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Vanilloid receptor loss is independent of the messenger plasticity that follows systemic resiniferatoxin administration
BRAIN RESEARCH ELSEVIER
Brain Research 719 (1996) 213-218
Short c o m m u n i c a t i o n
Vanilloid receptor loss is independent of the messenger plasticity that follows systemic resiniferatoxin administration T u n d e F a r k a s - S z a l l a s i a,b G a r y J. B e n n e t t c P e t e r M . B l u m b e r g a, T o m a s HiSkfelt b
Jan M. L u n d b e r g a, A r p a d Szallasi a, * a Department of Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden b Department ofNeuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden c National Institute of Dental Research, N.LH., Bethesda, MD 20892, USA d National Cancer Institute, N.LH., Bethesda, MD 20892, USA
Accepted 3 January 1996
Abstract
Resiniferatoxin (RTX) depletes vanilloid (capsaicin) receptors from lumbar dorsal root ganglia (DRG) of the rat. In addition, RTX causes changes in neuropeptide and nitric oxide synthase expression in lumbar DRG neurons, similar to those described following axotomy; this latter phenomenon is referred to as messenger plasticity. These findings suggested that vanilloid receptor loss may be part of the plasticity that follows RTX treatment. Here we show that vanilloid receptor expression, as detected by [3H]RTX autoradiography, is not changed in lumbar DRGs of axotomized rats, nor is it altered in a rat model (chronic constriction injury) of neuropathic pain. Thus, the in vivo expression of vanilloid receptors detected by specific [3H]RTX binding does not require the presence of intraaxonally transported trophic factors such as nerve growth factor. We conclude that messenger plasticity and vanilloid receptor loss are mediated by distinct mechanisms. Keywords: Messenger plasticity; Axotomy; Chronic constriction injury rat; Vanilloid receptor; Resiniferatoxin
Following peripheral axotomy, the expression of neuropeptides and the receptors at which they interact are altered in primary sensory neurons [14]. In general, excitatory neuropeptides such as substance P are down-regulated [16] whereas inhibitory neuropeptides (e.g., galanin or neuropeptide Y) are increased [13,36]. Axotomy also induces the expression of nitric oxide synthase (NOS) in these neurons [9,32,39]. These changes, collectively referred to as messenger plasticity, are suggested to promote neuronal survival and adaptation [14]. Although the mechanism(s) underlying this plasticity is (are) only beginning to be understood, a deprivation of trophic factors (e.g., nerve growth factor and leukemia inhibitory factor) made in tissues innervated by primary sensory neurons and transported retrogradely back to the cell bodies of these neurons is a very likely candidate mechanism [10,19,24,33]. A subset of these primary sensory neurons is excited
* Corresponding author. Fax: (46) (8) 33 22 78; e-mail: arpad.szallasi@ fyfa.ki.se Elsevier Science B.V. SSDI 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 0 6 5 - 0
and then subsequently desensitized by capsaicin, the pungent principle in hot pepper [6,12]. Capsaicin by an ill-defined mechanism blocks intraaxonal transport [17,22], leading to 'chemical nerve lesion' [15]. Thus, capsaicin may induce messenger plasticity similar to that described following mechanical nerve lesion. In fact, recently we have demonstrated increased levels of galanin, vasoactive intestinal polypeptide (VIP) and NOS in lumbar DRG of the rat [8] following systemic administration of resiniferatoxin (RTX), an ultrapotent analog of capsaicin [5,26]. Increased expression of neuronal NOS has also been described after systemic capsaicin administration [34]. Capsaicin- and RTX-like molecules are collectively referred to as vanilloids, thus, the receptors at which these compounds interact may be termed vanilloid receptors [27]. In vivo vanilloid treatment is followed by a loss of vanilloid receptors [11,28]. It is known that rat DRG neurons cultured in vitro respond to capsaicin only when the cells are maintained in the presence of nerve growth factor (NGF) [2,4,37,38]. A plausible prediction is that vanilloid receptor loss would thus be part of the plasticity
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associated with N G F deprivation. Furthermore, this model would predict a similar loss of vanilloid receptors on the cell bodies of primary sensory neurons following surgical nerve injury. Taking advantage of a recent autoradiographic method using [3H]RTX to visualize vanilloid receptors [30], this study aimed to examine vanilloid receptor expression in lumbar DRG of the rat following sectioning or chronic constriction injury (CCI) of the sciatic nerve [3], respectively. In addition, vanilloid receptor and neuropeptide (galanin and VIP) expression were evaluated using adjacent sections obtained from lumbar DRGs of rats given RTX subcutaneously• Adult female S p r a g u e - D a w l e y rats weighing 2 0 0 - 2 5 0 g received a single, s.c. dose of RTX (LC Laboratories), 100 / x g / k g injected in a volume of 100 /xl ethanol into the scruff of the neck while the the animals were under light ether anesthesia [28]. Alternatively, the animals underwent surgery as follows: briefly, sciatic nerves of the animals anesthesized with sodium pentobarbital (40 m g / k g i.p.) were exposed at mid-thigh on both sides and the left sciatic nerve was cut (axotomy). In other animals (male S p r a g u e - D a w l e y rats weighing 2 9 0 - 3 3 0 g at the time of the surgery), four ligatures were tied loosely about the exposed left sciatic nerve with approximately 1 mm spacing to induce so-called chronic constriction injury (CCI) as a result of the intraneural edema formation [3]; right sciatic nerves (exposed but not strangulated) served as sham-operated controls. From animals treated with RTX or axotomy, lumbar DRG ( L 3 - L 5 ) were collected from both sides 1 and 7 days as well as 2, 4 and 8 weeks after treatment• Rats with CCI were sacrificed on the 20th day post-operation• All animals were killed by an overdose of sodium pentobarbital and then perfused transcardially with ice-cold phosphate-buffered saline. Ganglia were collected into ice-cold saline and then multiple ganglia were frozen in saline on the same chuck so that treated and control DRGs could be processed on the same slides. Animal experimentation was carried out in accord with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1978), and was approved by the respective institutional Ethical Committees. For autoradiography with [3H]RTX (specific activity, 37 C i / m m o l ; synthesized by the Chemical Synthesis and Analysis Laboratory, NCI-FCRF) [30], 14 # M sections of the ganglia cut in a cryostat were thaw-mounted onto
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Fig. 1. Fihn [3H]resiniferatoxinautoradiograms of cryostat sections from lumbar dorsal root ganglia (L3-L5) of the rat, arranged in vertical groups, a: systemic resiniferatoxin (100 /xg/kg s.c.) treatment 14 days before: control, con: resiniferatoxin-treated, RTX. b: peripheral axotomy of the left sciatic nerve, sham operation on the right side: con, control: 1 w, post-operational day 7; 2 w, post-operational day 14. c: chronic constriction injury by loose ligatures on the left sciatic nervc: con, control: sham, sham operation; cci, chronic constriction injury. Bars indicate I ram.
