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Evidence for P2X3 receptors in the developing rat brain
Pergamon
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Neuroscience Vol. 87, No. 3, pp. 533–539, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00294-2
Letter to Neuroscience EVIDENCE FOR P2X3 RECEPTORS IN THE DEVELOPING RAT BRAIN E. J. KIDD,* K. J. MILLER, A. J. SANSUM and P. P. A. HUMPHREY Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K.
P2X receptor-mediated responses to the ATP analogue, á,â-methylene ATP, in rat brain1,6,10–12,14,15,17,23,24 cannot be accounted for by the receptor proteins known to be present.9,16,26 Such experiments are often performed on cells from neonates1,10,12,17,24 and, since differential developmental regulation of P2X1 and P2X2 receptor messenger RNAs has already been demonstrated,18 this is likely to be the case for other P2X receptors. This study was designed to address the possible existence of á,â-methylene ATP-sensitive P2X3 receptors in rat brains of various ages using a P2X3 receptor-selective antibody. P2X3 receptor protein was found in discrete regions of the embryonic (E16) and neonatal rat brain (P7 and P14) but was not detectable in adult animals. This is the first demonstration of the presence of these receptors in brains from various ages of rat and the differential expression of these receptors in neonates may account for some reported electrophysiological responses to á,â-methylene ATP. 1998 IBRO. Published by Elsevier Science Ltd. The distribution of the mRNA for the seven cloned P2X ligand-gated ion channel receptors for ATP in the CNS is well known.7,8,18 However, only P2X2 receptor protein has been found to be widespread in the adult rat brain,16,26 P2X3 receptor protein being confined to the nucleus tractus solitarius (NTS), the solitary tract and the spinal trigeminal nucleus27 and P2X1 receptor protein being undetectable.26 These findings do not satisfactorily explain the electrophysiological actions of á,â-methylene ATP *To whom correspondence should be addressed. Abbreviations: á,âMeATP, á,â-methylene ATP, VCA, anterior ventral cochlear nucleus, DRG, dorsal root ganglia, E, embryonic, IRt, intermediate reticular zone, Me5, mesencephalic trigeminal nucleus, P, neonatal, NTS, nucleus tractus solitarius, PBS, phosphate-buffered saline, PrH, prepositus hypoglossal nucleus, Rt, reticular thalamic nucleus, SFV, Semliki Forest virus, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sp5, spinal trigeminal tract, TBST, Tris-buffered saline with Tween-20.
(á,âMeATP) on central neurons1,6,10–12,14,15,17,23,24 as only P2X1 and P2X3 receptors are potently activated by this agonist,5,19,25 while P2X2 receptors are insensitive.3 Having previously shown differential developmental regulation of P2X1 and P2X2 receptor mRNAs in the rat brain,18 it is possible that other P2X receptors are also expressed in the young animals in which many of these electrophysiological recordings were made.1,10,12,17,24 The aim of this study was to determine whether P2X3 receptors are expressed in the brains of embryonic, neonatal and/or adult rats. A polyclonal antibody was raised to a nine-amino acid peptide identical to the carboxy-terminus of the rat P2X3 receptor.5,19 This antibody recognized a broad band with an apparent molecular weight of about 49–58,000, larger than the predicted size of the P2X3 receptor of 45,0005 and also detected two bands of about 50,000 and 54,000 in membranes containing the recombinant human P2X3 receptor (Fig. 1). The antibody was highly selective for the P2X3 receptor protein, no bands being seen for P2X1, P2X2 or P2X4 receptor proteins (Fig. 1). Following preadsorption of the antibody with the peptide against which it was raised, the specific band in the rat P2X3 receptor-containing membranes was no longer seen and the pre-immune serum taken from the rabbit before the first immunization also did not identify any bands in these membranes (Fig. 1). There were no available cell lines for rat P2X5, P2X6 and P2X7 receptors to check for cross-reactivity of the P2X3 receptor antibody. However, the peptide sequence used is not found in the predicted sequences for the other P2X receptors or for any other known receptor sequences and therefore it is unlikely that the antibody would recognize P2X5, P2X6 or P2X7 receptors. In addition, the distribution of the mRNA for these three receptors7,8 and the immunostaining described below are not similar, although the localization of the mRNA for P2X5 and P2X6 receptors was performed in three-week-old rats7 and that for P2X7 receptors in adult animals.8 Therefore it is not
