- Email: [email protected]
Caudal end of the rat spinal dorsal horn
Neuroscience Letters 445 (2008) 153–157
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Caudal end of the rat spinal dorsal horn Miklós Réthelyi ∗ , Erzsébet Horváth-Oszwald, Csaba Boros Szentágothai János Laboratory, Department of Anatomy, Histology and Embryology, Semmelweis University, Tuzoltó utca 58, Budapest, Hungary ˝
a r t i c l e
i n f o
Article history: Received 29 May 2008 Received in revised form 18 August 2008 Accepted 19 August 2008 Keywords: Spinal cord Dorsal horn Conus medullaris Substantia gelatinosa
a b s t r a c t We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons. © 2008 Elsevier Ireland Ltd. All rights reserved.
Textbook descriptions reveal that the spinal cord terminates with the cone-shaped conus medullaris. The filum terminale that follows the conus medullaris caudalwards is considered mostly as a vestigial portion of the central nervous system, without nerve cells. We demonstrated in two recent publications [3,19] that although the spinal cord in the rat terminates in the conus terminalis, the central area of the spinal grey matter continues in the filum terminale in an orderly way. The reduction of the spinal grey matter along the conus medullaris in the cat was described earlier [24], our observations in the rat supported Rexed’s findings. Proceeding caudally the motoneurons and with them the ventral horns discontinue first. This is followed more caudally by the reduction in size of the dorsal horns. The grey matter of the filum terminale seems to be mainly the continuation of the neuropil around the central canal. The dorsal horn, defined as laminae I–IV [17] is the termination site of the collaterals of various populations of myelinated and unmyelinated primary afferent fibers [14]. Coarse and fine myelinated cutaneous mechanoreceptor fibers terminate in the deeper half of the dorsal horn (laminae III and IV), while another population of the fine myelinated and the unmyelinated cutaneous sensory fibers terminate in the superficial dorsal horn (laminae I and II;
Abbreviations: CGRP, calcitonin gene-related peptide; GNDF, glial-derived neurotrophic factor; IB4, isolectin B4 from Bandeiraea simplicifolia; NeuN, neuronspecific nuclear protein; NGF, nerve growth factor; PKCgamma, proteinkinase gamma; VGLUT1, vesicular glutamate transporter 1. ∗ Corresponding author. Tel.: +36 1 218 4100; fax: +36 1 218 4100. E-mail address: [email protected] (M. Réthelyi). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.070
[12,13,25]). Lamina II, i.e. the transparent substantia gelatinosa (SG; [23]) seems to be the major termination site of unmyelinated sensory fibers [18]. It has been shown that the rostrocaudal course of the myelinated sensory fibers in the spinal cord is more extensive than that of the unmyelinated sensory fibers [12,22]. We presumed that this general rule should be valid also in the conus medullaris, where the dorsal horn discontinues. If it is, then in the caudalmost portion of the conus medullaris the dorsal horn should consists exclusively of myelinated primary sensory fibers, because the unmyelinated collaterals cease to arborize and terminate more cranially than the myelinated fibers do. We tested this hypothesis with the immunohistochemical detection of three groups of primary sensory fibers that terminate in large quantities in the dorsal horn. VGLUT1 immunoreaction shows the distribution of myelinated sensory fibers in laminae III and IV [8,30]. IB4 labeling in the dorsal horn shows the terminal ramification of unmyelinated sensory fibers [11] terminating in lamina II (SG). IB4 seems to label the population of sensory fibers that contain fluoride-resistant acid phosphatase [5]. Antibodies recognizing CGRP seem to label more than one group of sensory fibers that terminate mainly in laminae I and II. Individual CGRP immunoreacted fibers course also in the deep dorsal horn (laminae III and IV; [7,10]). All animal experiments were performed in accordance with European Communities Council Directive of 24 November 1986 (86/609/ECC) and were approved by the Committee on Animal Experiments, Semmelweis University, Budapest. All animals were housed in a room with a 12-h light/dark cycle, and they had access to food and water ad libitum. Adult Sprague–Dawley rats of both sexes (260–290 g b.w.) were included in the study. The animals
154
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
Table 1 Antibodies, dilutions and sources Antibody
Species
Dilution
Source
Calcitonin gene-related peptide (CGRP) Neuron-specific nuclear protein (NeuN) Protein kinase (PKC) gamma Vesicular glutamate transporter 1 (VGLUT1)
Goat Mouse Rabbit Guinea pig
1:1000 1:1000 1:1000 1:1000
Santa Cruz Biotechnology, Heidelberg, Germany Millipore (Chemicon), Budapest, Hungary Santa Cruz Biotechnology, Heidelberg, Germany Millipore (Chemicon), Budapest, Hungary
were deeply anaesthetized with a mixture of ketamine (calypsol) and xylazine (primazin) then perfused intracardially with a brief rinse with saline followed by a freshly prepared 4% paraformaldehyde solution in 0.1 M phosphate buffer. The caudal segments of the spinal cord were dissected and postfixed overnight in the same fixative at 4 ◦ C. In five animals the conus medullaris was embedded in 10% agar and 50–60-m thick transverse sections were cut with a Vibratome. The sections from two rats were treated with OsO4 vapor, then coverslipped. Selected transverse sections from the conus medullaris were processed for single and multiple immunohistochemistry. The specimens were treated with 50% ethanol (30 min) to enhance antibody penetration. Next, the sections were treated for an hour in phosphate buffer saline (PBS) containing 2% normal horse serum (NHS) and 0.3% Triton X. Sections were incubated in primary antibodies or cocktails of primary antibodies for 48 h. All antibodies used in this study were diluted in PBS with 0.1% Triton X. Sections were rinsed in PBS and incubated in solutions containing speciesspecific secondary antibodies (all raised in donkey and diluted 1:500) coupled to fluorophores (Alexa-488, Invitrogen-Molecular Probes, Budapest, Hungary) for 24 h. To examine the primary afferent collaterals in the conus medullaris antibodies against CGRP and VGLUT1 were used. The distribution of the perikarya in the dorsal horn was analyzed with antibodies raised against NeuN and PKCgamma. The primary antibodies used in this study are listed in Table 1. Since well-known markers were used and the results did not differ from the wellknown distribution of neurons, no specific control experiments were performed. Biotinylated isolectin IB4 from Bandeiraea simplicifolia (Sigma) was also used and was detected with a fluorophore conjugated to streptavidin (Streptavidin-Alexa 488). Sections then were mounted in an antifade medium (Vectashield, Vector Laboratories) and examined with a Bio-Rad Radience 2100 Rainbow confocal laser scanning system equipped with a Nicon Eclipse E800 microscope. The dorsal horn in the conus medullaris is square shaped and shows a laterally tilted position (Fig. 1a). Osmium-treated Vibratome sections revealed that the dorsal margin of the dorsal horn (lamina I) is hardly separated from the lateral surface of the