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218
slides that had been precoated with chrome a l u m / g e l a t i n and were then stored at - 7 0 ° C until assayed. On the day of the assay, sections equilibrated to room temperature were incubated in a humid chamber with 1 nM [3H]RTX
215
in a buffer containing (in mM) KC1 5, NaC1 5.8, CaC12 0.75, MgC12 2, sucrose 320, HEPES 10 (pH 7.4), supplemented with 1 m g / m l bovine serum albumin (Cohn fraction V). Non-specific binding (background) was deter-
Fig. 2. Dark-field micrographs of lumbar dorsal root ganglia (L3-L5) after hybridization with probes complementary to mRNAs for galanin (a,b,c) or VIP (d,e,f.) in control rats (a,d), in rats given 100 /xg/kg resiniferatoxin subcutaneously 14 days before (b,e) and in rats with sectioned left sciatic nerve (14th post operational day; c,f). Bar indicates 50 /~m.
216
T. Farkas-Szallasi et al./Brain Research 719 (1996) 213-218
mined in the presence of 1 /xM non-radioactive RTX. Following a 60 min incubation at 37°C, slides were rinsed with ice-cold 20 mM Tris-C1 buffer (pH 7.4), washed 3 times (10 min each) with the above buffer supplemented with 0.1% ( w / v ) bovine serum albumin, immersed into ice-cold distilled water, dried rapidly in a stream of cold air, and finally exposed to Amersham Hyperfilm-3H for 5 weeks. Autoradiograms were placed on a Northern Light precision illuminator (Imaging Research) equipped with a Nikon lens and a Quick Capture frame grabber board (Data Translation); a sampling square of 10 × 10 pixels was overlaid on the cellular part of the dorsal root ganglia for a total of 30 times for each experimental group; and images were analyzed using a Macintosh PC with the Image Software 1.52 written by Dr. Wayne Raspband (Natl. Inst. Mental Health, Bethesda, MD). For in situ hybridization with probes (Scandinavian Gene Synthesis AB) complementary to nucleotides 324371 of rat galanin [35] and to nucleotides 347-394 of rat VIP [23], 14 /xM sections of the ganglia cut in a cryostat were thaw-mounted onto 'Probe On' slides (Fisher Scientific). The probes were labelled at the 3' end with a - 3 5 S dATP (New England Nuclear) using the terminal deoxynucleotidyl transferase (Amersham) reaction and then purified through Nensorb-20 columns (New England Nuclear) to yield specific activities in the range of 1-4 × 106 c p m / n g oligonucleotide, as described previously [7]. Hybridization was carried out for 16-18 h at 42°C in humifled boxes in the presence of 106 cpm of labelled probe per 100 /xl hybridization cocktail according to published procedures [7]. The specificity of the labelling was confirmed by adding an excess (100-fold) of non-radioactive probe to the hybridization mixture. After hybridization, the sections were rinsed repeatedly, equilibrated to room temperature, dipped into distilled water, dehydrated, and, finally, dried in air. Slides immersed into NTB2 nuclear track emulsion (Kodak) were exposed for 4 weeks at - 2 0 ° C and then developed. Slides were examined in a Nikon Mikrophot-FX microscope equipped for darkfield analysis. RTX treatment (100 /xg/kg s.c.) depleted vanilloid receptors (specific [3H]RTX binding sites) from rat DRGs; this phenomenon was observed before [28,31] and is confirmed here (Fig. la). Quantitatively, a significant, 37% decrease was observed in the density of autoradiographic labelling of lumbar DRG (L3-L5) sections by [3H]RTX 2 weeks after treatment (Table 1). In adjacent sections, as expected [8], the number of neuron profiles positive for galanin a n d / o r VIP mRNA, respectively, showed a marked increase (Fig. 2). However, contrary to the most straightforward expectation, no change was observed in the [3H]RTX labelling of lumbar DRG sections of axotomized rats (Fig. lb and Table 1), although adjacent sections showed the anticipated changes [13,251 in galanin and VIP mRNA positivities (Fig. 2). We could not detect any significant change in the autoradiographic labelling by
Table I Effect of systemic resiniferatoxin treatment (14 days) and peripheral axotomy (14 days) or chronic constriction injury (20 days) of the sciatic nerve on autoradiographic labelling by [3H]resiniferatoxin of lumbar dorsal root ganglion neurons (L3-L5) of the rat A. Resiniferatoxin (100 / x g / k g s.c.) treatment: 64 +_ l I% B. Axotomy: ipsilateral side - - 105 + 12% Contralateral side - - 93 _+8% C. Chronic constriction injury: (a) right (control) side; sham operation - - 104 _+ 11% (b) left (injury) side; sham operation - - 92 _+ 13% chronic constriction injury - - 90_+ 12% Optical densities are expressed in % of the respective control values. Mean_+ S.D.; 30 measurements for each experimental group. * One-tailed P < 0.0001. Statistical analysis was performed using the unpaired t-test (resiniferatoxin treatment) and the Tukey-Kramer multiple comparisons test (axotomy as well as chronic constriction injury), respectively.