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Fig. 1. Specificity of the P2X3 receptor antibody as revealed by western blotting experiments. A peptide (DSGAYSIGH, single letter code) corresponding to the predicted amino acids 389–397 of the carboxyterminus of the rat P2X3 receptor5,19 was synthesized by the Regal Group (Surrey, U.K.). The peptide is identical in sequence to the rat P2X3 receptor but is found in no other protein sequence in the SwissProt and GenBank databases (Dec. 1995). Two milligrams of the peptide were coupled to Keyhole limpet hemocyanin. Twenty-five micrograms of the conjugate were suspended in Freund’s complete (for the initial injection) or incomplete adjuvant and injected intramuscularly into each of two rabbits. Six injections were performed over a nine-month period from the time of the first injection, with the first five injections occurring at three-weekly intervals and the sixth one week before the terminal bleed. Bleeds were taken one week after each three-weekly boost, the blood was clotted overnight at 4C and then centrifuged and the serum removed. Serum from the terminal bleed was used for all the experiments described here. Five micrograms of rat vas deferens membranes for P2X1 receptors (lane 1), 5 µg of membranes from HEK-293 cells stably expressing the recombinant rat P2X2 receptor26 (lane 2), 10 µl of membranes from CHO-K1 cells infected with a Semliki Forest virus (SFV) construct containing the rat P2X3 receptor20 (lane 3), 5 µg of membranes from HEK-293 cells stably expressing the recombinant human P2X3 receptor (the recombinant human P2X3 receptor cDNA was a generous gift from Prof. Stuhmer and Dr Soto, lane 4) and 5 µg of membranes from HEK-293 cells stably expressing the recombinant rat P2X4 receptor (K. J. Miller, unpublished observations, lane 5) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel. Protein concentrations could not be estimated for the SFV-infected CHO-K1 cells due to the final amount of total protein being very small, while the concentration of receptors is very high.20 There were no available cell lines for rat P2X5, P2X6 and P2X7 receptors to check for cross-reactivity of the P2X3 receptor antibody. It should be noted that, while the vas deferens is a rich source of P2X1 receptors, it has also been shown to contain both P2X2 and P2X4 receptor mRNAs.2,3 Gels were blotted onto 0.2 µm nitrocellulose membranes (Novex, Offenbach, Germany) using a semi-dry blotter (Pharmacia Biotech, St Albans, U.K.) for 60 min at 0.8 mA/cm2 of gel. Membranes were washed in Tris-buffered saline (TBS, 20 mM Tris-base, pH 7.5; 150 mM NaCl) and blocked overnight at 4C in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) buffer and supplemented with 5% (w/v) dried milk. After three washes in TBST buffer, the membranes were incubated in the primary antibody (1:1000 dilution) for 60 min at room temperature. The membranes were washed five times in TBST buffer and incubated in a 1:1000 dilution of a peroxidase-labelled secondary antibody (anti-rabbit IgG, Vector Laboratories, Peterborough, U.K.) for 60 min at room temperature. The membranes were then washed five more times in TBST buffer and immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham, Little Chalfont, U.K.). Mark12 mol. wt markers from Novex were used to calculate the sizes of the bands obtained. The arrowheads indicate the positions of the bands for lactate dehydrogenase (36,500 mol. wt), glutamic dehydrogenase (55,400 mol. wt) and bovine serum albumin (66,300 mol. wt) in the Mark12 markers. The antibody labelled a broad band (mol. wt about 49–58,000) in CHO-K1 cells containing the recombinant rat P2X3 receptor (lane 3) and two bands of about 50,000 and 54,000 in the recombinant human P2X3 receptor-expressing HEK-293 cell membranes (lane 4). Recognition of the recombinant human P2X3 receptor protein by the antibody was not unexpected as the corresponding amino acid sequence differs by only one amino acid (F instead of Y) from the rat receptor sequence.13 The antibody was specific as it did not cross-react with any of the other receptor proteins examined (lanes 1, 2 and 5). Control experiments, carried out using either preadsorbed P2X3 receptor antibody (dilution 1:1000) which had been incubated overnight at 4C with 15 µM of the P2X3 receptor peptide or the pre-immune serum, did not detect any bands in the CHO-K1 rat P2X3 receptor-containing membranes (lanes 6 and 7, respectively). All other chemicals were from Sigma (Poole, U.K.) or Merck (Lutterworth, U.K.) unless otherwise stated.
P2X3 receptors in developing rat brain
possible to eliminate completely any possible overlap between the labelling of receptor transcripts and proteins. The distribution of the receptor protein was examined in embryonic (E16), neonatal (P7, P14) and adult (64-day-old) Sprague–Dawley rats. The most intense labelling was seen in E16 animals where structures in the brain and surrounding tissues were stained. These areas were restricted to the hindbrain, namely the mesencephalic trigeminal nucleus (Me5; Fig. 2A,B), the superior and inferior olives (data not shown), the intermediate reticular zone (IRt), the spinal trigeminal tract (sp5) and the prepositus hypoglossal nucleus (PrH; Fig. 3B). Other areas labelled
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included the mandibular nerves to the teeth (data not shown), the auditory nerve from the inner ear (Fig. 3A) and the trigeminal ganglia and nerve (Fig. 3C,D). In P7 brains the labelling was confined to a few areas. A subpopulation of cells in the reticular thalamic nucleus (Rt) were stained with varying intensities (Fig. 3E). Fibres and processes were intensely labelled in the anterior ventral cochlear nucleus (VCA) where the staining was often punctate (Fig. 3F), possibly representing end bulbs of Held.28 A subpopulation of cells in Me5 and many processes were strongly stained (Fig. 2C,D) in contrast to the E16 brain where the majority of the cells in Me5 were
Fig. 2. (Caption overleaf).