spinal cord only by a thin layer of the white matter. The wide, transparent band immediately under the dorsal border represents the SG (lamina II). The uneven but sharp ventral border of the transparent band follows an oblique course. The deeper laminae of the dorsal horns (laminae III and IV) from both sides are separated by vertical bundles of thick nerve fibers coursing at both sides of the midline. At more caudal levels but still in the conus medullaris the size of the dorsal horn is slightly reduced, its shape did not show significant changes (Fig. 1b). The separation of the dorsal horns from the central grey matter is almost complete at this level. What it is remarkable is the lack of the transparent band on one side and its reduction in size and lateral dislocation on the contralateral side of the spinal cord. The transparent band is reduced to a small rectangular area in the lateral one-third of the dorsal horn. IB4-labeled nerve fibers were found in the ventral portion of the superficial dorsal horn corresponding to the transparent band at the cranial level of the conus medullaris (Fig. 2). More caudally IB4 labeling was completely lacking in the dorsal horn on one side, and it was reduced to a small rectangular area in the lateral edge of the superficial dorsal horn contralaterally (Fig. 2b). The identical distribution of the transparent area in Fig. 1b and the IB4 labeling in Fig. 2b is striking. In the cranial part of the conus medullaris CGRP immunoreactivity was found in the superficial layer of the superficial dorsal horn, in partial overlap with the IB4-labeled stripe (Fig. 2a). Single CGRPimmunoreactive fine fibers were seen also in the deep dorsal horn. More caudally dense CGRP immunoreactivity could be seen partially overlapping with the remaining IB4-labeled fibers (Fig. 2b). Single CGRP-immunoreactive fibers coursed in the marginal zone and in the deep dorsal horn in both sides of the spinal cord. VGLUT1-immunoreactive fibers densely filled the laminae of the deep dorsal horn in the cranial segments of the conus medullaris (Fig. 2a). The oblique border between IB4-labeled and VGLUT1immunoreactive territories was sharp and corresponded to the ventral border of the transparent band in Fig. 1a. More caudally VGLUT1 immunoreactivity occupied the entire dorsal horn on the side without IB4-labeled fibers (Fig. 2b). On the contralateral side VGLUT1 immunoreactivity respected the IB4 staining, but in the dorsomedial half of the grey matter VGLUT1 immunoreactivity reached the surface of the dorsal horn.
Fig. 1. (a) Osmium-treated Vibratome section from the cranial part of the conus medullaris. The relatively large-sized dorsal horns (DH) consist of a superficial transparent area (lamina I + substantia gelatinosa) and a deeper non-transparent area (laminae III + IV). Arrows indicate bundles of nerve fibers originating from the dorsal funiculus and separating the right and left DH. (b) Osmium-treated Vibratome section of the caudal part of the conus medullaris. The transparent region in the DH is restricted unilaterally to the ventrolateral portion of the DH (asterisk). Scales: 100 m.
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
155
Fig. 2. (a) Triple staining of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled nerve fibers. There is an overlap between the superficially located CGRP and the deeply distributed IB4 fibers (yellow). CGRP-immunoreactive fibers arborize also in the deeper portions of the dorsal horn (arrows). VGLUT1-immunoreactive fibers densely arborize in the deep portion of the dorsal horn, scattered immunoreactive fibers course into the intermediate zone. (b) Triple staining of the distal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled nerve fibers. While CGRP-immunoreactive fibers arborize bilaterally in the superficial and deep portions of the dorsal horn (arrows), IB4-labeled fibers are restricted unilaterally to the ventrolateral portion of the superficial dorsal horn (asterisk). The territorial overlap between the two kinds of primary afferent fibers (yellow) is still conspicuous. VGLUT1-immunoreactive fibers spread until the dorsal margin of the dorsal horn due to the lack of IB4 labeling. The identical distribution of the transparent area in Fig. 1b and IB4 labeling on this picture is striking. (c) Triple staining of the proximal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled fibers. The superficially located CGRP-immunoreactivity partially overlaps with the deep IB4 labeling (yellow). The immunoreaction in the superficial dorsal horn covers a smaller area on the right side, although the plane of the section is perpendicular to the axis of the conus terminalis. The asymmetry is especially evident in the IB4 staining. (d) Double immunostaining showing the distribution of the NeuN-immunostained neuronal perikarya (green) in the proximal part of the conus medullaris ((c) and (d) are adjacent sections). Double-labeled PKCgamma immunopositive neurons (red) are distributed mainly in the area showing IB4 labeling in (c), although PKCgammaimmunoreactive neurons can be found in the deeper part of the dorsal horn. Parallel with the IB4 labeling in (c) also the PKCgamma immunoreactivity is asymmetrical. (e and f) Triple staining of the distal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled fibers (both sections are from the same series as that in (c). IB4 labeling disappeared from the right dorsal horn and gradually reduces caudal wards. CGRP immunoreactivity persists both in the superficial and deep locations. VGLUT1 immunoreactivity expands over the entire dorsal horn. Scales: 100 m.
