal transport in maintaining central synaptic tatin-containing and substance P-containing primary afferent neu-connections, Exp. Brain Res. 24 (1975) 97–101. rons in the rat, Neuroscience 1 (1976) 131–136.

[8] P. Delree, D. Martin, C. Sadzot-Delvaux, B. Rogister, P. Leprince, P. [26] T. Hokfelt, J-O. Kellerth, G. Nilsson, B. Pernow, Experimental Robe, J.M. Rigo, P.P. Lefebvre, B. Malgrange, J. Schoenen, G. immunohistochemical studies on the localization and distribution of Moonen, In vitro and in vivo modulation of 5-hydroxytryptamine-, substance P in cat primary sensory neurons, Brain Res. 100 (1975) thyrotropin-releasing hormone- and calcitonin-gene related peptide- 235–252.

like immunoreactivities in adult rat sensory neurons, Neuroscience [27] L.C. Holford, P. Case, S.N. Lawson, Substance P, neurofilament, 51 (1992) 401–410. peripherin and SSEA4 immunocytochemistry of human dorsal root [9] A. Dray, Chemosensitivity of cultured dorsal root ganglia cells, ganglion neurons obtained from post-mortem tissue: a quantitative

(8)

[28] H.-Z. Hu, Z.-W. Li, J.-Q. Si, Evidence for the existence of substance [46] K. Noguchi, R. Dubner, M. De Leon, E. Senba, M.A. Ruda, P autoreceptor in the membrane of rat dorsal root ganglion neurons, Axotomy induces preprotachykinin gene expression in a subpopula-Neuroscience 77 (1997) 535–541. tion of dorsal root ganglion neurons, J. Neurosci. Res. 37 (1994) [29] J.L. Hylden, G.L. Wilcox, Intrathecal substance P elicits a caudally- 596–603.

directed biting and scratching behavior in mice, Brain Res. 217 [47] K. Noguchi, E. Senba, Y. Morita, M. Sato, M. Tohyama,a-CGRP (1981) 212–215. and b-CGRP mRNAs are differentially regulated in the rat spinal [30] A. Ishida-Yamamoto, E. Senba, Cell types and axonal sizes of cord and dorsal root ganglion, Brain Res. Mol. Brain Res. 7 (1990)

calcitonin gene-related peptide-containing primary sensory neurons 299–304.

of the rat, Brain Res. Bull. 24 (1990) 759–764. [48] F. Nothias, A. Tessler, M. Murray, Restoration of substance P and [31] T. Jessell, A. Tsunoo, I. Kanazawa, M. Otsuka, Substance P: calcitonin gene-related peptide in dorsal root ganglia and dorsal horn depletion in the dorsal horn of rat spinal cord after section of the after neonatal sciatic nerve lesion, J. Comp. Neurol. 334 (1993) peripheral processes of primary sensory neurons, Brain Res. 168 370–384.

(1979) 247–259. [49] M. Otsuka, K. Yoshioka, Neurotransmitter functions of mammalian ¨

[32] G. Ju, T. Hokfelt, E. Brodin, J. Fahrenkrug, J.A. Fischer, P. Frey, R. tachykinins, Physiol. Rev. 73 (1993) 229–308.

P, J.C. Elde, Brown, Primary sensory neurons of the rat showing [50] G.A. Phares, P.E. Lloyd, Purification, primary structure, and neuro-calcitonin gene-related peptide immunoreactivity and their relation nal localization of cerebral peptide 1 from Aplysia, Peptides 17 to substance P-, somatostatin-, galanin-, vasoactive intestinal poly- (1996) 753–761.

peptide- and cholecystokinin-immunoreactive ganglion cells, Cell [51] P.D. Ryu, G. Gerber, K. Murase, M. Randic, Calcitonin gene-related Tissue Res. 247 (1987) 417–431. peptide enhances calcium current of rat dorsal root ganglion neurons [33] I. Kangrga, M. Randic, Tachykinins and calcitonin gene-related and spinal excitatory synaptic transmission, Neurosci. Lett. 89

peptide enhance release of endogenous glutamate and aspartate from (1988) 305–312.