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labelled (Fig. 2A,B). In P14 animals, the staining in the brain had markedly decreased with only a few cells labelled in Me5 (Fig. 2E,F). Very weak staining of fibres and processes in the anterior ventral cochlear nucleus was seen, but there was no detectable labelling in the reticular thalamic nucleus (data not shown). There was no detectable staining in any region of the adult rat brain including Me5, the NTS, the solitary tract and the spinal trigeminal nucleus (data not shown). However, in agreement with previous reports,5,19,27 a subpopulation of adult dorsal root ganglia (DRG) cells were strongly labelled (Fig. 3G). No staining was seen in any area with the pre-immune serum (see Figs 2B,F and 3D). This study provides the first demonstration of the localization of a fast-desensitizing, á,âMeATPsensitive P2X receptor in brains from various ages of rat, namely the P2X3 receptor. The presence of labelling only in the brains of embryonic and neo-
natal rats and the absence of any detectable receptor protein in the adult brain, shows that the expression of this receptor is differentially regulated during development similarly to the P2X1 receptor where mRNA was only seen in the brains of young animals.18 P2X3 receptors are highly expressed in embryonic animals, then decline in P7 rats and in P14 animals there is little detectable P2X3 receptor protein. Cook et al.9 examined the presence of P2X3 receptor protein in the Me5 nucleus in the adult rat brain and the lack of detectable P2X3 receptor protein here confirms that study. Furthermore, Regenold et al.21 did not find ATP-sensitive Me5 neurons in adult rats. However, the findings reported here suggest that some of the electrophysiological responses to purinergic agonists described in areas such as Me5 in neonates17 may have been mediated by P2X3 receptors. The electrophysiological results obtained in other areas1,6,10–12,14,15,23,24 where no
Fig. 2. Immunoreactivity for P2X3 receptors in the trigeminal mesencephalic nucleus (Me5) of E16 (A,B), P7 (C,D) and P14 (E,F) rats. A female Sprague–Dawley rat (Glaxo Wellcome, Stevenage, U.K.), gestation day 16, counting the day of conception as day 1, was killed by an overdose of CO2 and the embryos were removed rapidly. The entire head of each embryo was removed and immersed in 2% (v/v) neutral-buffered paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and fixed for 24 h. P7, P14 and adult (64-day-old) male rats were anaesthetized with an intraperitoneal overdose of pentobarbital (Sagatal, Rhone Merieux, Dublin, Ireland), concentration 60 mg/kg and perfused with 0.1 M PBS and then with 2% (v/v) neutral-buffered paraformaldehyde in 0.1 M PBS. All efforts were made to minimize the suffering of the animals and to reduce the number of animals used. Brains and DRG (adult rats only) were dissected out and post-fixed overnight in 2% (v/v) paraformaldehyde at 4C. The tissues were subsequently transferred to 30% (w/v) sucrose for cryoprotection and, after they had fully absorbed the sucrose, were frozen at 30C in isopentane and subsequently stored at 80C until use. Twelve-micrometre coronal brain sections and 12 µm sections from DRG embedded in Cryo-M-Bed (Bright, Huntingdon, U.K.) were cut, thaw-mounted on gelatin-coated slides and frozen until use. Immunohistochemistry was essentially performed as described by Schindler et al.22 and all steps were carried out at room temperature unless otherwise specified. Briefly, sections were rinsed in 0.1 M PBS and endogenous peroxidase was inactivated by incubating the sections for 30 min in 20% (v/v) methanol and 1.5% (v/v) hydrogen peroxide (H2O2) in 0.1 M PBS. The sections were then washed three times in PBS and blocked in 0.1 M PBS containing 0.1% (v/v) Triton X-100 and 3% (v/v) normal goat serum (Vector Laboratories) for 10 min. Immunohistochemistry was performed using the P2X3 receptor antibody in 0.1 M PBS/0.1% Triton X-100/3% goat serum at a dilution of 1:2000 for 72 h at 4C under Parafilm (American National Can, Greenwich, U.S.A.) coverslips. Control adjacent sections were incubated with the pre-immune serum (1:2000) and processed in an identical manner to the sections exposed to the primary antibody. After three washes in PBS, sections were incubated in secondary antibody at a concentration of 1:270 (biotinylated anti-rabbit IgG, Vector Laboratories) for 2 h at room temperature. Sections were washed again in PBS, incubated for 45 min in the avidin-biotin complex reagent (Vector Laboratories) at room temperature, rinsed in PBS and colour development was carried out using 0.5mg/ml 3,3 -diaminobenzidine in a 0.0018% (v/v) H2O2 solution in PBS. The sections were counter-stained using Methyl Green (Vector Laboratories) for 30 s at 60C, rinsed in distilled water, dipped 10–15 times in acetone containing 0.05% (v/v) acetic acid, dehydrated rapidly, cleared in xylene and then mounted with Ralmount (Merck). Labelled cell bodies can be detected for the majority of the Me5 neurons in the E16 rat (A,B) while in the P7 (C,D) and P14 (E,F) rats a diminishing subpopulation of cells was labelled. Fibres and processes can clearly be seen in the P7 animal (arrowhead D). No staining was seen in any of the control sections incubated with the pre-immune serum in place of the P2X3 receptor antibody (B,F and data not shown). Scale bars=25 µm (A,B,D); 50 µm (C,E,F). Fig. 3. Immunoreactivity for P2X3 receptors in different regions of the E16 (A–D), P7 (E,F) day-old and adult (G) rat nervous systems. Sections were processed as described in Fig. 2. In the E16 embryo, intense labelling was seen in the auditory nerve (viii) around the inner ear (A) and in the hindbrain in the spinal trigeminal tract (sp5), prepositus hypoglossal nucleus (PrH) and intermediate reticular formation (IRt; B) and the trigeminal ganglia and nerve (v; C). Weak labelling was detected in cells of the reticular thalamic nucleus (Rt; E) in P7 rats while fibres and processes in the anterior ventral cochlear nucleus (VCA; F) were strongly stained. In adult animals staining was seen in the sensory nervous system in a subpopulation of cell bodies in the DRG (G). No staining was seen in any of the control sections incubated with the pre-immune serum (D and data not shown). Scale bars=25 µm (G); 50 µm (A,C–F); 100 µm (B).
