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Axon terminals possessing the α2c-adrenergic receptor in the rat dorsal horn are predominantly excitatory
Brain Research 965 (2003) 269–273 www.elsevier.com / locate / brainres
Short communication
Axon terminals possessing the a 2c -adrenergic receptor in the rat dorsal horn are predominantly excitatory M.J. Olave, D.J. Maxwell* Spinal Cord Group, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8 QQ , UK Accepted 27 November 2002
Abstract In this study we used confocal microscopy to show that most (83.67%) a 2C -adrenergic receptor (a 2C -AR)-immunoreactive terminals in the superficial dorsal horn contain the vesicular glutamate transporter 2 and hence are glutamatergic. Few (11.33%) terminals contain glutamic acid decarboxylase (a marker for GABAergic axons) and none were associated with the B subunit of cholera toxin (a marker for myelinated primary afferents) or the vesicular glutamate transporter 1. These data indicate that most dorsal horn axons possessing the a 2C -AR are excitatory and add further support to the suggestion that they originate principally from spinal interneurons. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Spinal cord; Noradrenaline; Antinociception; Immunocytochemistry
Pharmacological evidence indicates that antinociceptive actions of noradrenaline in the spinal cord are mediated principally by a 2 -adrenergic receptors (a 2 -ARs; [4,21,10]). Such receptors belong to the family of G-protein-coupled receptors [3] and induce membrane hyperpolarisation [15,13]. The a 2A and a 2C subtypes of a 2 -ARs are found predominantly in the spinal cord and, whilst the antinociceptive role of the a 2A subtype is now well established (e.g. see Ref. [5]), it has been shown only recently that the a 2C subtype also has an involvement in spinal analgesia [1]. Immunocytochemical studies reveal that a 2A -ARs, are present on the terminals of capsaicin-sensitive peptidergic primary afferents [14] but that immunoreactivity for the a 2C -AR is associated principally with spinal interneurons [12,14,9]. A dense plexus of a 2C -ARimmunoreactive axons is present in the superficial dorsal horn. In a previous study [9], we suggested that the majority of these axons were likely to have an excitatory action because few of them contained glutamic acid decarboxylase (GAD; the synthetic enzyme for GABA) *Corresponding author. Tel.: 144-141-339-8855x0994; fax: 144-141330-2868. E-mail address: [email protected] (D.J. Maxwell).
and most of them formed asymmetric synapses which are thought to mediate excitation [19]. These axons are not immunoreactive for calcitonin gene-related peptide (CGRP) or bind the lectin IB4 and therefore do not originate from peptidergic or the IB4 class of unmyelinated primary afferents. Although the majority of a 2C -AR axons undoubtedly originate from spinal interneurons [14,9], in situ hybridization studies indicate that mRNA encoding the a 2C -AR is present in some dorsal root ganglion cells [7,2] and therefore there is a possibility that the receptor is present on central terminals of primary afferents that are not identified by CGRP or IB4. We had two specific aims in this study. Firstly, we wanted to determine if a 2C -AR-immunoreactive axons are excitatory. We used confocal microscopy and quantitative immunocytochemistry to establish what proportion of axon terminals that possess a 2C -ARs is also immunoreactive for the vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) which are specific markers for glutamatergic axons [16,20]. Secondly, we investigated the possibility that a 2C -ARs are present on central terminals of myelinated primary afferents which were identified by labelling them with the B subunit of cholera toxin (CTb). Three male Sprague–Dawley rats (250 g; Harlan) were
0006-8993 / 02 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(02)04124-0
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M. J. Olave, D. J. Maxwell / Brain Research 965 (2003) 269–273