the rat spinal dorsal horn slice, J. Neurosci. 10 (1990) 2026–2038. [52] P.D. Ryu, K. Murase, G. Gerber, M. Randic, Actions of calcitonin [34] Y. Kashihara, M. Sakaguchi, M. Kuno, Axonal transport and gene-related peptide on rat sensory ganglion neurones, Physiol.

distribution of endogenous calcitonin gene-related peptide in rat Bohemoslovaca 37 (1988) 259–265.

peripheral nerve, J. Neurosci. 9 (1989) 3796–3802. [53] H.-S. Schaible, On the role of tachykinins and calcitonin gene-[35] P. Keen, A.J. Harmar, F. Spears, E. Winter, Biosynthesis, axonal related peptide in the spinal mechanisms of nociception and in the transport and turnover of neuronal substance P, Ciba Found. Symp. induction and maintenance of inflammation-evoked hyperexcitability 91 (1982) 145–164. in spinal cord neurons (with special reference to nociception in [36] J.A. Kessler, I.B. Black, Similarities in development of substance P joints), in: T. Kumazawa, L. Kruger, K. Mizumura (Eds.), The and somatostatin in peripheral sensory neurons: effects of capsaicin Polymodal Receptor: a Gateway to Pathological Pain, Progress in and nerve growth factor, Proc. Natl. Acad. Sci. USA 78 (1981) Brain Research, Vol. 113, Elsevier, Amsterdam, 1996, pp. 423–441. 4644–4647. [54] J. Schoenen, P. Delree, P. Leprince, G. Moonen, Neurotransmitter [37] L. Kruger, C. Sternini, N.C. Brecha, P.W. Mantyh, Distribution of phenotype plasticity in cultured dissociated adult rat dorsal root calcitonin gene-related peptide immunoreactivity in relation to the ganglia: an immunocytochemical study, J Neurosci. Res. 22 (1989) rat central somatosensory projection, J. Comp. Neurol. 273 (1988) 473–487.

149–162. [55] G. Segond von Banchet, M. Petersen, H.-G. Schaible, Expression of [38] J.-P. Lai, S.D. Douglas, W.-Z. Ho, Human lymphocytes express neurokinin-1 receptors on cultured dorsal root ganglion neurons

substance P and its receptor, J. Neuroimmunol. 86 (1998) 80–86. from the adult rat, Neuroscience 90 (1999) 677–684.

[39] Y. Lee, K. Takami, Y. Kawai, S. Girgis, C.J. Hillyard, I. MacIntyre, [56] E.W. Sommer, J. Kazimierczak, B. Droz, Neuronal subpopulations P.C. Emson, M. Tohyama, Distribution of calcitonin gene-related in the dorsal root ganglion of the mouse as characterized by peptide in the rat peripheral nervous system with reference to its combination of ultrastructural and cytochemical features, Brain Res. coexistence with substance P, Neuroscience 15 (1985) 1227–1237. 346 (1985) 310–326.

[40] F. Lembeck, P. Holzer, Substance P as neurogenic mediator of [57] C. Sternini, K. Anderson, Calcitonin gene-related peptide-containing antidromic vasodilation and neurogenic plasma extravasation, neurons supplying the rat digestive system: differential distribution Naunyn-Schmiedeberg’s Arch. Pharmacol. 310 (1979) 175–183. and expression pattern, Somatosensory Motor Res. 9 (1992) 45–59. [41] H.S. Li, Z.Q. Zhao, Small sensory neurons in the rat dorsal root [58] P. Szucs, E. Polgar, I. Spigelman, R. Porszasz, I. Nagy, Neurokinin-ganglia express functional NK-1 tachykinin receptor, Eur. J. Neuro- 1 receptor expression in dorsal root ganglion neurons of young rats, sci. 10 (1998) 1292–1299. J. Periph. Nerv. System. 4 (1999) 270–278.