P2X3 receptors in developing rat brain
Fig. 3.
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immunoreactivity for P2X3 receptors was found here could be explained by the presence of P2X1 receptors, as our own studies using the P2X1 receptor-selective antibody from Vulchanova et al.26 suggest that this receptor protein is also present in P7 brains (E. J. Kidd, unpublished observations). The absence of detectable staining for P2X3 receptors in the NTS, solitary tract and the spinal trigeminal nucleus, which contrasts with the results obtained by Vulchanova et al.,27 might be explained by the increased resolution of the confocal microscope used in that study. The staining of cell bodies in the trigeminal ganglia is in agreement with the
previously described presence of P2X3 receptors in tooth pulp and trigeminal ganglia.9 CONCLUSION
The results presented here show that fastdesensitizing, á,âMeATP-sensitive P2X receptors, namely P2X3 subunits, do exist in the rat brain but apparently only in embryonic or neonatal animals and further support the role of ATP as a fast neurotransmitter in the CNS.4 Clearly the regulation of the expression of P2X receptors in development and disease has potentially important physiological consequences and warrants further investigation.
REFERENCES
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Balachandran C. and Bennett M. R. (1996) ATP-activated cationic and anionic conductances in cultured rat hippocampal neurons. Neurosci. Lett. 204, 73–76. Bo X., Zhang Y., Nassar M., Burnstock G. and Schoepfer R. (1995) A P2X purinoceptor cDNA conferring a novel pharmacological profile. Fedn Eur. biochem. Socs Lett. 375, 129–133. Brake A. J., Wagenbach M. J. and Julius D. (1994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371, 519–523. Burnstock G. (1996) Purinergic neurotransmission. Semin. Neurosci. 8, 171–257. Chen C.-C., Akopian A. N., Sivilotti L., Colquhoun D., Burnstock G. and Wood J. N. (1995) A P2X purinoceptor expressed by a subset of sensory neurons. Nature 377, 428–431. Chessell I. P., Michel A. D. and Humphrey P. P. A. (1997) Functional evidence for multiple purinoceptor subtypes in the rat medial vestibular nucleus. Neuroscience 77, 783–791. Collo G., North R. A., Kawashima E., Merlo-Pich E., Neidhart S., Surprenant A. and Buell G. (1996) Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J. Neurosci. 16, 2495–2507. Collo G., Neidhart S., Kawashima E., Kosco-Vilbois M., North R. A. and Buell G. (1997) Tissue distribution of the P2X7 receptor. Neuropharmacology 36, 1277–1283. Cook S. P., Vulchanova L., Hargreaves K. M., Elde R., McCleskey E. W. (1997) Distinct ATP receptors on pain-sensing and stretch-sensing neurons. Nature 387, 505–508. Edwards F. A., Gibb A. J. and Colquhoun D. (1992) ATP receptor-mediated synaptic currents in the central nervous system. Nature 359, 144–147. Frohlich R., Boehm S. and Illes P. (1996) Pharmacological characterization of P2 purinoceptor types in rat locus coeruleus neurons. Eur. J. Pharmac. 315, 255–261. Furukawa K., Ishibashi H. and Akaike N. (1994) ATP-induced inward current in neurons freshly dissociated for the tuberomammillary nucleus. J. Neurophysiol. 71, 868–873. Garcia-Guzman M., Stuhmer W. and Soto F. (1997) Molecular characterization and pharmacological properties of the human P2X3 purinoceptor. Molec. Brain Res. 47, 59–66. Harms L., Finta E. P., Tschopl M. and Illes P. (1992) Depolarization of rat locus coeruleus neurons by adenosine 5 -triphosphate. Neuroscience 48, 941–952. Hiruma H. and Bourque C. W. (1995) P2 purinoceptor-mediated depolarization of rat supraoptic neurosecretory cells in vitro. J. Physiol. 489.3, 805–811. Kanjhan R., Housley G. D., Thorne P. R., Christie D. L., Palmer D. J., Luo L. and Ryan A. F. (1996) Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera. NeuroReport 7, 2665–2669. Khakh B. S., Humphrey P. P. A. and Henderson G. (1997) ATP-gated cation channels (P2X purinoceptors) in trigeminal mesencephalic nucleus neurones of the rat. J. Physiol. 498.3, 709–715. Kidd E. J., Grahames C. B. A., Simon J., Michel A. D., Barnard E. A. and Humphrey P. P. A. (1995) Localization of P2X purinoceptor transcripts in the rat nervous system. Molec. Pharmac. 