deeply anaesthetised with halothane and 4 ml 1% cholera toxin B subunit (CTb; List Laboratories, CA, USA) was injected into the left sciatic nerve to label terminations of myelinated primary afferents [6,11]. Then 3 days later, the animals were deeply anaesthetised with sodium pentobarbitone (1 ml of 60 mg / ml i.p.) and perfused through the left ventricle with saline followed by a fixative containing 4% formaldehyde in phosphate buffer pH 7.6. All procedures were carried out according to British Home Office legislation. The L4 lumbar segment was removed from each animal and placed in the same fixative solution for 8 h. Transverse sections (50 mm thick) were cut with a Vibratome. Sections were treated with 50% ethanol (30 min) to enhance antibody penetration, which was followed by blocking in 10% normal donkey serum for 1 h. Dual immunofluorescence was performed with a guinea pig anti-a 2c -AR antiserum (diluted 1:500; Neuromics, Minneapolis, USA.; see Ref. [14] for details) which was incubated for 48 h with each one of the following antibodies: rabbit anti-VGLUT2 (1:5000), rabbit antiVGLUT1 (1:5000) (both obtained from Synaptic Systems, ¨ Gottingen, Germany), goat anti-CTb (1:5000) (List Laboratories) and a sheep anti-GAD (1:400), which recognises both isoforms of the enzyme [8]. Sections were rinsed and incubated in solutions containing species-specific secondary antibodies (all raised in donkey and diluted 1:100) coupled to fluorescein isothyoanate, to identify a 2c -ARimmunoreactivity, or lissamine rhodamine to identify the other marker (Jackson ImmunoResearch, Luton, UK). Primary and secondary antibodies were diluted in phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 1% normal donkey serum. Once the sections were mounted, fields from the central region of the superficial dorsal horn (parts of lamina I and lamina II for VGLUT2 and GAD, Laminae II / III for VGLUT1 and lamina I and lamina III for CTb) were systematically scanned with a BioRad MRC 1024 confocal laser scanning microscope. Initially, the appropriate region of the dorsal horn was identified and a 2C -AR-immunoreactivity examined. The parameters were set for each type of immunofluorescence and fields (70370 mm) consisting of six pairs of optical sections gathered sequentially with a 340 oil-immersion lens at 0.5 mm intervals in the z-axis at a zoom factor of 3. Multiple fields were collected for each combination of
antibodies. For each marker, two fields (one from the right horn and one from the left horn except for CTb where fields were taken only from the side ipsilateral to the injection) were collected from five transverse sections for each animal. Thus a total of 10 fields per marker was collected from each of the three animals. In our previous study [9], we used an automated method to estimate the degree of overlap of various markers with immunoreactivity for a 2c -AR-immunoreactive axons. Whilst this method is excellent for comparing proportions of overlap of various markers, the values it provides are relative and not absolute. In the present work we decided to apply a visual-assessment method because we wanted to produce an accurate estimate of numbers of a 2c -AR-immunoreactive puncta that possess immunoreactivity for the four markers. For this purpose, 100 a 2c -AR-immunoreactive puncta per animal (i.e. 300 in total) were sampled from the scanned fields with the aid of NeuroLucida for Confocal software (MicroBrightField, Colchester, VT). For each animal, a random sample of 10 boutons per marker was extracted from the third and fourth optical sections from each of the 10 fields collected. Sampled a 2c -AR-IR boutons were assessed individually to determine if they contained -immunoreactivity for each one of the markers. The numbers of double-labelled a 2c -AR puncta were then counted for each animal and the final overall mean percentage value (6standard deviation) was calculated for the three animals. Immunoreactivity for the a 2c -AR was concentrated on axon terminals in the superficial laminae of the dorsal horn (Fig. 1e) and in the lateral spinal nucleus and had a similar pattern to that reported previously [14,9]. We used markers to label terminals containing excitatory (i.e. VGLUT1 and VGLUT2) or inhibitory (i.e. GAD) amino acid transmitters in order to estimate the proportion of a 2c -AR axons that are likely to have an excitatory or inhibitory action. Numerous a 2c -AR-immunoreactive puncta contained immunoreactivity for VGLUT2 (Fig. 1a). However the intensity of VGLUT2 labelling varied considerably in terminals from very intense to weak; all intensities of immunoreactivity were considered positive for the purposes of the quantitative analysis. A small number of a 2c -AR terminals were associated with GAD-immunoreactivity (Fig. 1b) but none of them were immunoreactive for VGLUT1 (Fig. 1c).