Fig. 2c, e and f demonstrates similar transformation of the dorsal horn in three steps in another animal. The density of the IB4-labeled fibers decreased already on the right side in the most cranial section (Fig. 2c). The gradual modification in the distribution of the three groups of fibers shows the same pattern as in Fig. 2a and b. On a section adjacent to that in Fig. 2c the neuronal perikarya and among them the PKCgamma immunopositive neuronal perikarya are shown (Fig. 2d). PKCgamma-immunoreactive perikarya cluster in the transparent band, although some PKCgamma-positive neurons were also seen in the deep dorsal horn. Parallel with the disappearance of the IB4 labeling, also PKCgamma immunoreactivity became weaker on the right side in the dorsal horn than contralaterally. The question raised in the text, whether the myelinated and unmyelinated sensory fibers discontinue in the dorsal horn of the
conus medullaris at different rostrocaudal levels can be answered affirmatively. The transparent band – SG or lamina II – and populations of primary afferent fibers discontinue more cranially than the dorsal horn itself. The way how the dorsal horn and its components end allows us to speculate further and draw further conclusions. The stepwise caudal elimination of the spinal grey matter as it was outlined in our previous [19] and present papers supported the earlier observations and interpretation of one of us (M.R.) about the architecture of the spinal grey matter. Golgi studies made on adult cats [17] indicated that the spinal grey matter consists of a central core as well as dorsal, ventral and lateral appendages (horns) attached to the central core. We demonstrated recently that in the conus medullaris the elimination of the motoneurons in the ventral horn is followed more caudally by the elimination of the dorsal horn defined as laminae I through IV. The remaining portion of the grey
156
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
matter, the central core, as described by Réthelyi [17] continues in the filum terminale. The central core and appendages architecture of the spinal grey matter was elaborated in review papers [21,29]. The elimination pattern of the various fiber and neuron populations of the dorsal horn in the conus medullaris allows us to consider two structurally and functionally related groups of neurons: one group of neurons linked to the SG and another group of SG-independent neurons: 1. The disappearance of the SG, the IB4-labeled sensory fibers, the PKCgamma-immunoreactive neurons and a significant portion of the CGRP-immunoreactive sensory fibers are synchronized. These neurons and sensory fibers create the SG-linked components of the dorsal horn. It is probable that the transparency of lamina II is structurally linked to the terminal arborization of the IB4-labeled unmyelinated sensory fibers. IB4 and PKCgamma immunoreaction disappears simultaneously suggesting that PKCgamma-labeled neurons in the superficial dorsal horn [16] may be the prime recipients of the impulses carried by the IB4-labeled sensory neurons. Likewise, IB4 labeling and a significant portion of the CGRP immunoreactivity disappear simultaneously in lamina II. Two distinct populations of unmyelinated sensory fibers could be demonstrated with the above two kinds of immunoreactions [2]. IB4-labeled fibers represent the non-peptidergic and GDNFdependent population, while CGRP immunoreaction labels the peptidergic, NGF-dependent, substance-P containing population of sensory fibers. The ultrastructure of the axon endings and the physiological features of the peptidergic and non-peptidergic fibers vary significantly [6,27]. This diversity in morphological features suggests that IB4 labeling and CGRP immunoreactivity identify different arborizations in lamina II. As a contradiction, terminal axonal varicosities in lamina II were reported showing unequivocal colocalization of IB4 and CGRP positivity [9]. Identical colocalization was found also in our experiments. The surprising asymmetry in the SG-linked neurons could be elucidated by the asymmetry in the number of the neurons in the dorsal root ganglia. In the three sacral and the first two coccygeal dorsal root ganglia in the cat the number of the neurons were 170,016, 8224, 6256, 4128 and 4016 on the right side and 14,032, 8968, 5304, 5136 and 4000 on the left side, respectively. The number of the nuclei was counted in every sixth paraffin section [28]. Although the difference in the total number of the neurons in the five ganglia between right and left sides is 5.55%, the differences between the corresponding ganglia 17.54% (S1 segment, tilted to the right) and 24.42% (Coc1 segment, tilted to the left). In a parallel study, the number of the myelinated fibers was counted in the dorsal roots of the same segments on toluidine blue stained plastic sections. In contrast to the neurons, the total number of the myelinated fibers in the five segments varied less than 1%, and the segmental differences remained under 5% (Szarijanni and Réthelyi, unpublished observations). The above data seem to indicate that in the cat, the segmental distribution of the neurons in the caudal dorsal root ganglia with unmyelinated fibers is random. The same distribution may prevail in the rat spinal cord resulting in the asymmetrical discontinuation of the dorsal horn. Caudal from the disappearance of the SG (lamina II), the dorsal horn consists of lamina I as well as lamina III and IV. It is interesting that lamina II can be eliminated from the dorsal horn with neonatal capsaicin treatment. It was shown with various neurohistological techniques that in the spinal cord of adult rats treated with capsaicin soon after birth the thick myelinated fibers from the deeper laminae of the dorsal horn sprout dorsally through a territory that encompassed lamina II [15,20]. Redu-