[42] J.E. Maggio, J.C. Hunter, Regional distribution of kassinin-like [59] G.S. von Banchet, H-G. Schaible, Localization of the neurokinin 1 immunoreactivity in rat central andperipheral tissues and the effect receptor on a subset of substance P-positive and isolectin B4-of capsaicin, Brain Res. 307 (1984) 370–373. negative dorsal root ganglion neurons of the rat, Neurosci. Lett. 274 [43] P.W. McCarthy, S.N. Lawson, Cell type and conduction velocity of (1999) 175–178.

rat primary sensory neurons with calcitonin gene-related peptide-like [60] S.J. Wimalawansa, Calcitonin gene-related peptide and its receptors: immunoreactivity, Neuroscience 34 (1990) 623–632. molecular genetics, physiology, pathophysiology, and therapeutic [44] P.K. Mulderry, M.A. Ghatei, R.A. Spokes, P.M. Jones, A.M. potentials, Endocr. Rev. 17 (1996) 533–585.

Pierson, Q.A. Hamid, S. Kanse, S.G. Amara, J.M. Burrin, S. Legon, [61] L. Xing, J. Guo, J. Tang, Y. Tang, X. Wang, Morphological evidence J.M. Polak, S.R. Bloom, Differential expression of a-CGRP and for the location of calcitonin gene-related peptide (CGRP)

immuno-b-CGRP by primary sensory neurons and enteric autonomic neurons reactivity in rat lymphocytes, Cell Vision 5 (1998) 8–12. of the rat, Neuroscience 25 (1988) 195–205. [62] X-F. Zhou, W-P. Gai, R-A. Rush, CGRP immunoreactive neurons in [45] R.A. Nicoll, C. Schenker, S.E. Leeman, Substance P as a transmitter rat dorsal root ganglia do not all contain low-affinity NGF receptor

(1)

186 H. Hiruma et al. / Brain Research 883 (2000) 184 –191

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

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 m

m; width,

$

1.0 m

m) 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 m

m). In control extracellular medium (Hepes-buffered

3. Results

saline, pH 7.3, 37

8 C), mean numbers of particles (per min)

transported in anterograde and retrograde directions were

3.1. Morphological and immunocytochemical properties

63.3

6 16.3 (mean

6 S.D., n

5 19) and 63.1

6 25.3 (n

5 19),

respectively. The instantaneous velocity of moving

par-Most (

.

98%) of isolated and cultured adult mouse

ticles varied from 0.03 to 2.48

m

m / s in control medium.

DRG neurons were small (diameter,

,

25 m

m) and medium

(diameter, 25–40

m

m) 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

(2)

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 (n

5 5) (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; n

5 4). 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).

(3)

188 H. Hiruma et al. / Brain Research 883 (2000) 184 –191

4. Discussion

CGRP is modified by culture [8,54], influences of culture

are also considerable factors.

In the present study, we clarified the effects of sensory

We applied video-enhanced microscopy to investigate

neuropeptides, substance P and CGRP, on axonal transport

the effects of neuropeptides on axonal transport in such

in isolated and cultured adult mouse DRG neurons. These

small- and medium-sized DRG neurons. Application of

neurons were small and medium in size, and 89.5% of the

substance P decreased the number and instantaneous

DRG neurons were immunoreactive for substance P and

velocity of particles transported in anterograde and

retro-66.0% of neurons expressed immunoreactivity for CGRP.

grade directions. In contrast,

a

-CGRP increased the

num-In the intact DRG, it has been reported that 10–25% of

ber and velocity of particles transported bidirectionally.

neurons

are

immunoreactive

for

substance

P

Application of

b

-CGRP had no effect on axonal transport.