Fig. 1. (a–c) Confocal microscope images of a 2c -AR-immunoreactivity (green) and immunorectivity for the markers (red) used in the study. Three sets of single optical sections are shown. In each set, the first image shows immunoreactivity for the a 2c -AR (a, b, c), the second image shows labelling for the vesicular glutamate transporter 2 (VGLUT2), glutamic acid decarboxylase (GAD) and the vesicular glutamate transporter 1 (VGLUT1) (a9, b9 and c9, respectively) and the third image is the result of merging both images (a0, b0, c0). The presence of yellow profiles in a0 and b0 indicates overlap of immunoreactivity. Arrows indicate selected a 2c -AR-immunoreactive puncta that are double-labelled with intense VGLUT2 and arrowheads show terminals that are associated with weak VGLUT2 in series a. Arrows indicate selected structures that are double labelled with GAD in series b [Scale bars510 mm]. (d) A low-power image showing the pattern of labelling in the dorsal horn with the B subunit of cholera toxin following injection into the sciatic nerve [Scale bar5100 mm]; (e) A magnified view of the dorsolateral area of the dorsal horn illustrated in (d) showing the relationship between immunoreactivities for the a 2c -AR (A2C, green) and CTb (red) [Scale bar550 mm]. (f) Details of the boxed area in (e) showing CTb labeling in lamina I (red) and immunoreactivity for the a 2c -AR (green). Note that there is no overlap of CTb and a 2c -AR puncta [Scale bar510 mm]. Series g shows immunolabelling for the a 2c -AR (g) and CTb (g9) in lamina III. In the merged image (g0) it can be seen that a 2c -AR and CTb puncta do not overlap. [Scale bar510 mm].
M. J. Olave, D. J. Maxwell / Brain Research 965 (2003) 269–273
Quantitative analysis confirmed that 83.6762.52% of a 2c AR axons contain VGLUT2 but that only 11.3361.53% contain GAD and that they do not contain VGLUT1 (Fig. 2). We could find no evidence to show that CTb-labelled
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myelinated primary afferent terminals in lamina I or in lamina III possess the receptor (Figs. 1d–g and 2) even though numerous CTb-labelled axons were present in these regions (Fig. 1d).
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Fig. 2. A histogram showing mean percentage values of numbers of a 2c -AR immunoreactive puncta that were double-labelled with the markers. A substantial number of a 2c -AR puncta were immunoreactive for the vesicular glutamate transporter 2 (VGLUT2) and a small number were immunoreactive for glutamic acid decarboxylase (GAD) but none were associated with the vesicular glutamate transporter 1 (VGLUT1) or with CTb in lamina I (CTb-LI) or lamina III (CTb-LIII). The data were obtained from three adult rats. Error bars5standard deviation.
The principal finding of the present study was that many terminals (greater than 80%) in the superficial dorsal horn that possess the a 2c -AR also contain VGLUT2. This evidence suggests that a 2c -ARs are associated with axons that predominantly have an excitatory action. We also confirmed that only a small proportion (approximately 11%) of these axons contain GAD. Most GABAergic neurons in the superficial dorsal horn contain GABA or a mixture of GABA and glycine [17] and the GAD marker would be predicted to label the entire population of inhibitory axons that release classical inhibitory amino acid transmitters. Therefore it is likely that only a small proportion of a 2c -AR-immunoreactive axons have an inhibitory action. The conclusion that most a 2c -AR-immunoreactive terminals are excitatory is also supported by findings from electron microscopic investigations which show that more than 75% of a 2c -AR-immunoreactive terminals form asymmetric axo-dendritic synapses [9].