48, 569–573. Lewis C., Neidhart S., Holy C., North R. A., Buell G. and Surprenant A. (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 377, 432–435. Lundstrom K., Michel A., Blasey H., Bernard A. R., Hovius R., Vogel H. and Surprenant A. (1997) Expression of ligand-gated ion channels with the Semliki Forest virus expression system. J. Rec. Sig. Trans. Res. 17, 115–126. Regenold J. T., Haas H. L. and Illes P. (1988) Effects of purinoceptor agonists on electrophysiological properties of rat mesencephalic trigeminal neurones in vitro. Neurosci. Lett. 92, 347–350. Schindler M., Sellers L. A., Humphrey P. P. A. and Emson P. C. (1997) Immunohistochemical localization of the somatostatin sst2(A) receptor in the rat brain and spinal cord. Neuroscience 76, 225–240. Sun M.-K., Wahlestedt C. and Reis D. J. (1992) Action of externally applied ATP on rat reticulospinal vasomotor neurons. Eur. J. Pharmac. 224, 93–96. Ueno S., Harata N., Inoue K. and Akaike N. (1992) ATP-gated current in dissociated rat nucleus solitarii neurons. J. Neurophysiol. 68, 778–785. Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A. and Buell G. (1994) A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP. Nature 371, 516–519. Vulchanova L., Arvidsson U., Riedl M., Wang J., Buell G., Surprenant A., North R. A. and Elde R. (1996) Differential distribution of two ATP-gated ion channels (P2X receptors) determined by immunocytochemistry. Proc. natn. Acad. Sci. U.S.A. 93, 8063–8067.
P2X3 receptors in developing rat brain 27.
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Vulchanova L., Riedl M. S., Shuster S. J., Buell G., Surprenant A., North R. A. and Elde R. (1997) Immunohistochemical study of the P2X2 and P2X3 receptor subunits in rat and monkey sensory neurons and their central terminals. Neuropharmacology 36, 1229–1242. 28. Webster W. R. (1995) Auditory system. In The Rat Nervous System (ed. Paxinos G.), 2nd edn, chap. 31, pp. 797–831. Academic, New York. (Accepted 18 May 1998)
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Neuroscience Vol. 87, No. 3, pp. 533–539, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00294-2
Letter to Neuroscience EVIDENCE FOR P2X3 RECEPTORS IN THE DEVELOPING RAT BRAIN E. J. KIDD,* K. J. MILLER, A. J. SANSUM and P. P. A. HUMPHREY Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K.
P2X receptor-mediated responses to the ATP analogue, á,â-methylene ATP, in rat brain1,6,10–12,14,15,17,23,24 cannot be accounted for by the receptor proteins known to be present.9,16,26 Such experiments are often performed on cells from neonates1,10,12,17,24 and, since differential developmental regulation of P2X1 and P2X2 receptor messenger RNAs has already been demonstrated,18 this is likely to be the case for other P2X receptors. This study was designed to address the possible existence of á,â-methylene ATP-sensitive P2X3 receptors in rat brains of various ages using a P2X3 receptor-selective antibody. P2X3 receptor protein was found in discrete regions of the embryonic (E16) and neonatal rat brain (P7 and P14) but was not detectable in adult animals. This is the first demonstration of the presence of these receptors in brains from various ages of rat and the differential expression of these receptors in neonates may account for some reported electrophysiological responses to á,â-methylene ATP. 1998 IBRO. Published by Elsevier Science Ltd. The distribution of the mRNA for the seven cloned P2X ligand-gated ion channel receptors for ATP in the CNS is well known.7,8,18 However, only P2X2 receptor protein has been found to be widespread in the adult rat brain,16,26 P2X3 receptor protein being confined to the nucleus tractus solitarius (NTS), the solitary tract and the spinal trigeminal nucleus27 and P2X1 receptor protein being undetectable.26 These findings do not satisfactorily explain the electrophysiological actions of á,â-methylene ATP *To whom correspondence should be addressed. Abbreviations: á,âMeATP, á,â-methylene ATP, VCA, anterior ventral cochlear nucleus, DRG, dorsal root ganglia, E, embryonic, IRt, intermediate reticular zone, Me5, mesencephalic trigeminal nucleus, P, neonatal, NTS, nucleus tractus solitarius, PBS, phosphate-buffered saline, PrH, prepositus hypoglossal nucleus, Rt, reticular thalamic nucleus, SFV, Semliki Forest virus, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sp5, spinal trigeminal tract, TBST, Tris-buffered saline with Tween-20.