[3H]RTX of lumbar DRG sections of rats with CCI either (Fig. l c and Table 1). Given the therapeutic potential of vanilloids to relieve neuropathic pain [20,29], this finding may be of practical importance. A simple interpretation of these findings is that vanilloid receptor expression in vivo, unlike that in vitro [2,4,37,38], does not depend on intraaxonally transported trophic factors such as NGF. It might likewise explain why we could not detect enhanced vanilloid receptor expression in lumbar DRG or spinal cord of rats with peripheral tissue inflammation (Ji, HiSkfelt and Szallasi, unpublished observation), known to up-regulate NGF production [18]. This interpretation, however, conflicts with the findings of Bevan and Winter [4] who observed an attenuated 45Ca2+ uptake in response to capsaicin by lumbar DRG neurons obtained from rats with sectioned sciatic nerve. Recently, Blumberg and coworkers [1] have suggested the existence of two vanilloid receptor classes with distinct structure-activity relations, detected by [3H]RTX binding and by calcium uptake assays, respectively. These different findings would be reconciled if these putative vanilloid receptor subclasses also differed in NGF-sensitivity. If this assumption holds true, axotomy, by selectively eliminating the NGF-sensitive receptors, may be a useful tool to study the function of these hypothetical vanilloid receptor types. Finally, the mechanism by which vanilloids cause receptor depletion remains to be resolved. Receptor loss by vanilloids in vivo is maximal as early as 24 h after treatment [28] which contrasts with the much slower, gradual loss of vanilloid sensitivity (with a half time of 3 days) in vitro [37]. Whereas the latter phenomenon might reflect a decreased transcription/translation of the receptor protein, the rapid in vivo receptor loss probably has a different underlying mechanism. When examined 6 h after treatment, no loss of specific [3H]RTX binding sites could be detected [28]; thus, this receptor loss does not seem to be rapid enough to reflect receptor internalization [21] either. Regardless of its mechanism, the presence/absence
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218
of vanilloid receptors appears to be an important biochemical difference between RTX-treated animals, on the one hand, and rats with sectioned sciatic nerve or CCI, on the other hand.
Acknowledgements This study was supported by the Swedish MRC (14X6554; O4X-2887) and the Magnus Bergvalls Stiftelse. The expert technical assistance of Mrs. Siv Nilsson and Ms. Katarina Aman is appreciated.
References [1] Acs, G., Lee, J., Marquez, V. and Blumberg, P.M., Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes, Mol. Brain Res., 35 (1996) 173182. [2] Aguayo, L.G. and White, G., Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons, Brain Res., 570 (1992) 61-67. [3] Bennett, G.J. and Xie Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107. [4] Bevan, S. and Winter, J., Nerve growth factor (NGF) differentially regulates the chemosensitivity of adult rat cultured sensory neurons, J. Neurosci., 15 (1995) 4918-4926. [5] Blumberg, P.M., Szallasi, A. and Acs, G., Resiniferatoxin- an ultrapotent capsaicin analogue. In J.N. Wood (Ed.), Capsaicin in the Study of Pain, Academic Press, New York, 1993, pp. 45-62. [6] Buck, T.F. and Burks, S.H., The neuropharmacology of capsaicin: a review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. [7] Dagerlind, A., Friberg, K., Bean, A.J. and H~Skfelt, T., Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry, Histochemistry, 98 (1992) 39-49. [8] Farkas-Szallasi, T., Lundberg, J.M., Wiesenfeld-Hallin, Z., Htikfelt, T. and Szallasi, A., Increased levels of GMAP, VIP and nitric oxide synthase, and their mRNAs, in lumbar dorsal root ganglia of the rat following systemic resiniferatoxin treatment, NeuroReport. 6 (1995) 2230-2234. [9] Fiallos-Estrada, C.E., Herdegen, T., Kummer, W., Mayer, B., Bravo, R. and Zimmermann, M., Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JU co-expression in rat dorsal root ganglion neurons following sciatic nerve transection, Neurosci. Lett., 150 (1993) 169-173. [10] Fitzgerald, M., Wall, P.D., Goedert, M. and Emson, P.C., Nerve growth factor counteracts the neurophysiological and neurochemical effects of chronic sciatic nerve section, Brain Res., 332 (1985) 131-141. [11] Goso, C., Piovacari, G. and Szallasi, A., Resiniferatoxin-induced loss of vanilloid receptors is reversible in the urinary bladder but not in the spinal cord of the rat, Neurosci. Lett., 162 (1993) 197-200. [12] Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 144-201. [13] Htikfelt, T., Wiesenfeld-Hallin, Z., Villar, M. and Melander, T., Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy, Neurosci. Lett., 83 (1987) 217-220.