Terminals forming this type of synapse are predicted to have an excitatory action [19]. Many a 2c -AR-immunoreactive terminals also contain enkephalin, somatostatin or neurotensin [9,14]. It has recently been shown that all of these types of peptidergic terminal are associated with high proportions of VGLUT2-immunoreactivity [18]. These findings also support our suggestion that the majority of a 2c -AR-immunoreactive terminals contain glutamate and have an excitatory action. It was concluded previously that most a 2c -AR-immunoreactive axons originate from spinal interneurons [14,9]. Immunoreactivity for the a 2c -AR is not significantly reduced following rhizotomy or neonatal capsaicin treatment and is not associated with markers such as CGRP or IB4. Therefore it is presumed that the a 2c -AR-immunoreactive axons in the superficial dorsal horn are not terminals of primary afferents. Indeed some a 2c -AR-immunoreactive axons contain enkephalin and neurotensin which are markers that are specifically found in terminals of spinal interneurons. Nevertheless, in situ hybridisation studies indicate that both large and small dorsal root ganglion cells contain a 2c -AR mRNA [7,2] and therefore this receptor would be expected to be present on central terminals of primary afferent fibres. In the present study we have shown that myelinated primary afferent axons that take-up and transport CTb are not associated with a 2c -ARimmunoreactivity. The receptor was also not found on terminals labeled with VGLUT1, a marker that is associated mainly with large myelinated primary afferents [20]. Taken together with the previous findings, our evidence suggests that few, if any, a 2c -AR-immunoreactive axons in the superficial dorsal horn originate from primary afferents. It is difficult to reconcile this conclusion with the findings of the in situ hybridization studies but it is possible that a 2c -ARs manufactured in dorsal root ganglion cells are not trafficked to central terminations and, in this respect, may be similar to the neuropeptide Y Y1 receptor [22]. Alternatively some a 2c -ARs may be present on a subgroup of primary afferents that are not labelled by CTb, IB4 or CGRP. In conclusion, we have shown that the majority of axon terminals in the superficial dorsal horn that are immunoreactive for the a 2c -AR are likely to be excitatory. It is also probable that most of these axons are terminals of spinal interneurons. In our previous study [9] we observed that many a 2c -AR-immunoreactive terminals in lamina I clustered around proximal dendrites and cell bodies in this region. We tentatively suggested that such cells might be projection neurons that have an involvement in nociception. The evidence obtained in the present study suggests that noradrenaline could weaken excitatory synaptic transmission to spinal neurons by acting through a 2c -ARs on the terminals of interneurons. This could be one of the mechanisms that underlies the antinociceptive action of noradrenaline in the spinal cord.
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Acknowledgements We wish to thank Robert Kerr for excellent technical assistance. M.J.O. is supported by a University of Glasgow Postgraduate Scholarship.
References [1] C.A. Fairbanks, L.S. Stone, K.F. Kitto, H.O. Nguyen, I.J. Posthumus, G.L. Wilcox, alpha(2C)-Adrenergic receptors mediate spinal analgesia and adrenergic-opioid synergy, J. Pharmacol. Exp. Ther. 300 (2002) 282–290. [2] M.S. Gold, S. Dastmalchi, J.D. Levine, a 2 -adrenergic receptor subtypes in rat dorsal root and superior cervical ganglion neurons, Pain 69 (1997) 179–190. [3] K. Hoehn, A. Reid, J. Sawynok, Pertussis toxin inhibits antinociception produced by intrathecal injection of morphine, noradrenaline and baclofen, Eur. J. Pharmacol. 146 (1988) 65–72. [4] J.R. Howe, J.Y. Wang, T.L. Yaksh, Selective antagonism of the antinociceptive effect of intrathecally applied alpha adrenergic agonists by intrathecal prazosin and intrathecal yohimbine, J. Pharmacol. Exp. Ther. 224 (1983) 552–558. [5] J.W. Kable, L.C. Murrin, D.B. Bylund, In vivo gene modification elucidates subtype-specific functions of alpha(2)-adrenergic receptors, J. Pharmacol. Exp. Ther. 293 (2000) 1–7. [6] C.C. LaMotte, S.E. Kapadia, C.M. Shapiro, Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoid-HRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP), J. Comp. Neurol. 311 (1991) 546–562. ¨ [7] A.P. Nicholas, V. Pieribone, T. Hokfelt, Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study, J. Comp. Neurol. 328 (1993) 575–594. [8] W.H. Oertel, D.E. Schmechel, E. Mugnaini, M.L. Tappaz, I.J. Kopin, Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum, Neuroscience 6 (1981) 2715–2735. [9] M.J. Olave, D.J. Maxwell, An investigation of neurons that possess the a 2c -adrenergic receptor in the rat dorsal horn, Neuroscience 115 (2002) 31–40. [10] H.K. Proudfit, Pharmacologic evidence for the modulation of nociception by noradrenergic neurons, Prog. Brain Res. 77 (1988) 357–370.