[2,6,21,25,27,48] and 25–100% of neurons are immuno-

These responses occurred in all the DRG neurons tested.

reactive for CGRP [6,22,43,48,62]. Thus, the expression

The immunocytochemical staining revealed that the

neu-ratios of these neuropeptides in isolated and cultured DRG

rons examined were positive as well as negative to

neurons are different from those in DRG neurons in situ. It

substance P and CGRP, although positive neurons were

has been described that immunoreactivity for substance P

predominant in accord with the proportion of positive

is present mainly in small- and medium-sized DRG

cells. From these findings, it is suggested that substance P

neurons [25], whereas CGRP immunoreactivity distributes

and

a

-CGRP can modulate axonal transport in most of

in small to large-sized cells [15,30,32,37,39]. Therefore,

isolated and cultured DRG neurons, regardless of whether

the difference in expression ratios may be due to the size

or not the cells contain these neuropeptides. Supporting

distribution of cells. In addition, because the percentage of

our findings, several experimental methods have

demon-adult DRG neurons immunostaining for substance P and

strated that substance P and CGRP receptors are expressed

(4)

in DRG neurons. Substance P receptor (neurokinin-1

and

b

-CGRP can both couple all CGRP receptor subtypes

receptor) immunoreactivities and binding sites have been

[60]. Therefore, one possibility for explaining our results is

found in isolated and cultured rat DRG neurons [55,59] as

that mouse DRG neurons may express CGRP receptor

well as in the intact rat DRG [58]. Neurokinin-1 receptor

subtypes exclusively selective to

a

-CGRP.

mRNA has also been identified in the mouse DRG by

It has been well documented that substance P and

using reverse transcription-polymerase chain reaction (RT-

mRNA encoding the precursor for substance P are present

PCR) [1]. The neurokinin-1 receptor seems to be present

in the rat DRG [3,25,46]. Both

a

-CGRP and

b

-CGRP and

mostly in small- and medium-sized DRG cells [41,55].

their mRNAs are also expressed in rat DRG neurons

Several electrophysiological studies have revealed that the

[16,44,47,57], but

a

-CGRP is predominant [16,44,57].

majority (90%) of acutely isolated rat DRG neurons are

Thus, axonal transport in sensory neurons may be

con-sensitive to exogenously applied substance P [10,28],

trolled by substance P and

a

-CGRP which can be released

suggesting that substance P receptor is expressed on the

from its own and adjacent sensory neurons, through

cell membrane of most isolated DRG cells. Although

autocrine and paracrine mechanisms. In addition, recent

CGRP receptors have not yet been identified in DRG

evidence has shown that substance P and CGRP are

neurons, electrophysiological studies have shown that

synthesized and released by lymphocytes [38,61]. Thus,

CGRP elicits excitatory responses in rat DRG neurons in

these neuropeptides derived from lymphocytes may also be

situ [51,52], suggesting that DRG neurons functionally

involved in the modification of axonal transport.

express CGRP receptors. As described above, we found

Substance P and CGRP are released from sensory

that

a

-CGRP but not

b

-CGRP was effective to modify

neurons simultaneously in response to inflammation [13].

axonal transport. In this regard, it is described that there

These neuropeptides cooperatively induce vasodilatation

may be some subtypes of CGRP receptors with different

and extravasation in peripheral tissue [4,14,20]. In a

selectivity to

a

-CGRP and

b

-CGRP, although

a

-CGRP

similar way, nociceptive transmission is modulated by

(5)

190 H. Hiruma et al. / Brain Research 883 (2000) 184 –191

[10] A. Dray, R.D. Pinnock, Effects of substance P on adult rat sensory

synergic action of these neuropeptides [33,49]. However,

ganglion neurones in vitro, Neurosci. Lett. 33 (1982) 61–66.