217
[14] Htikfelt, T., Zhang, X. and Wiesenfeld-Hallin, Z., Messenger plasticity in primary sensory neurons following axotomy and its functional implications, Trends Neurosci., 17 (1994) 22-30. [15] Jancso, G., Pathobiological reactions of C-fibre primary sensory neurones to peripheral nerve injury. Exp. Physiol., 77 (1992) 405431. [16] Jessell, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P: Depletion in the dorsal horn of the rat spinal cord after section of the peripheral processes of primary sensory neurons, Brain Res., 168 (1979) 247-259. [17] Kawakami, T., Hikawa, N., Kusakabe, T., Kano, M., Bandou, Y., Gotoh, H. and Takenaka, T., Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture, J. Neurobiol., 24 (1993) 545-551. [18] Lewin, G.R., Ritter, A.M. and Mendel, L.M., Nerve growth factorinduced hyperalgesia in the neonatal and adult rat, J. Neurosci., 13 (1993) 2136-2148. [19] Lindsay, R.M. and Harmar, A.J., Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurones, Nature, 337 (1989) 362-364. [20] Lynn, B., Capsaicin: actions on nociceptive C-fibers and therapeutic potential, Pain, 41 (1990) 61-69. [21] Mantyh, P.W., DeMaster, E., Malhotra, A., Ghilardi, J.R., Rogers, S.D., Mantyh, C.R., Liu, H., Basbaum, A.I., Vigna, S.R., Maggio, J.E. and Simone, D.A., Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation, Science, 268 (1995) 1629-1632. [22] Miller, M.S., Buck, S.H., Sipes, I.G., Yamamura, H.I. and Burks, T.F., Regulation of substance P by nerve growth factor: disruption by capsaicin, Brain Res., 250 (1982) 193-196. [23] Nishizawa, M., Hayakawa, Y., Yanaihara, N. and Okamoto, H., Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat, FEBS Lett., 183 (1985) 55-59. [24] Rao, M.S., Sun, Y., Escary, J.L., Perreau, J., Tresser, S., Patterson, P.H., Zigmond, R.E., Brulet, P. and Landis, S.C., Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons, Neuron, 11 (1993) 1175-1185. [25] Shehab, S.A. and Atkinson, M.E., Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons, Brain Res., 372 (1986) 37-44. [26] Szallasi, A. and Blumberg, P.M., Resiniferatoxin, a phorbol-related diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper, Neuroscience, 30 (1989) 515-520. [27] Szallasi, A. and Blumberg, P.M., Resiniferatoxin and its analogs provide novel insights into the pharmacology of the vanilloid (capsaicin) receptor, Life Sci., 47 (1990) 1399-1408. [28] Szallasi, A. and Blumberg, P.M., Vanilloid receptor loss in rat sensory ganglia associated with long term desensitization to resiniferatoxin, Neurosci. Lett., 136 (1992) 51-54. [29] Szallasi, A. and Blumberg, P.M., Mechanisms and therapeutic potential of vanilloids (capsaicin-like molecules), Adv. Pharmacol., 24 (1993) 123-155. [30] Szallasi, A., Blumberg, P.M., Nilsson, S., H0kfelt, T. and Lundberg, J.M., Visualization by [3H]resiniferatoxin autoradiography of capsaicin-sensitive neurons in the rat, pig and man, Eur. Z Pharmacol., 264 (1994) 217-221. [31] Szallasi, A., Nilsson, S., Farkas-Szallasi, T., Blumberg, P.M., HiSkfelt, T. and Lundberg, J.M., Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment, Brain Res., 703 (1995) 175-183. [32] Verge, V.M.K., Zhang, X., Xu, X.-J., Wiesenfeld-Hallin, Z. and H~Skfelt, T., Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization
218
[33]
[34]
[35]
[36]
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218 and functional studies, Proc. Natl. Acad. Sci. USA, 89 (1992) 11617-11621. Verge, V.M.K., Richardson, P.M., Wiesenfeld-Hallin, Z. and H~Skfelt, T., Differential influence of nerve growth factor on neuropeptide expression in vivo - - a novel role in peptide suppression in adult sensory neurons, J. Neurosci., 15 (1995) 2081-2096. Vizzard, M.A., Erdman, S.L. and De Groat, W.C., Increased expression of neuronal nitric oxide synthase in dorsal root ganglion neurons after systemic capsaicin administration, Neuroscience, 67 (1995) 1-5. Vrontakis, M.E., Peden, L.M., Duckworth, M.L. and Friesen, H.G., Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA, J. Biol. Chem., 262 (35) 16755-16758. Wakisaka, S., Kajander, K.C. and Bennett, G.J., Increased neuropep-
tide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy, Neurosci. Lett., 124 (1991) 200-203. [37] Winter, J., Forbes, C.A., Sternberg, J. and Lindsay, R.M., Nerve growth factor (NGF) regulates adult rat dorsal root ganglion neuron responses to capsaicin, Neuron, 1 (1988) 973-981. [38] Winter, J., Walpole, C.S.J., Bevan, S. and James, I.F., Characterization of resiniferatoxin binding sites on sensory neurons: co-regulation of resiniferatoxin binding and capsaicin-sensitivity in adult rat dorsal root ganglia, Neuroscience, 57 (1993) 747-757. [39] Zhang, X., Verge, V.M.K., Wiesenfeld-Hallin, Z., Ju, G., Bredt, D., Snyder, S.H. and H/Skfelt, T., Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy, J. Comp. Neurol., 335 (1993) 563-575.