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[11] C. Rivero-Melian, G. Grant, Choleragenoid horseradish peroxidase used for studying projections of some hindlimb cutaneous nerves and plantar foot afferents to the dorsal horn and Clarke’s column in the rat, Exp. Brain Res. 84 (1991) 125–132. [12] D.L. Rosin, E.M. Talley, A. Lee, R.L. Stornetta, B.D. Gaylinn, P.G. Guyenet, K.R. Lynch, Distribution of Alpha 2c adrenergic receptor like immunoreactivity in the rat central nervous system, J. Comp. Neurol. 372 (1996) 135–165. [13] K.-Z. Shen, R.A. North, A. Surprenant, Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea-pig submucosal neurons, J. Physiol. (Lond.) 445 (1992) 581–599. [14] L.S. Stone, C. Broberger, L. Vulchanova, G.L. Wilcox, T. Hokfelt, M.S. Riedl, R. Elde, Differential distribution of alpha2A and alpha2C adrenergic receptor immunoreactivity in the rat spinal cord, J. Neurosci. 18 (1998) 5928–5937. [15] A. Surprenant, K.-Z. Shen, R.A. North, H. Tatsumi, Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurones, J. Physiol. (Lond.) 431 (1990) 585–608. [16] S. Takamori, J.S. Rhee, C. Rosenmund, R. Jahn, Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons, Nature 407 (2000) 189–194. [17] A.J. Todd, R.C. Spike, The localization of classical transmitters and neuropeptides within neurons in laminae I–III of the mammalian spinal dorsal horn, Prog. Neurobiol. 41 (1993) 609–638. ´ G.G. Nagy, M. Mackie, O.P. [18] A.J. Todd, D.I. Hughes, E. Polgar, Ottersen, D.J. Maxwell, The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically-defined axonal populations in the rat spinal cord with emphasis on the dorsal horn, Eur. J. Neurosci. (in press). [19] K. Uchizono, Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature 209 (4997) (1965) 642–643. [20] H. Varoqui, M.K. Schafer, H. Zhu, E. Weihe, J.D. Erickson, Identification of the differentiation-associated Na 1 / PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses, J. Neurosci. 22 (2002) 142–155. [21] T.L. Yaksh, Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing, Pharmacol. Biochem. Behav. 22 (1985) 845–858. [22] X. Zhang, L. Bao, Z.-Q. Xu, J. Kopp, U. Arvidsson, R. Elde, T. ¨ Hokfelt, Localization of neuropeptide Y Y1 receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons, Proc. Natl Acad. Sci. USA 91 (1994) 11738–11742.
Short communication
Axon terminals possessing the a 2c -adrenergic receptor in the rat dorsal horn are predominantly excitatory M.J. Olave, D.J. Maxwell* Spinal Cord Group, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8 QQ , UK Accepted 27 November 2002
Abstract In this study we used confocal microscopy to show that most (83.67%) a 2C -adrenergic receptor (a 2C -AR)-immunoreactive terminals in the superficial dorsal horn contain the vesicular glutamate transporter 2 and hence are glutamatergic. Few (11.33%) terminals contain glutamic acid decarboxylase (a marker for GABAergic axons) and none were associated with the B subunit of cholera toxin (a marker for myelinated primary afferents) or the vesicular glutamate transporter 1. These data indicate that most dorsal horn axons possessing the a 2C -AR are excitatory and add further support to the suggestion that they originate principally from spinal interneurons. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Spinal cord; Noradrenaline; Antinociception; Immunocytochemistry
Pharmacological evidence indicates that antinociceptive actions of noradrenaline in the spinal cord are mediated principally by a 2 -adrenergic receptors (a 2 -ARs; [4,21,10]). Such receptors belong to the family of G-protein-coupled receptors [3] and induce membrane hyperpolarisation [15,13]. The a 2A and a 2C subtypes of a 2 -ARs are found predominantly in the spinal cord and, whilst the antinociceptive role of the a 2A subtype is now well established (e.g. see Ref. [5]), it has been shown only recently that the a 2C subtype also has an involvement in spinal analgesia [1]. Immunocytochemical studies reveal that a 2A -ARs, are present on the terminals of capsaicin-sensitive peptidergic primary afferents [14] but that immunoreactivity for the a 2C -AR is associated principally with spinal interneurons [12,14,9]. A dense plexus of a 2C -ARimmunoreactive axons is present in the superficial dorsal horn. In a previous study [9], we suggested that the majority of these axons were likely to have an excitatory action because few of them contained glutamic acid decarboxylase (GAD; the synthetic enzyme for GABA) *Corresponding author. Tel.: 144-141-339-8855x0994; fax: 144-141330-2868. E-mail address: [email protected] (D.J. Maxwell).