plication of the new classical experiments with neurochemical markers could demonstrate the validity and also the capsaicinsensitive character of the SG-linked neuron population. 2. VGLUT1 and CGRP immunoreaction, as components of the SGindependent neurons, remains in the most caudal portion of the conus medullaris. VGLUT1 seems to label various kinds of thick, myelinated cutaneous sensory fibers. The best known of them are the hair follicle afferents described as flame-shaped arborizations [4,26]. The CGRP-labeled fibers in lamina I and scattered in the deep dorsal horn are the best candidates for the fine myelinated A-delta fibers, although no CGRP immunoreactivity could be demonstrated in the terminals of HRP labeled, physiologically identified A-delta fibers [1]. VGLUT1 and CGRP immunoreactivity could be followed more caudally in the filum terminale. CGRP-immunoreactive fine fibers course in a small midline nucleus dorsal to the central canal, while VGLUT1 immunoreactivity could be seen scattered in the entire neuropil of the filum terminale [3]. The present observations and interpretation reinforce also the widely accepted notion that the SG is the termination site of the unmyelinated sensory fibers, both IB4- and CGRP-positive fibers terminate in this lamina. Moreover, they suggest that CGRP immunoreactivity labels more than one population of primary afferent fibers, and postulate on indirect evidence that A-delta nociceptor terminals are CGRP positive in the rat. References [1] F.J. Alvarez, A.M. Kavookjian, A.R. Light, Ultrastructural morphology, synaptic relationships, and CGRP immunoreactivity of physiologically identified C-fiber terminals in the monkey spinal cord, J. Comp. Neurol. 329 (1993) 472–490. [2] A.L. Bailey, A. Ribeiro-da-Silva, Transient loss of terminals from non-peptidergic nociceptive fibers in the substantia gelatinosa of spinal cord following chronic constriction injury of the sciatic nerve, Neuroscience 138 (2006) 675–690. [3] Cs. Boros, E. Lukácsi, E. Horváth-Oszwald, M. Rethelyi, Neurochemical architecture of the filum terminale in the rat, Brain Res. 1209 (2008) 105–114. [4] A.G. Brown, Organization in the Spinal Cord, Springer, 1981. [5] A. Coimbra, B.P. Sodre-Borges, M.M. Magalhaes, The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section, J. Neurocytol. 3 (1974) 199–217. [6] X. Fang, L. Djouhri, S. McMullan, C. Berry, S.G. Waxman, K. Okuse, S.N. Lawson, Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression, J. Neurosci. 26 (2006) 7281–7292. [7] S.J. Gibson, J.M. Polak, S.R. Bloom, I.M. Sabate, P.M. Mulderry, M.A. Ghatei, G.P. McGregor, J.F.B. Morrison, J.S. Kelly, R.M. Evans, M.G. Rosenfeld, Calcitonin generelated peptide immonoreactivity in the spinal cord of man and of eight other species, J. Neurosci. 4 (1984) 3101–3111. [8] D.I. Hughes, E. Polgár, S.A.S. Shehab, A.J. Todd, Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord, Brain Res. 1017 (2004) 69–76. [9] S.J. Hwang, J.M. Oh, J.G. Valtschanoff, The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive, Brain Res. 1062 (2005) 86–91. [10] G. Jakab, I. Salamon, P. Petrusz, M. Rethelyi, Termination patterns of calscitonin gene-related peptide-immunoreactive nerve fibers in the dorsal horn of the human spinal cord, Exp. Brain Res. 80 (1990) 609–617. [11] P.D. Kitchener, P. Wilson, P.J. Snow, Selective labelling of primary sensory afferent terminals in lamina II of the dorsal horn by injection of Bandeiraea simplicifolia isolectin B4 into peripheral nerves, Neuroscience 54 (1993) 545–551. [12] 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. [13] A.R. Light, E.R. Perl, Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers, J. Comp. Neurol. 186 (1979) 133–150. [14] D.J. Maxwell, M. Rethelyi, Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord, Trends Neurosci. 10 (1987) 117–123. [15] J.I. Nagy, S.P. Hunt, The termination of primary afferents within the rat dorsal horn: evidence for rearrangement following capsaicin treatment, J. Comp. Neurol. 218 (1983) 145–158. [16] E. Polgár, J.H. Fowler, M.M. McGill, A.J. Todd, The types of neuron which contain protein kinase C gamma in rat spinal cord, Brain Res. 833 (1999) 71–80. [17] M. Réthelyi, Central core in the spinal gray matter, Acta Morph. Acad. Sci. Hung. 24 (1976) 63–70.
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157 [18] M. Réthelyi, Preterminal and terminal axon arborizations in the substantia gelatinosa of cat’s spinal cord, J. Comp. Neurol. 172 (1977) 511–528. [19] M. Réthelyi, E. Lukácsi, Cs. Boross, The caudal end of the rat spinal cord: transformation to and ultrastructure of the filum terminale, Brain Res. 1028 (2004) 133–139. [20] M. Réthelyi, M.Z. Salim, G. Jancsó, Altered distribution of dorsal root fibers in the rat following neonatal capsaicin treatment, Neuroscience 18 (1986) 749– 761. [21] M. Réthelyi, J. Szentágothai, in: A. Iggo (Ed.), Distribution and Connections of Afferent Fibers in the Spinal Cord. Handbook of Sensory Physiology, vol. II, Springer, 1973, pp. 237–252. [22] M. Réthelyi, D.L. Trevino, E.R. Perl, Distribution of primary afferent fibers within the sacrococcygeal dorsal horn: an autoradiographic study, J. Comp. Neurol. 185 (1979) 603–622. [23] B. Rexed, The cytoarchitectonic organization of the spinal cord in the cat, J. Comp. Neurol. 96 (1952) 415–496. [24] B. Rexed, Cytoarchitectonic atlas of the spinal cord in the cat, J. Comp. Neurol. 100 (1954) 297–379.
157
[25] B. Robertson, G. Grant, A comparison between wheat germ agglutinin- and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat, Neuroscience 14 (1985) 895–905. [26] M.E. Scheibel, A.B. Scheibel, Terminal axonal patterns in cat spinal cord. II. The dorsal horn, Brain Res. 9 (1968) 32–58. [27] Ch.L. Stucky, G.R. Lewin, Isolectin B4-positive and -negative nociceptors are functionally distinct, J. Neurosci. 19 (1999) 6497–6505. [28] N. Szarijanni, M. Réthelyi, Differential distribution of small and large neurons in the sacrococcygeal dorsal root ganglia, Acta Morph. Acad. Sci. Hung. 27 (1979) 25–35. [29] J. Szentágothai, M. Réthelyi, Cyto- and neuropil architecture of the spinal cord, in: J.E. Desmedt (Ed.), New Developments in Electromyography and Clinical Neurophysiology, vol. 3, 1973, pp. 2–37. [30] A.J. Todd, D.I. Hughes, E. Polgár, G.G. Nagy, M. Mackie, O.P. Ottersen, D.J. Maxwell, The expression of vesicular glutamate transporter VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn, Eur. J. Neurosci. 17 (2003) 13–27.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Caudal end of the rat spinal dorsal horn Miklós Réthelyi ∗ , Erzsébet Horváth-Oszwald, Csaba Boros Szentágothai János Laboratory, Department of Anatomy, Histology and Embryology, Semmelweis University, Tuzoltó utca 58, Budapest, Hungary ˝
a r t i c l e
i n f o
Article history: Received 29 May 2008 Received in revised form 18 August 2008 Accepted 19 August 2008 Keywords: Spinal cord Dorsal horn Conus medullaris Substantia gelatinosa
a b s t r a c t We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons. © 2008 Elsevier Ireland Ltd. All rights reserved.