Brain Research 719 (1996) 213-218
Short c o m m u n i c a t i o n
Vanilloid receptor loss is independent of the messenger plasticity that follows systemic resiniferatoxin administration T u n d e F a r k a s - S z a l l a s i a,b G a r y J. B e n n e t t c P e t e r M . B l u m b e r g a, T o m a s HiSkfelt b
Jan M. L u n d b e r g a, A r p a d Szallasi a, * a Department of Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden b Department ofNeuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden c National Institute of Dental Research, N.LH., Bethesda, MD 20892, USA d National Cancer Institute, N.LH., Bethesda, MD 20892, USA
Accepted 3 January 1996
Abstract
Resiniferatoxin (RTX) depletes vanilloid (capsaicin) receptors from lumbar dorsal root ganglia (DRG) of the rat. In addition, RTX causes changes in neuropeptide and nitric oxide synthase expression in lumbar DRG neurons, similar to those described following axotomy; this latter phenomenon is referred to as messenger plasticity. These findings suggested that vanilloid receptor loss may be part of the plasticity that follows RTX treatment. Here we show that vanilloid receptor expression, as detected by [3H]RTX autoradiography, is not changed in lumbar DRGs of axotomized rats, nor is it altered in a rat model (chronic constriction injury) of neuropathic pain. Thus, the in vivo expression of vanilloid receptors detected by specific [3H]RTX binding does not require the presence of intraaxonally transported trophic factors such as nerve growth factor. We conclude that messenger plasticity and vanilloid receptor loss are mediated by distinct mechanisms. Keywords: Messenger plasticity; Axotomy; Chronic constriction injury rat; Vanilloid receptor; Resiniferatoxin
Following peripheral axotomy, the expression of neuropeptides and the receptors at which they interact are altered in primary sensory neurons [14]. In general, excitatory neuropeptides such as substance P are down-regulated [16] whereas inhibitory neuropeptides (e.g., galanin or neuropeptide Y) are increased [13,36]. Axotomy also induces the expression of nitric oxide synthase (NOS) in these neurons [9,32,39]. These changes, collectively referred to as messenger plasticity, are suggested to promote neuronal survival and adaptation [14]. Although the mechanism(s) underlying this plasticity is (are) only beginning to be understood, a deprivation of trophic factors (e.g., nerve growth factor and leukemia inhibitory factor) made in tissues innervated by primary sensory neurons and transported retrogradely back to the cell bodies of these neurons is a very likely candidate mechanism [10,19,24,33]. A subset of these primary sensory neurons is excited
* Corresponding author. Fax: (46) (8) 33 22 78; e-mail: arpad.szallasi@ fyfa.ki.se Elsevier Science B.V. SSDI 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 0 6 5 - 0
and then subsequently desensitized by capsaicin, the pungent principle in hot pepper [6,12]. Capsaicin by an ill-defined mechanism blocks intraaxonal transport [17,22], leading to 'chemical nerve lesion' [15]. Thus, capsaicin may induce messenger plasticity similar to that described following mechanical nerve lesion. In fact, recently we have demonstrated increased levels of galanin, vasoactive intestinal polypeptide (VIP) and NOS in lumbar DRG of the rat [8] following systemic administration of resiniferatoxin (RTX), an ultrapotent analog of capsaicin [5,26]. Increased expression of neuronal NOS has also been described after systemic capsaicin administration [34]. Capsaicin- and RTX-like molecules are collectively referred to as vanilloids, thus, the receptors at which these compounds interact may be termed vanilloid receptors [27]. In vivo vanilloid treatment is followed by a loss of vanilloid receptors [11,28]. It is known that rat DRG neurons cultured in vitro respond to capsaicin only when the cells are maintained in the presence of nerve growth factor (NGF) [2,4,37,38]. A plausible prediction is that vanilloid receptor loss would thus be part of the plasticity
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associated with N G F deprivation. Furthermore, this model would predict a similar loss of vanilloid receptors on the cell bodies of primary sensory neurons following surgical nerve injury. Taking advantage of a recent autoradiographic method using [3H]RTX to visualize vanilloid receptors [30], this study aimed to examine vanilloid receptor expression in lumbar DRG of the rat following sectioning or chronic constriction injury (CCI) of the sciatic nerve [3], respectively. In addition, vanilloid receptor and neuropeptide (galanin and VIP) expression were evaluated using adjacent sections obtained from lumbar DRGs of rats given RTX subcutaneously• Adult female S p r a g u e - D a w l e y rats weighing 2 0 0 - 2 5 0 g received a single, s.c. dose of RTX (LC Laboratories), 100 / x g / k g injected in a volume of 100 /xl ethanol into the scruff of the neck while the the animals were under light ether anesthesia [28]. Alternatively, the animals underwent surgery as follows: briefly, sciatic nerves of the animals anesthesized with sodium pentobarbital (40 m g / k g i.p.) were exposed at mid-thigh on both sides and the left sciatic nerve was cut (axotomy). In other animals (male S p r a g u e - D a w l e y rats weighing 2 9 0 - 3 3 0 g at the time of the surgery), four ligatures were tied loosely about the exposed left sciatic nerve with approximately 1 mm spacing to induce so-called chronic constriction injury (CCI) as a result of the intraneural edema formation [3]; right sciatic nerves (exposed but not strangulated) served as sham-operated controls. From animals treated with RTX or axotomy, lumbar DRG ( L 3 - L 5 ) were collected from both sides 1 and 7 days as well as 2, 4 and 8 weeks after treatment• Rats with CCI were sacrificed on the 20th day post-operation• All animals were killed by an overdose of sodium pentobarbital and then perfused transcardially with ice-cold phosphate-buffered saline. Ganglia were collected into ice-cold saline and then multiple ganglia were frozen in saline on the same chuck so that treated and control DRGs could be processed on the same slides. Animal experimentation was carried out in accord with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1978), and was approved by the respective institutional Ethical Committees. For autoradiography with [3H]RTX (specific activity, 37 C i / m m o l ; synthesized by the Chemical Synthesis and Analysis Laboratory, NCI-FCRF) [30], 14 # M sections of the ganglia cut in a cryostat were thaw-mounted onto
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Fig. 1. Fihn [3H]resiniferatoxinautoradiograms of cryostat sections from lumbar dorsal root ganglia (L3-L5) of the rat, arranged in vertical groups, a: systemic resiniferatoxin (100 /xg/kg s.c.) treatment 14 days before: control, con: resiniferatoxin-treated, RTX. b: peripheral axotomy of the left sciatic nerve, sham operation on the right side: con, control: 1 w, post-operational day 7; 2 w, post-operational day 14. c: chronic constriction injury by loose ligatures on the left sciatic nervc: con, control: sham, sham operation; cci, chronic constriction injury. Bars indicate I ram.