(á,âMeATP) on central neurons1,6,10–12,14,15,17,23,24 as only P2X1 and P2X3 receptors are potently activated by this agonist,5,19,25 while P2X2 receptors are insensitive.3 Having previously shown differential developmental regulation of P2X1 and P2X2 receptor mRNAs in the rat brain,18 it is possible that other P2X receptors are also expressed in the young animals in which many of these electrophysiological recordings were made.1,10,12,17,24 The aim of this study was to determine whether P2X3 receptors are expressed in the brains of embryonic, neonatal and/or adult rats. A polyclonal antibody was raised to a nine-amino acid peptide identical to the carboxy-terminus of the rat P2X3 receptor.5,19 This antibody recognized a broad band with an apparent molecular weight of about 49–58,000, larger than the predicted size of the P2X3 receptor of 45,0005 and also detected two bands of about 50,000 and 54,000 in membranes containing the recombinant human P2X3 receptor (Fig. 1). The antibody was highly selective for the P2X3 receptor protein, no bands being seen for P2X1, P2X2 or P2X4 receptor proteins (Fig. 1). Following preadsorption of the antibody with the peptide against which it was raised, the specific band in the rat P2X3 receptor-containing membranes was no longer seen and the pre-immune serum taken from the rabbit before the first immunization also did not identify any bands in these membranes (Fig. 1). There were no available cell lines for rat P2X5, P2X6 and P2X7 receptors to check for cross-reactivity of the P2X3 receptor antibody. However, the peptide sequence used is not found in the predicted sequences for the other P2X receptors or for any other known receptor sequences and therefore it is unlikely that the antibody would recognize P2X5, P2X6 or P2X7 receptors. In addition, the distribution of the mRNA for these three receptors7,8 and the immunostaining described below are not similar, although the localization of the mRNA for P2X5 and P2X6 receptors was performed in three-week-old rats7 and that for P2X7 receptors in adult animals.8 Therefore it is not
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Fig. 1. Specificity of the P2X3 receptor antibody as revealed by western blotting experiments. A peptide (DSGAYSIGH, single letter code) corresponding to the predicted amino acids 389–397 of the carboxyterminus of the rat P2X3 receptor5,19 was synthesized by the Regal Group (Surrey, U.K.). The peptide is identical in sequence to the rat P2X3 receptor but is found in no other protein sequence in the SwissProt and GenBank databases (Dec. 1995). Two milligrams of the peptide were coupled to Keyhole limpet hemocyanin. Twenty-five micrograms of the conjugate were suspended in Freund’s complete (for the initial injection) or incomplete adjuvant and injected intramuscularly into each of two rabbits. Six injections were performed over a nine-month period from the time of the first injection, with the first five injections occurring at three-weekly intervals and the sixth one week before the terminal bleed. Bleeds were taken one week after each three-weekly boost, the blood was clotted overnight at 4C and then centrifuged and the serum removed. Serum from the terminal bleed was used for all the experiments described here. Five micrograms of rat vas deferens membranes for P2X1 receptors (lane 1), 5 µg of membranes from HEK-293 cells stably expressing the recombinant rat P2X2 receptor26 (lane 2), 10 µl of membranes from CHO-K1 cells infected with a Semliki Forest virus (SFV) construct containing the rat P2X3 receptor20 (lane 3), 5 µg of membranes from HEK-293 cells stably expressing the recombinant human P2X3 receptor (the recombinant human P2X3 receptor cDNA was a generous gift from Prof. Stuhmer and Dr Soto, lane 4) and 5 µg of membranes from HEK-293 cells stably expressing the recombinant rat P2X4 receptor (K. J. Miller, unpublished observations, lane 5) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel. Protein concentrations could not be estimated for the SFV-infected CHO-K1 cells due to the final amount of total protein being very small, while the concentration of receptors is very high.20 There were no available cell lines for rat P2X5, P2X6 and P2X7 receptors to check for cross-reactivity of the P2X3 receptor antibody. It should be noted that, while the vas deferens is a rich source of P2X1 receptors, it has also been shown to contain both P2X2 and P2X4 receptor mRNAs.2,3 Gels were blotted onto 0.2 µm nitrocellulose membranes (Novex, Offenbach, Germany) using a semi-dry blotter (Pharmacia Biotech, St Albans, U.K.) for 60 min at 0.8 mA/cm2 of gel. Membranes were washed in Tris-buffered saline (TBS, 20 mM Tris-base, pH 7.5; 150 mM NaCl) and blocked overnight at 4C in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) buffer and supplemented with 5% (w/v) dried milk. After three washes in TBST buffer, the membranes were incubated in the primary antibody (1:1000 dilution) for 60 min at room temperature. The membranes were washed five times in TBST buffer and incubated in a 1:1000 dilution of a peroxidase-labelled secondary antibody (anti-rabbit IgG, Vector Laboratories, Peterborough, U.K.) for 60 min at room temperature. The membranes were then washed five more times in TBST buffer and immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham, Little Chalfont, U.K.). Mark12 mol. wt markers from Novex were used to calculate the sizes of the bands obtained. The arrowheads indicate the positions of the bands for lactate dehydrogenase (36,500 mol. wt), glutamic dehydrogenase (55,400 mol. wt) and bovine serum albumin (66,300 mol. wt) in the Mark12 markers. The antibody labelled a broad band (mol. wt about 49–58,000) in CHO-K1 cells containing the recombinant rat P2X3 receptor (lane 3) and two bands of about 50,000 and 54,000 in the recombinant human P2X3 receptor-expressing HEK-293 cell membranes (lane 4). Recognition of the recombinant human P2X3 receptor protein by the antibody was not unexpected as the corresponding amino acid sequence differs by only one amino acid (F instead of Y) from the rat receptor sequence.13 The antibody was specific as it did not cross-react with any of the other receptor proteins examined (lanes 1, 2 and 5). Control experiments, carried out using either preadsorbed P2X3 receptor antibody (dilution 1:1000) which had been incubated overnight at 4C with 15 µM of the P2X3 receptor peptide or the pre-immune serum, did not detect any bands in the CHO-K1 rat P2X3 receptor-containing membranes (lanes 6 and 7, respectively). All other chemicals were from Sigma (Poole, U.K.) or Merck (Lutterworth, U.K.) unless otherwise stated.