Textbook descriptions reveal that the spinal cord terminates with the cone-shaped conus medullaris. The filum terminale that follows the conus medullaris caudalwards is considered mostly as a vestigial portion of the central nervous system, without nerve cells. We demonstrated in two recent publications [3,19] that although the spinal cord in the rat terminates in the conus terminalis, the central area of the spinal grey matter continues in the filum terminale in an orderly way. The reduction of the spinal grey matter along the conus medullaris in the cat was described earlier [24], our observations in the rat supported Rexed’s findings. Proceeding caudally the motoneurons and with them the ventral horns discontinue first. This is followed more caudally by the reduction in size of the dorsal horns. The grey matter of the filum terminale seems to be mainly the continuation of the neuropil around the central canal. The dorsal horn, defined as laminae I–IV [17] is the termination site of the collaterals of various populations of myelinated and unmyelinated primary afferent fibers [14]. Coarse and fine myelinated cutaneous mechanoreceptor fibers terminate in the deeper half of the dorsal horn (laminae III and IV), while another population of the fine myelinated and the unmyelinated cutaneous sensory fibers terminate in the superficial dorsal horn (laminae I and II;
Abbreviations: CGRP, calcitonin gene-related peptide; GNDF, glial-derived neurotrophic factor; IB4, isolectin B4 from Bandeiraea simplicifolia; NeuN, neuronspecific nuclear protein; NGF, nerve growth factor; PKCgamma, proteinkinase gamma; VGLUT1, vesicular glutamate transporter 1. ∗ Corresponding author. Tel.: +36 1 218 4100; fax: +36 1 218 4100. E-mail address: [email protected] (M. Réthelyi). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.070
[12,13,25]). Lamina II, i.e. the transparent substantia gelatinosa (SG; [23]) seems to be the major termination site of unmyelinated sensory fibers [18]. It has been shown that the rostrocaudal course of the myelinated sensory fibers in the spinal cord is more extensive than that of the unmyelinated sensory fibers [12,22]. We presumed that this general rule should be valid also in the conus medullaris, where the dorsal horn discontinues. If it is, then in the caudalmost portion of the conus medullaris the dorsal horn should consists exclusively of myelinated primary sensory fibers, because the unmyelinated collaterals cease to arborize and terminate more cranially than the myelinated fibers do. We tested this hypothesis with the immunohistochemical detection of three groups of primary sensory fibers that terminate in large quantities in the dorsal horn. VGLUT1 immunoreaction shows the distribution of myelinated sensory fibers in laminae III and IV [8,30]. IB4 labeling in the dorsal horn shows the terminal ramification of unmyelinated sensory fibers [11] terminating in lamina II (SG). IB4 seems to label the population of sensory fibers that contain fluoride-resistant acid phosphatase [5]. Antibodies recognizing CGRP seem to label more than one group of sensory fibers that terminate mainly in laminae I and II. Individual CGRP immunoreacted fibers course also in the deep dorsal horn (laminae III and IV; [7,10]). All animal experiments were performed in accordance with European Communities Council Directive of 24 November 1986 (86/609/ECC) and were approved by the Committee on Animal Experiments, Semmelweis University, Budapest. All animals were housed in a room with a 12-h light/dark cycle, and they had access to food and water ad libitum. Adult Sprague–Dawley rats of both sexes (260–290 g b.w.) were included in the study. The animals
154
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
Table 1 Antibodies, dilutions and sources Antibody
Species
Dilution
Source
Calcitonin gene-related peptide (CGRP) Neuron-specific nuclear protein (NeuN) Protein kinase (PKC) gamma Vesicular glutamate transporter 1 (VGLUT1)
Goat Mouse Rabbit Guinea pig
1:1000 1:1000 1:1000 1:1000
Santa Cruz Biotechnology, Heidelberg, Germany Millipore (Chemicon), Budapest, Hungary Santa Cruz Biotechnology, Heidelberg, Germany Millipore (Chemicon), Budapest, Hungary
were deeply anaesthetized with a mixture of ketamine (calypsol) and xylazine (primazin) then perfused intracardially with a brief rinse with saline followed by a freshly prepared 4% paraformaldehyde solution in 0.1 M phosphate buffer. The caudal segments of the spinal cord were dissected and postfixed overnight in the same fixative at 4 ◦ C. In five animals the conus medullaris was embedded in 10% agar and 50–60-m thick transverse sections were cut with a Vibratome. The sections from two rats were treated with OsO4 vapor, then coverslipped. Selected transverse sections from the conus medullaris were processed for single and multiple immunohistochemistry. The specimens were treated with 50% ethanol (30 min) to enhance antibody penetration. Next, the sections were treated for an hour in phosphate buffer saline (PBS) containing 2% normal horse serum (NHS) and 0.3% Triton X. Sections were incubated in primary antibodies or cocktails of primary antibodies for 48 h. All antibodies used in this study were diluted in PBS with 0.1% Triton X. Sections were rinsed in PBS and incubated in solutions containing speciesspecific secondary antibodies (all raised in donkey and diluted 1:500) coupled to fluorophores (Alexa-488, Invitrogen-Molecular Probes, Budapest, Hungary) for 24 h. To examine the primary afferent collaterals in the conus medullaris antibodies against CGRP and VGLUT1 were used. The distribution of the perikarya in the dorsal horn was analyzed with antibodies raised against NeuN and PKCgamma. The primary antibodies used in this study are listed in Table 1. Since well-known markers were used and the results did not differ from the wellknown distribution of neurons, no specific control experiments were performed. Biotinylated isolectin IB4 from Bandeiraea simplicifolia (Sigma) was also used and was detected with a fluorophore conjugated to streptavidin (Streptavidin-Alexa 488). Sections then were mounted in an antifade medium (Vectashield, Vector Laboratories) and examined with a Bio-Rad Radience 2100 Rainbow confocal laser scanning system equipped with a Nicon Eclipse E800 microscope. The dorsal horn in the conus medullaris is square shaped and shows a laterally tilted position (Fig. 1a). Osmium-treated Vibratome sections revealed that the dorsal margin of the dorsal horn (lamina I) is hardly separated from the lateral surface of the