and most of them formed asymmetric synapses which are thought to mediate excitation [19]. These axons are not immunoreactive for calcitonin gene-related peptide (CGRP) or bind the lectin IB4 and therefore do not originate from peptidergic or the IB4 class of unmyelinated primary afferents. Although the majority of a 2C -AR axons undoubtedly originate from spinal interneurons [14,9], in situ hybridization studies indicate that mRNA encoding the a 2C -AR is present in some dorsal root ganglion cells [7,2] and therefore there is a possibility that the receptor is present on central terminals of primary afferents that are not identified by CGRP or IB4. We had two specific aims in this study. Firstly, we wanted to determine if a 2C -AR-immunoreactive axons are excitatory. We used confocal microscopy and quantitative immunocytochemistry to establish what proportion of axon terminals that possess a 2C -ARs is also immunoreactive for the vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) which are specific markers for glutamatergic axons [16,20]. Secondly, we investigated the possibility that a 2C -ARs are present on central terminals of myelinated primary afferents which were identified by labelling them with the B subunit of cholera toxin (CTb). Three male Sprague–Dawley rats (250 g; Harlan) were
0006-8993 / 02 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(02)04124-0
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deeply anaesthetised with halothane and 4 ml 1% cholera toxin B subunit (CTb; List Laboratories, CA, USA) was injected into the left sciatic nerve to label terminations of myelinated primary afferents [6,11]. Then 3 days later, the animals were deeply anaesthetised with sodium pentobarbitone (1 ml of 60 mg / ml i.p.) and perfused through the left ventricle with saline followed by a fixative containing 4% formaldehyde in phosphate buffer pH 7.6. All procedures were carried out according to British Home Office legislation. The L4 lumbar segment was removed from each animal and placed in the same fixative solution for 8 h. Transverse sections (50 mm thick) were cut with a Vibratome. Sections were treated with 50% ethanol (30 min) to enhance antibody penetration, which was followed by blocking in 10% normal donkey serum for 1 h. Dual immunofluorescence was performed with a guinea pig anti-a 2c -AR antiserum (diluted 1:500; Neuromics, Minneapolis, USA.; see Ref. [14] for details) which was incubated for 48 h with each one of the following antibodies: rabbit anti-VGLUT2 (1:5000), rabbit antiVGLUT1 (1:5000) (both obtained from Synaptic Systems, ¨ Gottingen, Germany), goat anti-CTb (1:5000) (List Laboratories) and a sheep anti-GAD (1:400), which recognises both isoforms of the enzyme [8]. Sections were rinsed and incubated in solutions containing species-specific secondary antibodies (all raised in donkey and diluted 1:100) coupled to fluorescein isothyoanate, to identify a 2c -ARimmunoreactivity, or lissamine rhodamine to identify the other marker (Jackson ImmunoResearch, Luton, UK). Primary and secondary antibodies were diluted in phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 1% normal donkey serum. Once the sections were mounted, fields from the central region of the superficial dorsal horn (parts of lamina I and lamina II for VGLUT2 and GAD, Laminae II / III for VGLUT1 and lamina I and lamina III for CTb) were systematically scanned with a BioRad MRC 1024 confocal laser scanning microscope. Initially, the appropriate region of the dorsal horn was identified and a 2C -AR-immunoreactivity examined. The parameters were set for each type of immunofluorescence and fields (70370 mm) consisting of six pairs of optical sections gathered sequentially with a 340 oil-immersion lens at 0.5 mm intervals in the z-axis at a zoom factor of 3. Multiple fields were collected for each combination of