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218
slides that had been precoated with chrome a l u m / g e l a t i n and were then stored at - 7 0 ° C until assayed. On the day of the assay, sections equilibrated to room temperature were incubated in a humid chamber with 1 nM [3H]RTX
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in a buffer containing (in mM) KC1 5, NaC1 5.8, CaC12 0.75, MgC12 2, sucrose 320, HEPES 10 (pH 7.4), supplemented with 1 m g / m l bovine serum albumin (Cohn fraction V). Non-specific binding (background) was deter-
Fig. 2. Dark-field micrographs of lumbar dorsal root ganglia (L3-L5) after hybridization with probes complementary to mRNAs for galanin (a,b,c) or VIP (d,e,f.) in control rats (a,d), in rats given 100 /xg/kg resiniferatoxin subcutaneously 14 days before (b,e) and in rats with sectioned left sciatic nerve (14th post operational day; c,f). Bar indicates 50 /~m.
216
T. Farkas-Szallasi et al./Brain Research 719 (1996) 213-218
mined in the presence of 1 /xM non-radioactive RTX. Following a 60 min incubation at 37°C, slides were rinsed with ice-cold 20 mM Tris-C1 buffer (pH 7.4), washed 3 times (10 min each) with the above buffer supplemented with 0.1% ( w / v ) bovine serum albumin, immersed into ice-cold distilled water, dried rapidly in a stream of cold air, and finally exposed to Amersham Hyperfilm-3H for 5 weeks. Autoradiograms were placed on a Northern Light precision illuminator (Imaging Research) equipped with a Nikon lens and a Quick Capture frame grabber board (Data Translation); a sampling square of 10 × 10 pixels was overlaid on the cellular part of the dorsal root ganglia for a total of 30 times for each experimental group; and images were analyzed using a Macintosh PC with the Image Software 1.52 written by Dr. Wayne Raspband (Natl. Inst. Mental Health, Bethesda, MD). For in situ hybridization with probes (Scandinavian Gene Synthesis AB) complementary to nucleotides 324371 of rat galanin [35] and to nucleotides 347-394 of rat VIP [23], 14 /xM sections of the ganglia cut in a cryostat were thaw-mounted onto 'Probe On' slides (Fisher Scientific). The probes were labelled at the 3' end with a - 3 5 S dATP (New England Nuclear) using the terminal deoxynucleotidyl transferase (Amersham) reaction and then purified through Nensorb-20 columns (New England Nuclear) to yield specific activities in the range of 1-4 × 106 c p m / n g oligonucleotide, as described previously [7]. Hybridization was carried out for 16-18 h at 42°C in humifled boxes in the presence of 106 cpm of labelled probe per 100 /xl hybridization cocktail according to published procedures [7]. The specificity of the labelling was confirmed by adding an excess (100-fold) of non-radioactive probe to the hybridization mixture. After hybridization, the sections were rinsed repeatedly, equilibrated to room temperature, dipped into distilled water, dehydrated, and, finally, dried in air. Slides immersed into NTB2 nuclear track emulsion (Kodak) were exposed for 4 weeks at - 2 0 ° C and then developed. Slides were examined in a Nikon Mikrophot-FX microscope equipped for darkfield analysis. RTX treatment (100 /xg/kg s.c.) depleted vanilloid receptors (specific [3H]RTX binding sites) from rat DRGs; this phenomenon was observed before [28,31] and is confirmed here (Fig. la). Quantitatively, a significant, 37% decrease was observed in the density of autoradiographic labelling of lumbar DRG (L3-L5) sections by [3H]RTX 2 weeks after treatment (Table 1). In adjacent sections, as expected [8], the number of neuron profiles positive for galanin a n d / o r VIP mRNA, respectively, showed a marked increase (Fig. 2). However, contrary to the most straightforward expectation, no change was observed in the [3H]RTX labelling of lumbar DRG sections of axotomized rats (Fig. lb and Table 1), although adjacent sections showed the anticipated changes [13,251 in galanin and VIP mRNA positivities (Fig. 2). We could not detect any significant change in the autoradiographic labelling by
Table I Effect of systemic resiniferatoxin treatment (14 days) and peripheral axotomy (14 days) or chronic constriction injury (20 days) of the sciatic nerve on autoradiographic labelling by [3H]resiniferatoxin of lumbar dorsal root ganglion neurons (L3-L5) of the rat A. Resiniferatoxin (100 / x g / k g s.c.) treatment: 64 +_ l I% B. Axotomy: ipsilateral side - - 105 + 12% Contralateral side - - 93 _+8% C. Chronic constriction injury: (a) right (control) side; sham operation - - 104 _+ 11% (b) left (injury) side; sham operation - - 92 _+ 13% chronic constriction injury - - 90_+ 12% Optical densities are expressed in % of the respective control values. Mean_+ S.D.; 30 measurements for each experimental group. * One-tailed P < 0.0001. Statistical analysis was performed using the unpaired t-test (resiniferatoxin treatment) and the Tukey-Kramer multiple comparisons test (axotomy as well as chronic constriction injury), respectively.