P2X3 receptors in developing rat brain
possible to eliminate completely any possible overlap between the labelling of receptor transcripts and proteins. The distribution of the receptor protein was examined in embryonic (E16), neonatal (P7, P14) and adult (64-day-old) Sprague–Dawley rats. The most intense labelling was seen in E16 animals where structures in the brain and surrounding tissues were stained. These areas were restricted to the hindbrain, namely the mesencephalic trigeminal nucleus (Me5; Fig. 2A,B), the superior and inferior olives (data not shown), the intermediate reticular zone (IRt), the spinal trigeminal tract (sp5) and the prepositus hypoglossal nucleus (PrH; Fig. 3B). Other areas labelled
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included the mandibular nerves to the teeth (data not shown), the auditory nerve from the inner ear (Fig. 3A) and the trigeminal ganglia and nerve (Fig. 3C,D). In P7 brains the labelling was confined to a few areas. A subpopulation of cells in the reticular thalamic nucleus (Rt) were stained with varying intensities (Fig. 3E). Fibres and processes were intensely labelled in the anterior ventral cochlear nucleus (VCA) where the staining was often punctate (Fig. 3F), possibly representing end bulbs of Held.28 A subpopulation of cells in Me5 and many processes were strongly stained (Fig. 2C,D) in contrast to the E16 brain where the majority of the cells in Me5 were
Fig. 2. (Caption overleaf).
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labelled (Fig. 2A,B). In P14 animals, the staining in the brain had markedly decreased with only a few cells labelled in Me5 (Fig. 2E,F). Very weak staining of fibres and processes in the anterior ventral cochlear nucleus was seen, but there was no detectable labelling in the reticular thalamic nucleus (data not shown). There was no detectable staining in any region of the adult rat brain including Me5, the NTS, the solitary tract and the spinal trigeminal nucleus (data not shown). However, in agreement with previous reports,5,19,27 a subpopulation of adult dorsal root ganglia (DRG) cells were strongly labelled (Fig. 3G). No staining was seen in any area with the pre-immune serum (see Figs 2B,F and 3D). This study provides the first demonstration of the localization of a fast-desensitizing, á,âMeATPsensitive P2X receptor in brains from various ages of rat, namely the P2X3 receptor. The presence of labelling only in the brains of embryonic and neo-
natal rats and the absence of any detectable receptor protein in the adult brain, shows that the expression of this receptor is differentially regulated during development similarly to the P2X1 receptor where mRNA was only seen in the brains of young animals.18 P2X3 receptors are highly expressed in embryonic animals, then decline in P7 rats and in P14 animals there is little detectable P2X3 receptor protein. Cook et al.9 examined the presence of P2X3 receptor protein in the Me5 nucleus in the adult rat brain and the lack of detectable P2X3 receptor protein here confirms that study. Furthermore, Regenold et al.21 did not find ATP-sensitive Me5 neurons in adult rats. However, the findings reported here suggest that some of the electrophysiological responses to purinergic agonists described in areas such as Me5 in neonates17 may have been mediated by P2X3 receptors. The electrophysiological results obtained in other areas1,6,10–12,14,15,23,24 where no
Fig. 2. Immunoreactivity for P2X3 receptors in the trigeminal mesencephalic nucleus (Me5) of E16 (A,B), P7 (C,D) and P14 (E,F) rats. A female Sprague–Dawley rat (Glaxo Wellcome, Stevenage, U.K.), gestation day 16, counting the day of conception as day 1, was killed by an overdose of CO2 and the embryos were removed rapidly. The entire head of each embryo was removed and immersed in 2% (v/v) neutral-buffered paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and fixed for 24 h. P7, P14 and adult (64-day-old) male rats were anaesthetized with an intraperitoneal overdose of pentobarbital (Sagatal, Rhone Merieux, Dublin, Ireland), concentration 60 mg/kg and perfused with 0.1 M PBS and then with 2% (v/v) neutral-buffered paraformaldehyde in 0.1 M PBS. All efforts were made to minimize the suffering of the animals and to reduce the number of animals used. Brains and DRG (adult rats only) were dissected out and post-fixed overnight in 2% (v/v) paraformaldehyde at 4C. The tissues were subsequently transferred to 30% (w/v) sucrose for cryoprotection and, after they had fully absorbed the sucrose, were frozen at 30C in isopentane