spinal cord only by a thin layer of the white matter. The wide, transparent band immediately under the dorsal border represents the SG (lamina II). The uneven but sharp ventral border of the transparent band follows an oblique course. The deeper laminae of the dorsal horns (laminae III and IV) from both sides are separated by vertical bundles of thick nerve fibers coursing at both sides of the midline. At more caudal levels but still in the conus medullaris the size of the dorsal horn is slightly reduced, its shape did not show significant changes (Fig. 1b). The separation of the dorsal horns from the central grey matter is almost complete at this level. What it is remarkable is the lack of the transparent band on one side and its reduction in size and lateral dislocation on the contralateral side of the spinal cord. The transparent band is reduced to a small rectangular area in the lateral one-third of the dorsal horn. IB4-labeled nerve fibers were found in the ventral portion of the superficial dorsal horn corresponding to the transparent band at the cranial level of the conus medullaris (Fig. 2). More caudally IB4 labeling was completely lacking in the dorsal horn on one side, and it was reduced to a small rectangular area in the lateral edge of the superficial dorsal horn contralaterally (Fig. 2b). The identical distribution of the transparent area in Fig. 1b and the IB4 labeling in Fig. 2b is striking. In the cranial part of the conus medullaris CGRP immunoreactivity was found in the superficial layer of the superficial dorsal horn, in partial overlap with the IB4-labeled stripe (Fig. 2a). Single CGRPimmunoreactive fine fibers were seen also in the deep dorsal horn. More caudally dense CGRP immunoreactivity could be seen partially overlapping with the remaining IB4-labeled fibers (Fig. 2b). Single CGRP-immunoreactive fibers coursed in the marginal zone and in the deep dorsal horn in both sides of the spinal cord. VGLUT1-immunoreactive fibers densely filled the laminae of the deep dorsal horn in the cranial segments of the conus medullaris (Fig. 2a). The oblique border between IB4-labeled and VGLUT1immunoreactive territories was sharp and corresponded to the ventral border of the transparent band in Fig. 1a. More caudally VGLUT1 immunoreactivity occupied the entire dorsal horn on the side without IB4-labeled fibers (Fig. 2b). On the contralateral side VGLUT1 immunoreactivity respected the IB4 staining, but in the dorsomedial half of the grey matter VGLUT1 immunoreactivity reached the surface of the dorsal horn.
Fig. 1. (a) Osmium-treated Vibratome section from the cranial part of the conus medullaris. The relatively large-sized dorsal horns (DH) consist of a superficial transparent area (lamina I + substantia gelatinosa) and a deeper non-transparent area (laminae III + IV). Arrows indicate bundles of nerve fibers originating from the dorsal funiculus and separating the right and left DH. (b) Osmium-treated Vibratome section of the caudal part of the conus medullaris. The transparent region in the DH is restricted unilaterally to the ventrolateral portion of the DH (asterisk). Scales: 100 m.
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
155
Fig. 2. (a) Triple staining of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled nerve fibers. There is an overlap between the superficially located CGRP and the deeply distributed IB4 fibers (yellow). CGRP-immunoreactive fibers arborize also in the deeper portions of the dorsal horn (arrows). VGLUT1-immunoreactive fibers densely arborize in the deep portion of the dorsal horn, scattered immunoreactive fibers course into the intermediate zone. (b) Triple staining of the distal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled nerve fibers. While CGRP-immunoreactive fibers arborize bilaterally in the superficial and deep portions of the dorsal horn (arrows), IB4-labeled fibers are restricted unilaterally to the ventrolateral portion of the superficial dorsal horn (asterisk). The territorial overlap between the two kinds of primary afferent fibers (yellow) is still conspicuous. VGLUT1-immunoreactive fibers spread until the dorsal margin of the dorsal horn due to the lack of IB4 labeling. The identical distribution of the transparent area in Fig. 1b and IB4 labeling on this picture is striking. (c) Triple staining of the proximal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled fibers. The superficially located CGRP-immunoreactivity partially overlaps with the deep IB4 labeling (yellow). The immunoreaction in the superficial dorsal horn covers a smaller area on the right side, although the plane of the section is perpendicular to the axis of the conus terminalis. The asymmetry is especially evident in the IB4 staining. (d) Double immunostaining showing the distribution of the NeuN-immunostained neuronal perikarya (green) in the proximal part of the conus medullaris ((c) and (d) are adjacent sections). Double-labeled PKCgamma immunopositive neurons (red) are distributed mainly in the area showing IB4 labeling in (c), although PKCgammaimmunoreactive neurons can be found in the deeper part of the dorsal horn. Parallel with the IB4 labeling in (c) also the PKCgamma immunoreactivity is asymmetrical. (e and f) Triple staining of the distal part of the conus medullaris showing the distribution of CGRP (red)-, VGLUT1 (blue)-immunoreactive and IB4 (green)-labeled fibers (both sections are from the same series as that in (c). IB4 labeling disappeared from the right dorsal horn and gradually reduces caudal wards. CGRP immunoreactivity persists both in the superficial and deep locations. VGLUT1 immunoreactivity expands over the entire dorsal horn. Scales: 100 m.