the effects of substance P and

a

-CGRP observed in this

[11] H.A. Fernandez, J.A. Donoso, Axonal transport involvement in

study were opposite, the former was inhibitory and the

_{long-lasting synaptic modifications in Blatta orientalis, J. Neurobiol.}

latter was stimulatory. In addition, the duration of the

14 (1983) 61–75.

effect of CGRP was transient whereas the effect of

[12] H.L. Fernandez, C.A. Hodges-Savola, Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as a function

substance P was prolonged. Thus, sensory neuropeptides

of age, Neurochem. Res. 19 (1994) 1369–1377.

may contribute to the fine and balanced regulation of

[13] M.T. Galeazza, M.G. Garry, H.J. Yost, K.A. Strait, K.M.

Har-axonal transport in sensory neurons.

_{greaves, V.S. Seybold, Plasticity in the synthesis and storage of}

In the present study, we demonstrated that substance P

_{substance P and calcitonin gene-related peptide in primary afferent}

inhibited and

a

-CGRP stimulated axonal transport in

neurons during peripheral inflammation, Neuroscience 66 (1995)

443–458.

sensory neurons. Axonal transport is fundamental to

[14] R. Gamse, A. Saria, Potentiation of tachykinin-induced plasma

neuronal formation and synaptic transmission, therefore,

protein extravasation by calcitonin gene-related peptide, Eur. J.

these neuropeptides can modify axonal transport to affect

_{Pharmacol. 114 (1985) 61–66.}

development and function of the sensory nervous system.

_{[15] S.J. Gibson, J.M. Polak, S.R. Bloom, I.M. Sabate, P.M. Mulderry,}

M.A. Ghatei, G.P. McGregor, J.F. Morrison, J.S. Kelly, R.M. Evans, M.G. Rosenfeld, Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci. 4

Acknowledgements

(1984) 3101–3111.

[16] S.J. Gibson, J.M. Polak, A. Giaid, Q.A. Hamid, S. Kar, P.M. Jones,

This work was partly supported by the Academic

_{P. Denny, S. Legon, S.G. Amara, R.K. Craig, S.R. Bloom, R.J.S.}

Frontier Project from the Ministry of Education, Science,

Penketh, C. Rodek, N.B.N. Ibrahim, A. Dawson, Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of

Sports and Culture, Japan; a Grand-in-Aid for Scientific

the dorsal root ganglia and also in spinal motoneurones in man and

Research (No. 11670638) from the Ministry of Education,

rat, Neurosci. Lett. 91 (1988) 283–288.

Science, Sports and Culture, Japan; and Comprehensive

[17] M.S. Gold, S. Dastmalchi, J.D. Levine, Co-expression of nociceptor

Research on Aging and Health (H10-Aging and Health-

_{properties in dorsal root ganglion neurons from the adult rat in vitro,}

008), Health Science Research Grants, from the Ministry

Neuroscience 71 (1996) 265–275.

of Health and Welfare, Japan.

[18] A. Gorio, The role of axoplasmic transport in the restoration of synaptic transmission and in the process of sprouting during nerve regeneration, Adv. Exp. Med. Biol. 209 (1987) 41–49.

[19] B. Grafstein, D.W. Burmeister, C.M. McGuinness, G.W. Perry, J.R.

References

_{Sparrow, Role of fast axonal transport in regeneration of goldfish} optic axons, in: Neural Regeneration, F.J. Seil, E. Herbert, B.M. [1] T. Andoh, T. Nagasawa, Y. Kuraishi, Expression of tachykinin NK1 Carlson (Eds.), Progress in Brain Research, Vol. 71, Elsevier,

receptor mRNA in dorsal root ganglia of the mouse, Brain Res. Mol. Amsterdam, 1987, pp. 113–120.

in dorsal root ganglion neurons of the rat and monkey, J. Comp. Neuroscience 50 (1992) 745–749.