and subsequently stored at 80C until use. Twelve-micrometre coronal brain sections and 12 µm sections from DRG embedded in Cryo-M-Bed (Bright, Huntingdon, U.K.) were cut, thaw-mounted on gelatin-coated slides and frozen until use. Immunohistochemistry was essentially performed as described by Schindler et al.22 and all steps were carried out at room temperature unless otherwise specified. Briefly, sections were rinsed in 0.1 M PBS and endogenous peroxidase was inactivated by incubating the sections for 30 min in 20% (v/v) methanol and 1.5% (v/v) hydrogen peroxide (H2O2) in 0.1 M PBS. The sections were then washed three times in PBS and blocked in 0.1 M PBS containing 0.1% (v/v) Triton X-100 and 3% (v/v) normal goat serum (Vector Laboratories) for 10 min. Immunohistochemistry was performed using the P2X3 receptor antibody in 0.1 M PBS/0.1% Triton X-100/3% goat serum at a dilution of 1:2000 for 72 h at 4C under Parafilm (American National Can, Greenwich, U.S.A.) coverslips. Control adjacent sections were incubated with the pre-immune serum (1:2000) and processed in an identical manner to the sections exposed to the primary antibody. After three washes in PBS, sections were incubated in secondary antibody at a concentration of 1:270 (biotinylated anti-rabbit IgG, Vector Laboratories) for 2 h at room temperature. Sections were washed again in PBS, incubated for 45 min in the avidin-biotin complex reagent (Vector Laboratories) at room temperature, rinsed in PBS and colour development was carried out using 0.5mg/ml 3,3 -diaminobenzidine in a 0.0018% (v/v) H2O2 solution in PBS. The sections were counter-stained using Methyl Green (Vector Laboratories) for 30 s at 60C, rinsed in distilled water, dipped 10–15 times in acetone containing 0.05% (v/v) acetic acid, dehydrated rapidly, cleared in xylene and then mounted with Ralmount (Merck). Labelled cell bodies can be detected for the majority of the Me5 neurons in the E16 rat (A,B) while in the P7 (C,D) and P14 (E,F) rats a diminishing subpopulation of cells was labelled. Fibres and processes can clearly be seen in the P7 animal (arrowhead D). No staining was seen in any of the control sections incubated with the pre-immune serum in place of the P2X3 receptor antibody (B,F and data not shown). Scale bars=25 µm (A,B,D); 50 µm (C,E,F). Fig. 3. Immunoreactivity for P2X3 receptors in different regions of the E16 (A–D), P7 (E,F) day-old and adult (G) rat nervous systems. Sections were processed as described in Fig. 2. In the E16 embryo, intense labelling was seen in the auditory nerve (viii) around the inner ear (A) and in the hindbrain in the spinal trigeminal tract (sp5), prepositus hypoglossal nucleus (PrH) and intermediate reticular formation (IRt; B) and the trigeminal ganglia and nerve (v; C). Weak labelling was detected in cells of the reticular thalamic nucleus (Rt; E) in P7 rats while fibres and processes in the anterior ventral cochlear nucleus (VCA; F) were strongly stained. In adult animals staining was seen in the sensory nervous system in a subpopulation of cell bodies in the DRG (G). No staining was seen in any of the control sections incubated with the pre-immune serum (D and data not shown). Scale bars=25 µm (G); 50 µm (A,C–F); 100 µm (B).
P2X3 receptors in developing rat brain
Fig. 3.
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immunoreactivity for P2X3 receptors was found here could be explained by the presence of P2X1 receptors, as our own studies using the P2X1 receptor-selective antibody from Vulchanova et al.26 suggest that this receptor protein is also present in P7 brains (E. J. Kidd, unpublished observations). The absence of detectable staining for P2X3 receptors in the NTS, solitary tract and the spinal trigeminal nucleus, which contrasts with the results obtained by Vulchanova et al.,27 might be explained by the increased resolution of the confocal microscope used in that study. The staining of cell bodies in the trigeminal ganglia is in agreement with the
previously described presence of P2X3 receptors in tooth pulp and trigeminal ganglia.9 CONCLUSION
The results presented here show that fastdesensitizing, á,âMeATP-sensitive P2X receptors, namely P2X3 subunits, do exist in the rat brain but apparently only in embryonic or neonatal animals and further support the role of ATP as a fast neurotransmitter in the CNS.4 Clearly the regulation of the expression of P2X receptors in development and disease has potentially important physiological consequences and warrants further investigation.
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