Fig. 2c, e and f demonstrates similar transformation of the dorsal horn in three steps in another animal. The density of the IB4-labeled fibers decreased already on the right side in the most cranial section (Fig. 2c). The gradual modification in the distribution of the three groups of fibers shows the same pattern as in Fig. 2a and b. On a section adjacent to that in Fig. 2c the neuronal perikarya and among them the PKCgamma immunopositive neuronal perikarya are shown (Fig. 2d). PKCgamma-immunoreactive perikarya cluster in the transparent band, although some PKCgamma-positive neurons were also seen in the deep dorsal horn. Parallel with the disappearance of the IB4 labeling, also PKCgamma immunoreactivity became weaker on the right side in the dorsal horn than contralaterally. The question raised in the text, whether the myelinated and unmyelinated sensory fibers discontinue in the dorsal horn of the
conus medullaris at different rostrocaudal levels can be answered affirmatively. The transparent band – SG or lamina II – and populations of primary afferent fibers discontinue more cranially than the dorsal horn itself. The way how the dorsal horn and its components end allows us to speculate further and draw further conclusions. The stepwise caudal elimination of the spinal grey matter as it was outlined in our previous [19] and present papers supported the earlier observations and interpretation of one of us (M.R.) about the architecture of the spinal grey matter. Golgi studies made on adult cats [17] indicated that the spinal grey matter consists of a central core as well as dorsal, ventral and lateral appendages (horns) attached to the central core. We demonstrated recently that in the conus medullaris the elimination of the motoneurons in the ventral horn is followed more caudally by the elimination of the dorsal horn defined as laminae I through IV. The remaining portion of the grey
156
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157
matter, the central core, as described by Réthelyi [17] continues in the filum terminale. The central core and appendages architecture of the spinal grey matter was elaborated in review papers [21,29]. The elimination pattern of the various fiber and neuron populations of the dorsal horn in the conus medullaris allows us to consider two structurally and functionally related groups of neurons: one group of neurons linked to the SG and another group of SG-independent neurons: 1. The disappearance of the SG, the IB4-labeled sensory fibers, the PKCgamma-immunoreactive neurons and a significant portion of the CGRP-immunoreactive sensory fibers are synchronized. These neurons and sensory fibers create the SG-linked components of the dorsal horn. It is probable that the transparency of lamina II is structurally linked to the terminal arborization of the IB4-labeled unmyelinated sensory fibers. IB4 and PKCgamma immunoreaction disappears simultaneously suggesting that PKCgamma-labeled neurons in the superficial dorsal horn [16] may be the prime recipients of the impulses carried by the IB4-labeled sensory neurons. Likewise, IB4 labeling and a significant portion of the CGRP immunoreactivity disappear simultaneously in lamina II. Two distinct populations of unmyelinated sensory fibers could be demonstrated with the above two kinds of immunoreactions [2]. IB4-labeled fibers represent the non-peptidergic and GDNFdependent population, while CGRP immunoreaction labels the peptidergic, NGF-dependent, substance-P containing population of sensory fibers. The ultrastructure of the axon endings and the physiological features of the peptidergic and non-peptidergic fibers vary significantly [6,27]. This diversity in morphological features suggests that IB4 labeling and CGRP immunoreactivity identify different arborizations in lamina II. As a contradiction, terminal axonal varicosities in lamina II were reported showing unequivocal colocalization of IB4 and CGRP positivity [9]. Identical colocalization was found also in our experiments. The surprising asymmetry in the SG-linked neurons could be elucidated by the asymmetry in the number of the neurons in the dorsal root ganglia. In the three sacral and the first two coccygeal dorsal root ganglia in the cat the number of the neurons were 170,016, 8224, 6256, 4128 and 4016 on the right side and 14,032, 8968, 5304, 5136 and 4000 on the left side, respectively. The number of the nuclei was counted in every sixth paraffin section [28]. Although the difference in the total number of the neurons in the five ganglia between right and left sides is 5.55%, the differences between the corresponding ganglia 17.54% (S1 segment, tilted to the right) and 24.42% (Coc1 segment, tilted to the left). In a parallel study, the number of the myelinated fibers was counted in the dorsal roots of the same segments on toluidine blue stained plastic sections. In contrast to the neurons, the total number of the myelinated fibers in the five segments varied less than 1%, and the segmental differences remained under 5% (Szarijanni and Réthelyi, unpublished observations). The above data seem to indicate that in the cat, the segmental distribution of the neurons in the caudal dorsal root ganglia with unmyelinated fibers is random. The same distribution may prevail in the rat spinal cord resulting in the asymmetrical discontinuation of the dorsal horn. Caudal from the disappearance of the SG (lamina II), the dorsal horn consists of lamina I as well as lamina III and IV. It is interesting that lamina II can be eliminated from the dorsal horn with neonatal capsaicin treatment. It was shown with various neurohistological techniques that in the spinal cord of adult rats treated with capsaicin soon after birth the thick myelinated fibers from the deeper laminae of the dorsal horn sprout dorsally through a territory that encompassed lamina II [15,20]. Redu-