[3H]RTX of lumbar DRG sections of rats with CCI either (Fig. l c and Table 1). Given the therapeutic potential of vanilloids to relieve neuropathic pain [20,29], this finding may be of practical importance. A simple interpretation of these findings is that vanilloid receptor expression in vivo, unlike that in vitro [2,4,37,38], does not depend on intraaxonally transported trophic factors such as NGF. It might likewise explain why we could not detect enhanced vanilloid receptor expression in lumbar DRG or spinal cord of rats with peripheral tissue inflammation (Ji, HiSkfelt and Szallasi, unpublished observation), known to up-regulate NGF production [18]. This interpretation, however, conflicts with the findings of Bevan and Winter [4] who observed an attenuated 45Ca2+ uptake in response to capsaicin by lumbar DRG neurons obtained from rats with sectioned sciatic nerve. Recently, Blumberg and coworkers [1] have suggested the existence of two vanilloid receptor classes with distinct structure-activity relations, detected by [3H]RTX binding and by calcium uptake assays, respectively. These different findings would be reconciled if these putative vanilloid receptor subclasses also differed in NGF-sensitivity. If this assumption holds true, axotomy, by selectively eliminating the NGF-sensitive receptors, may be a useful tool to study the function of these hypothetical vanilloid receptor types. Finally, the mechanism by which vanilloids cause receptor depletion remains to be resolved. Receptor loss by vanilloids in vivo is maximal as early as 24 h after treatment [28] which contrasts with the much slower, gradual loss of vanilloid sensitivity (with a half time of 3 days) in vitro [37]. Whereas the latter phenomenon might reflect a decreased transcription/translation of the receptor protein, the rapid in vivo receptor loss probably has a different underlying mechanism. When examined 6 h after treatment, no loss of specific [3H]RTX binding sites could be detected [28]; thus, this receptor loss does not seem to be rapid enough to reflect receptor internalization [21] either. Regardless of its mechanism, the presence/absence
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218
of vanilloid receptors appears to be an important biochemical difference between RTX-treated animals, on the one hand, and rats with sectioned sciatic nerve or CCI, on the other hand.
Acknowledgements This study was supported by the Swedish MRC (14X6554; O4X-2887) and the Magnus Bergvalls Stiftelse. The expert technical assistance of Mrs. Siv Nilsson and Ms. Katarina Aman is appreciated.
References [1] Acs, G., Lee, J., Marquez, V. and Blumberg, P.M., Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes, Mol. Brain Res., 35 (1996) 173182. [2] Aguayo, L.G. and White, G., Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons, Brain Res., 570 (1992) 61-67. [3] Bennett, G.J. and Xie Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107. [4] Bevan, S. and Winter, J., Nerve growth factor (NGF) differentially regulates the chemosensitivity of adult rat cultured sensory neurons, J. Neurosci., 15 (1995) 4918-4926. [5] Blumberg, P.M., Szallasi, A. and Acs, G., Resiniferatoxin- an ultrapotent capsaicin analogue. In J.N. Wood (Ed.), Capsaicin in the Study of Pain, Academic Press, New York, 1993, pp. 45-62. [6] Buck, T.F. and Burks, S.H., The neuropharmacology of capsaicin: a review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. [7] Dagerlind, A., Friberg, K., Bean, A.J. and H~Skfelt, T., Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry, Histochemistry, 98 (1992) 39-49. [8] Farkas-Szallasi, T., Lundberg, J.M., Wiesenfeld-Hallin, Z., Htikfelt, T. and Szallasi, A., Increased levels of GMAP, VIP and nitric oxide synthase, and their mRNAs, in lumbar dorsal root ganglia of the rat following systemic resiniferatoxin treatment, NeuroReport. 6 (1995) 2230-2234. [9] Fiallos-Estrada, C.E., Herdegen, T., Kummer, W., Mayer, B., Bravo, R. and Zimmermann, M., Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JU co-expression in rat dorsal root ganglion neurons following sciatic nerve transection, Neurosci. Lett., 150 (1993) 169-173. [10] Fitzgerald, M., Wall, P.D., Goedert, M. and Emson, P.C., Nerve growth factor counteracts the neurophysiological and neurochemical effects of chronic sciatic nerve section, Brain Res., 332 (1985) 131-141. [11] Goso, C., Piovacari, G. and Szallasi, A., Resiniferatoxin-induced loss of vanilloid receptors is reversible in the urinary bladder but not in the spinal cord of the rat, Neurosci. Lett., 162 (1993) 197-200. [12] Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 144-201. [13] Htikfelt, T., Wiesenfeld-Hallin, Z., Villar, M. and Melander, T., Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy, Neurosci. Lett., 83 (1987) 217-220.