antibodies. For each marker, two fields (one from the right horn and one from the left horn except for CTb where fields were taken only from the side ipsilateral to the injection) were collected from five transverse sections for each animal. Thus a total of 10 fields per marker was collected from each of the three animals. In our previous study [9], we used an automated method to estimate the degree of overlap of various markers with immunoreactivity for a 2c -AR-immunoreactive axons. Whilst this method is excellent for comparing proportions of overlap of various markers, the values it provides are relative and not absolute. In the present work we decided to apply a visual-assessment method because we wanted to produce an accurate estimate of numbers of a 2c -AR-immunoreactive puncta that possess immunoreactivity for the four markers. For this purpose, 100 a 2c -AR-immunoreactive puncta per animal (i.e. 300 in total) were sampled from the scanned fields with the aid of NeuroLucida for Confocal software (MicroBrightField, Colchester, VT). For each animal, a random sample of 10 boutons per marker was extracted from the third and fourth optical sections from each of the 10 fields collected. Sampled a 2c -AR-IR boutons were assessed individually to determine if they contained -immunoreactivity for each one of the markers. The numbers of double-labelled a 2c -AR puncta were then counted for each animal and the final overall mean percentage value (6standard deviation) was calculated for the three animals. Immunoreactivity for the a 2c -AR was concentrated on axon terminals in the superficial laminae of the dorsal horn (Fig. 1e) and in the lateral spinal nucleus and had a similar pattern to that reported previously [14,9]. We used markers to label terminals containing excitatory (i.e. VGLUT1 and VGLUT2) or inhibitory (i.e. GAD) amino acid transmitters in order to estimate the proportion of a 2c -AR axons that are likely to have an excitatory or inhibitory action. Numerous a 2c -AR-immunoreactive puncta contained immunoreactivity for VGLUT2 (Fig. 1a). However the intensity of VGLUT2 labelling varied considerably in terminals from very intense to weak; all intensities of immunoreactivity were considered positive for the purposes of the quantitative analysis. A small number of a 2c -AR terminals were associated with GAD-immunoreactivity (Fig. 1b) but none of them were immunoreactive for VGLUT1 (Fig. 1c).
Fig. 1. (a–c) Confocal microscope images of a 2c -AR-immunoreactivity (green) and immunorectivity for the markers (red) used in the study. Three sets of single optical sections are shown. In each set, the first image shows immunoreactivity for the a 2c -AR (a, b, c), the second image shows labelling for the vesicular glutamate transporter 2 (VGLUT2), glutamic acid decarboxylase (GAD) and the vesicular glutamate transporter 1 (VGLUT1) (a9, b9 and c9, respectively) and the third image is the result of merging both images (a0, b0, c0). The presence of yellow profiles in a0 and b0 indicates overlap of immunoreactivity. Arrows indicate selected a 2c -AR-immunoreactive puncta that are double-labelled with intense VGLUT2 and arrowheads show terminals that are associated with weak VGLUT2 in series a. Arrows indicate selected structures that are double labelled with GAD in series b [Scale bars510 mm]. (d) A low-power image showing the pattern of labelling in the dorsal horn with the B subunit of cholera toxin following injection into the sciatic nerve [Scale bar5100 mm]; (e) A magnified view of the dorsolateral area of the dorsal horn illustrated in (d) showing the relationship between immunoreactivities for the a 2c -AR (A2C, green) and CTb (red) [Scale bar550 mm]. (f) Details of the boxed area in (e) showing CTb labeling in lamina I (red) and immunoreactivity for the a 2c -AR (green). Note that there is no overlap of CTb and a 2c -AR puncta [Scale bar510 mm]. Series g shows immunolabelling for the a 2c -AR (g) and CTb (g9) in lamina III. In the merged image (g0) it can be seen that a 2c -AR and CTb puncta do not overlap. [Scale bar510 mm].
M. J. Olave, D. J. Maxwell / Brain Research 965 (2003) 269–273
Quantitative analysis confirmed that 83.6762.52% of a 2c AR axons contain VGLUT2 but that only 11.3361.53% contain GAD and that they do not contain VGLUT1 (Fig. 2). We could find no evidence to show that CTb-labelled
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myelinated primary afferent terminals in lamina I or in lamina III possess the receptor (Figs. 1d–g and 2) even though numerous CTb-labelled axons were present in these regions (Fig. 1d).