plication of the new classical experiments with neurochemical markers could demonstrate the validity and also the capsaicinsensitive character of the SG-linked neuron population. 2. VGLUT1 and CGRP immunoreaction, as components of the SGindependent neurons, remains in the most caudal portion of the conus medullaris. VGLUT1 seems to label various kinds of thick, myelinated cutaneous sensory fibers. The best known of them are the hair follicle afferents described as flame-shaped arborizations [4,26]. The CGRP-labeled fibers in lamina I and scattered in the deep dorsal horn are the best candidates for the fine myelinated A-delta fibers, although no CGRP immunoreactivity could be demonstrated in the terminals of HRP labeled, physiologically identified A-delta fibers [1]. VGLUT1 and CGRP immunoreactivity could be followed more caudally in the filum terminale. CGRP-immunoreactive fine fibers course in a small midline nucleus dorsal to the central canal, while VGLUT1 immunoreactivity could be seen scattered in the entire neuropil of the filum terminale [3]. The present observations and interpretation reinforce also the widely accepted notion that the SG is the termination site of the unmyelinated sensory fibers, both IB4- and CGRP-positive fibers terminate in this lamina. Moreover, they suggest that CGRP immunoreactivity labels more than one population of primary afferent fibers, and postulate on indirect evidence that A-delta nociceptor terminals are CGRP positive in the rat. References [1] F.J. Alvarez, A.M. Kavookjian, A.R. Light, Ultrastructural morphology, synaptic relationships, and CGRP immunoreactivity of physiologically identified C-fiber terminals in the monkey spinal cord, J. Comp. Neurol. 329 (1993) 472–490. [2] A.L. Bailey, A. Ribeiro-da-Silva, Transient loss of terminals from non-peptidergic nociceptive fibers in the substantia gelatinosa of spinal cord following chronic constriction injury of the sciatic nerve, Neuroscience 138 (2006) 675–690. [3] Cs. Boros, E. Lukácsi, E. Horváth-Oszwald, M. Rethelyi, Neurochemical architecture of the filum terminale in the rat, Brain Res. 1209 (2008) 105–114. [4] A.G. Brown, Organization in the Spinal Cord, Springer, 1981. [5] A. Coimbra, B.P. Sodre-Borges, M.M. Magalhaes, The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section, J. Neurocytol. 3 (1974) 199–217. [6] X. Fang, L. Djouhri, S. McMullan, C. Berry, S.G. Waxman, K. Okuse, S.N. Lawson, Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression, J. Neurosci. 26 (2006) 7281–7292. [7] S.J. Gibson, J.M. Polak, S.R. Bloom, I.M. Sabate, P.M. Mulderry, M.A. Ghatei, G.P. McGregor, J.F.B. Morrison, J.S. Kelly, R.M. Evans, M.G. Rosenfeld, Calcitonin generelated peptide immonoreactivity in the spinal cord of man and of eight other species, J. Neurosci. 4 (1984) 3101–3111. [8] D.I. Hughes, E. Polgár, S.A.S. Shehab, A.J. Todd, Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord, Brain Res. 1017 (2004) 69–76. [9] S.J. Hwang, J.M. Oh, J.G. Valtschanoff, The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive, Brain Res. 1062 (2005) 86–91. [10] G. Jakab, I. Salamon, P. Petrusz, M. Rethelyi, Termination patterns of calscitonin gene-related peptide-immunoreactive nerve fibers in the dorsal horn of the human spinal cord, Exp. Brain Res. 80 (1990) 609–617. [11] P.D. Kitchener, P. Wilson, P.J. Snow, Selective labelling of primary sensory afferent terminals in lamina II of the dorsal horn by injection of Bandeiraea simplicifolia isolectin B4 into peripheral nerves, Neuroscience 54 (1993) 545–551. [12] 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. [13] A.R. Light, E.R. Perl, Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers, J. Comp. Neurol. 186 (1979) 133–150. [14] D.J. Maxwell, M. Rethelyi, Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord, Trends Neurosci. 10 (1987) 117–123. [15] J.I. Nagy, S.P. Hunt, The termination of primary afferents within the rat dorsal horn: evidence for rearrangement following capsaicin treatment, J. Comp. Neurol. 218 (1983) 145–158. [16] E. Polgár, J.H. Fowler, M.M. McGill, A.J. Todd, The types of neuron which contain protein kinase C gamma in rat spinal cord, Brain Res. 833 (1999) 71–80. [17] M. Réthelyi, Central core in the spinal gray matter, Acta Morph. Acad. Sci. Hung. 24 (1976) 63–70.
M. Réthelyi et al. / Neuroscience Letters 445 (2008) 153–157 [18] M. Réthelyi, Preterminal and terminal axon arborizations in the substantia gelatinosa of cat’s spinal cord, J. Comp. Neurol. 172 (1977) 511–528. [19] M. Réthelyi, E. Lukácsi, Cs. Boross, The caudal end of the rat spinal cord: transformation to and ultrastructure of the filum terminale, Brain Res. 1028 (2004) 133–139. [20] M. Réthelyi, M.Z. Salim, G. Jancsó, Altered distribution of dorsal root fibers in the rat following neonatal capsaicin treatment, Neuroscience 18 (1986) 749– 761. [21] M. Réthelyi, J. Szentágothai, in: A. Iggo (Ed.), Distribution and Connections of Afferent Fibers in the Spinal Cord. Handbook of Sensory Physiology, vol. II, Springer, 1973, pp. 237–252. [22] M. Réthelyi, D.L. Trevino, E.R. Perl, Distribution of primary afferent fibers within the sacrococcygeal dorsal horn: an autoradiographic study, J. Comp. Neurol. 185 (1979) 603–622. [23] B. Rexed, The cytoarchitectonic organization of the spinal cord in the cat, J. Comp. Neurol. 96 (1952) 415–496. [24] B. Rexed, Cytoarchitectonic atlas of the spinal cord in the cat, J. Comp. Neurol. 100 (1954) 297–379.
157
[25] B. Robertson, G. Grant, A comparison between wheat germ agglutinin- and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat, Neuroscience 14 (1985) 895–905. [26] M.E. Scheibel, A.B. Scheibel, Terminal axonal patterns in cat spinal cord. II. The dorsal horn, Brain Res. 9 (1968) 32–58. [27] Ch.L. Stucky, G.R. Lewin, Isolectin B4-positive and -negative nociceptors are functionally distinct, J. Neurosci. 19 (1999) 6497–6505. [28] N. Szarijanni, M. Réthelyi, Differential distribution of small and large neurons in the sacrococcygeal dorsal root ganglia, Acta Morph. Acad. Sci. Hung. 27 (1979) 25–35. [29] J. Szentágothai, M. Réthelyi, Cyto- and neuropil architecture of the spinal cord, in: J.E. Desmedt (Ed.), New Developments in Electromyography and Clinical Neurophysiology, vol. 3, 1973, pp. 2–37. [30] A.J. Todd, D.I. Hughes, E. Polgár, G.G. Nagy, M. Mackie, O.P. Ottersen, D.J. Maxwell, The expression of vesicular glutamate transporter VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn, Eur. J. Neurosci. 17 (2003) 13–27.