10 (1989) 1179–1194. [22] U. Hanesch, U. Pfrommer, B.D. Grubb, H.G. Schaible, Acute and

[4] S.D. Brain, T.J. Williams, Inflammatory oedema induced by syner- chronic phases of unilateral inflammation in rat’s ankle are associ-gism between calcitonin gene-related peptide (CGRP) and mediators ated with an increase in the proportion of calcitonin gene-related of increased vascular permeability, Br. J. Pharmacol. 86 (1985) peptide-immunoreactive dorsal root ganglion cells, Eur. J. Neurosci.

855–860. 5 (1993) 154–161.

afferent renal nerve soma of the rat, Neurosci. Lett. 173 (1994) [25] T. Hokfelt, R. Elde, O. Johansson, R. Luft, G. Nilsson, A. Arimura,

87–93. Immunohistochemical evidence for separate populations of

somatos-ˆ

like immunoreactivities in adult rat sensory neurons, Neuroscience [27] L.C. Holford, P. Case, S.N. Lawson, Substance P, neurofilament,

51 (1992) 401–410. peripherin and SSEA4 immunocytochemistry of human dorsal root

[9] A. Dray, Chemosensitivity of cultured dorsal root ganglia cells, ganglion neurons obtained from post-mortem tissue: a quantitative News Physiol. Sci. 11 (1996) 288–292. morphometric analysis, J. Neurocytol. 23 (1994) 577–589.

(6)

directed biting and scratching behavior in mice, Brain Res. 217 [47] K. Noguchi, E. Senba, Y. Morita, M. Sato, M. Tohyama,a-CGRP

(1981) 212–215. and b-CGRP mRNAs are differentially regulated in the rat spinal

[30] A. Ishida-Yamamoto, E. Senba, Cell types and axonal sizes of cord and dorsal root ganglion, Brain Res. Mol. Brain Res. 7 (1990) calcitonin gene-related peptide-containing primary sensory neurons 299–304.

(1979) 247–259. [49] M. Otsuka, K. Yoshioka, Neurotransmitter functions of mammalian

[32] G. Ju, T. Hokfelt, E. Brodin, J. Fahrenkrug, J.A. Fischer, P. Frey, R. tachykinins, Physiol. Rev. 73 (1993) 229–308.

peptide enhance release of endogenous glutamate and aspartate from (1988) 305–312.

distribution of endogenous calcitonin gene-related peptide in rat Bohemoslovaca 37 (1988) 259–265.

91 (1982) 145–164. in spinal cord neurons (with special reference to nociception in

[36] J.A. Kessler, I.B. Black, Similarities in development of substance P joints), in: T. Kumazawa, L. Kruger, K. Mizumura (Eds.), The and somatostatin in peripheral sensory neurons: effects of capsaicin Polymodal Receptor: a Gateway to Pathological Pain, Progress in and nerve growth factor, Proc. Natl. Acad. Sci. USA 78 (1981) Brain Research, Vol. 113, Elsevier, Amsterdam, 1996, pp. 423–441.

4644–4647. [54] J. Schoenen, P. Delree, P. Leprince, G. Moonen, Neurotransmitter

[37] L. Kruger, C. Sternini, N.C. Brecha, P.W. Mantyh, Distribution of phenotype plasticity in cultured dissociated adult rat dorsal root calcitonin gene-related peptide immunoreactivity in relation to the ganglia: an immunocytochemical study, J Neurosci. Res. 22 (1989) rat central somatosensory projection, J. Comp. Neurol. 273 (1988) 473–487.

149–162. [55] G. Segond von Banchet, M. Petersen, H.-G. Schaible, Expression of

[38] J.-P. Lai, S.D. Douglas, W.-Z. Ho, Human lymphocytes express neurokinin-1 receptors on cultured dorsal root ganglion neurons substance P and its receptor, J. Neuroimmunol. 86 (1998) 80–86. from the adult rat, Neuroscience 90 (1999) 677–684.

sci. 10 (1998) 1292–1299. J. Periph. Nerv. System. 4 (1999) 270–278.