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M. J. Olave, D. J. Maxwell / Brain Research 965 (2003) 269–273
Fig. 2. A histogram showing mean percentage values of numbers of a 2c -AR immunoreactive puncta that were double-labelled with the markers. A substantial number of a 2c -AR puncta were immunoreactive for the vesicular glutamate transporter 2 (VGLUT2) and a small number were immunoreactive for glutamic acid decarboxylase (GAD) but none were associated with the vesicular glutamate transporter 1 (VGLUT1) or with CTb in lamina I (CTb-LI) or lamina III (CTb-LIII). The data were obtained from three adult rats. Error bars5standard deviation.
The principal finding of the present study was that many terminals (greater than 80%) in the superficial dorsal horn that possess the a 2c -AR also contain VGLUT2. This evidence suggests that a 2c -ARs are associated with axons that predominantly have an excitatory action. We also confirmed that only a small proportion (approximately 11%) of these axons contain GAD. Most GABAergic neurons in the superficial dorsal horn contain GABA or a mixture of GABA and glycine [17] and the GAD marker would be predicted to label the entire population of inhibitory axons that release classical inhibitory amino acid transmitters. Therefore it is likely that only a small proportion of a 2c -AR-immunoreactive axons have an inhibitory action. The conclusion that most a 2c -AR-immunoreactive terminals are excitatory is also supported by findings from electron microscopic investigations which show that more than 75% of a 2c -AR-immunoreactive terminals form asymmetric axo-dendritic synapses [9].
Terminals forming this type of synapse are predicted to have an excitatory action [19]. Many a 2c -AR-immunoreactive terminals also contain enkephalin, somatostatin or neurotensin [9,14]. It has recently been shown that all of these types of peptidergic terminal are associated with high proportions of VGLUT2-immunoreactivity [18]. These findings also support our suggestion that the majority of a 2c -AR-immunoreactive terminals contain glutamate and have an excitatory action. It was concluded previously that most a 2c -AR-immunoreactive axons originate from spinal interneurons [14,9]. Immunoreactivity for the a 2c -AR is not significantly reduced following rhizotomy or neonatal capsaicin treatment and is not associated with markers such as CGRP or IB4. Therefore it is presumed that the a 2c -AR-immunoreactive axons in the superficial dorsal horn are not terminals of primary afferents. Indeed some a 2c -AR-immunoreactive axons contain enkephalin and neurotensin which are markers that are specifically found in terminals of spinal interneurons. Nevertheless, in situ hybridisation studies indicate that both large and small dorsal root ganglion cells contain a 2c -AR mRNA [7,2] and therefore this receptor would be expected to be present on central terminals of primary afferent fibres. In the present study we have shown that myelinated primary afferent axons that take-up and transport CTb are not associated with a 2c -ARimmunoreactivity. The receptor was also not found on terminals labeled with VGLUT1, a marker that is associated mainly with large myelinated primary afferents [20]. Taken together with the previous findings, our evidence suggests that few, if any, a 2c -AR-immunoreactive axons in the superficial dorsal horn originate from primary afferents. It is difficult to reconcile this conclusion with the findings of the in situ hybridization studies but it is possible that a 2c -ARs manufactured in dorsal root ganglion cells are not trafficked to central terminations and, in this respect, may be similar to the neuropeptide Y Y1 receptor [22]. Alternatively some a 2c -ARs may be present on a subgroup of primary afferents that are not labelled by CTb, IB4 or CGRP. In conclusion, we have shown that the majority of axon terminals in the superficial dorsal horn that are immunoreactive for the a 2c -AR are likely to be excitatory. It is also probable that most of these axons are terminals of spinal interneurons. In our previous study [9] we observed that many a 2c -AR-immunoreactive terminals in lamina I clustered around proximal dendrites and cell bodies in this region. We tentatively suggested that such cells might be projection neurons that have an involvement in nociception. The evidence obtained in the present study suggests that noradrenaline could weaken excitatory synaptic transmission to spinal neurons by acting through a 2c -ARs on the terminals of interneurons. This could be one of the mechanisms that underlies the antinociceptive action of noradrenaline in the spinal cord.
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Acknowledgements We wish to thank Robert Kerr for excellent technical assistance. M.J.O. is supported by a University of Glasgow Postgraduate Scholarship.
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