217
[14] Htikfelt, T., Zhang, X. and Wiesenfeld-Hallin, Z., Messenger plasticity in primary sensory neurons following axotomy and its functional implications, Trends Neurosci., 17 (1994) 22-30. [15] Jancso, G., Pathobiological reactions of C-fibre primary sensory neurones to peripheral nerve injury. Exp. Physiol., 77 (1992) 405431. [16] Jessell, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P: Depletion in the dorsal horn of the rat spinal cord after section of the peripheral processes of primary sensory neurons, Brain Res., 168 (1979) 247-259. [17] Kawakami, T., Hikawa, N., Kusakabe, T., Kano, M., Bandou, Y., Gotoh, H. and Takenaka, T., Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture, J. Neurobiol., 24 (1993) 545-551. [18] Lewin, G.R., Ritter, A.M. and Mendel, L.M., Nerve growth factorinduced hyperalgesia in the neonatal and adult rat, J. Neurosci., 13 (1993) 2136-2148. [19] Lindsay, R.M. and Harmar, A.J., Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurones, Nature, 337 (1989) 362-364. [20] Lynn, B., Capsaicin: actions on nociceptive C-fibers and therapeutic potential, Pain, 41 (1990) 61-69. [21] Mantyh, P.W., DeMaster, E., Malhotra, A., Ghilardi, J.R., Rogers, S.D., Mantyh, C.R., Liu, H., Basbaum, A.I., Vigna, S.R., Maggio, J.E. and Simone, D.A., Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation, Science, 268 (1995) 1629-1632. [22] Miller, M.S., Buck, S.H., Sipes, I.G., Yamamura, H.I. and Burks, T.F., Regulation of substance P by nerve growth factor: disruption by capsaicin, Brain Res., 250 (1982) 193-196. [23] Nishizawa, M., Hayakawa, Y., Yanaihara, N. and Okamoto, H., Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat, FEBS Lett., 183 (1985) 55-59. [24] Rao, M.S., Sun, Y., Escary, J.L., Perreau, J., Tresser, S., Patterson, P.H., Zigmond, R.E., Brulet, P. and Landis, S.C., Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons, Neuron, 11 (1993) 1175-1185. [25] Shehab, S.A. and Atkinson, M.E., Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons, Brain Res., 372 (1986) 37-44. [26] Szallasi, A. and Blumberg, P.M., Resiniferatoxin, a phorbol-related diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper, Neuroscience, 30 (1989) 515-520. [27] Szallasi, A. and Blumberg, P.M., Resiniferatoxin and its analogs provide novel insights into the pharmacology of the vanilloid (capsaicin) receptor, Life Sci., 47 (1990) 1399-1408. [28] Szallasi, A. and Blumberg, P.M., Vanilloid receptor loss in rat sensory ganglia associated with long term desensitization to resiniferatoxin, Neurosci. Lett., 136 (1992) 51-54. [29] Szallasi, A. and Blumberg, P.M., Mechanisms and therapeutic potential of vanilloids (capsaicin-like molecules), Adv. Pharmacol., 24 (1993) 123-155. [30] Szallasi, A., Blumberg, P.M., Nilsson, S., H0kfelt, T. and Lundberg, J.M., Visualization by [3H]resiniferatoxin autoradiography of capsaicin-sensitive neurons in the rat, pig and man, Eur. Z Pharmacol., 264 (1994) 217-221. [31] Szallasi, A., Nilsson, S., Farkas-Szallasi, T., Blumberg, P.M., HiSkfelt, T. and Lundberg, J.M., Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment, Brain Res., 703 (1995) 175-183. [32] Verge, V.M.K., Zhang, X., Xu, X.-J., Wiesenfeld-Hallin, Z. and H~Skfelt, T., Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization
218
[33]
[34]
[35]
[36]
T. Farkas-Szallasi et al. / Brain Research 719 (1996) 213-218 and functional studies, Proc. Natl. Acad. Sci. USA, 89 (1992) 11617-11621. Verge, V.M.K., Richardson, P.M., Wiesenfeld-Hallin, Z. and H~Skfelt, T., Differential influence of nerve growth factor on neuropeptide expression in vivo - - a novel role in peptide suppression in adult sensory neurons, J. Neurosci., 15 (1995) 2081-2096. Vizzard, M.A., Erdman, S.L. and De Groat, W.C., Increased expression of neuronal nitric oxide synthase in dorsal root ganglion neurons after systemic capsaicin administration, Neuroscience, 67 (1995) 1-5. Vrontakis, M.E., Peden, L.M., Duckworth, M.L. and Friesen, H.G., Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA, J. Biol. Chem., 262 (35) 16755-16758. Wakisaka, S., Kajander, K.C. and Bennett, G.J., Increased neuropep-
tide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy, Neurosci. Lett., 124 (1991) 200-203. [37] Winter, J., Forbes, C.A., Sternberg, J. and Lindsay, R.M., Nerve growth factor (NGF) regulates adult rat dorsal root ganglion neuron responses to capsaicin, Neuron, 1 (1988) 973-981. [38] Winter, J., Walpole, C.S.J., Bevan, S. and James, I.F., Characterization of resiniferatoxin binding sites on sensory neurons: co-regulation of resiniferatoxin binding and capsaicin-sensitivity in adult rat dorsal root ganglia, Neuroscience, 57 (1993) 747-757. [39] Zhang, X., Verge, V.M.K., Wiesenfeld-Hallin, Z., Ju, G., Bredt, D., Snyder, S.H. and H/Skfelt, T., Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy, J. Comp. Neurol., 335 (1993) 563-575.