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TISSUE-SPECIFIC STEM CELLS

New GABAergic Interneurons Supported by Myelin-Specific T Cells Are Formed in Intact Adult Spinal Cord

Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel

Key Words. Neuroimmune • Neural stem cell • Autoimmunity • Stromal-derived factor-1 • Neural differentiation

Correspondence: Michal Schwartz, Ph.D., Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel. Telephone: 972-8-934-2467; Fax: 972-8-934-6018; e-mail: michal.schwartz{at}weizmann.ac.il

Received on November 6, 2006; accepted for publication on May 16, 2007.

First published online in STEM CELLS EXPRESS  May 31, 2007.

	ABSTRACT

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Neural stem/progenitor cells are known to exist in the intact spinal cord, but the presence of newly formed neurons during adulthood has not been documented there to date. Here, we report the appearance of newly formed neurons under normal physiological conditions. These neurons are immature, express a GABAergic phenotype, and are primarily located in the dorsal part of the spinal cord. This localization appeared to be mediated by stromal-derived factor-1/CXC-chemokine receptor-4 signaling in the dorsal region. The extent of spinal cord neurogenesis was found to be greatly influenced by immune system integrity and in particular by myelin-specific T cells. These observations provide evidence for in vivo spinal cord neurogenesis under nonpathological conditions and introduce novel mechanisms regulating adult spinal cord plasticity.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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The adult spinal cord contains neural stem cells that can be isolated and induced to self-renew and differentiate in vitro into all three neural lineages: neurons, astrocytes, and oligodendrocytes [1]. Unlike in the classic neurogenic areas of the brain (the hippocampal dentate gyrus and the subventricular zone of the lateral ventricles), stem/progenitor cells in the intact spinal cord were found to give rise only to glial cells (astrocytes and oligodendrocytes) [2]. However, cultured spinal cord-derived neural stem cells have been shown to differentiate to neurons upon transplantation to the dentate gyrus, a neurogenic region [1].

Adult neural stem/progenitor cells in the spinal cord were shown to differentiate into neurons under pathological conditions [3–5]. This neurogenesis was attributed to pathology-induced generation of an ectopic neurogenic environment. It is possible, however, to view repair as an extreme manifestation of physiological maintenance processes. We therefore considered the possibility that some forms of neurogenesis may occur, albeit to a lesser extent, in the intact spinal cord. In the present study, we wished to first determine whether neurogenesis takes place in the adult spinal cord and, if so, to what extent it is influenced, similarly to the hippocampal neurogenesis, by the peripheral adaptive immune system [6].

	MATERIALS AND METHODS

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Animals
Male C57Bl/6J and BALB/c/OLA wild-type and severe combined immune deficiency (SCID) mice and B10.PL wild-type and T_MBP-transgenic mice [6, 7], all aged 8–12 weeks, were supplied by the Animal Breeding Center of The Weizmann Institute of Science.

5-Bromo-2-deoxyuridine Administration and Tissue Preparation
We dissolved 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) by sonication in phosphate-buffered saline (PBS). In the first experiment, BrdU (75 mg/kg body weight) was injected twice daily for 5 days and animals were killed either 1 or 28 days after the last injection. In the second experiment, a short pulse of BrdU was administered (200 mg/kg body weight, three injections over 24 hours), and mice were killed 1, 7, or 28 days after the first BrdU injection. Since the rate of cell proliferation in the spinal cord is lower than in other neurogenic areas of the central nervous system (CNS) (i.e., the subventricular zone and the dentate gyrus), a higher BrdU dose was administered. This dosage was shown by others to be nontoxic and efficient in labeling slowly proliferating cells within the CNS [8, 9]. Spinal cord sections were incubated in 2 N HCl at 37°C for 30 minutes following microwave treatment, as described below.

Immunohistochemistry
Following perfusion with cold PBS, spinal cords were removed, postfixed with Bouin's fixative (75% saturated picric acid, 25% formaldehyde, 5% glacial acetic acid; Sigma-Aldrich) for 48 hours, and then transferred to 70% ethanol (EtOH). The tissues were dehydrated through a gradient of 70%, 95%, and 100% EtOH in xylene and paraffin and were then embedded in paraffin. Paraffin sections, 6-µm thick, were used throughout the study. The paraffin was removed by successive rinsing of slides for 15 minutes with each of the following: xylene and then EtOH 100%, 95%, 70%, and 50% and PBS. Antigen retrieval was maximized by heating the slides to the boiling point in 10 mM sodium citrate pH 6.0 or in 100 mM Tris pH 9.0 in a microwave oven then heating them at 20% microwave power for an additional 10 minutes. The slides were then blocked with blocking solution (PBS containing 20% normal horse serum and 0.2% Triton X-100 or PBS containing mouse immunoglobulin blocking reagent obtained from Vector Laboratories [Burlingame, CA, http://www.vectorlabs.com]) and stained for 48 hours with specified combinations of the following primary antibodies: rat anti-BrdU (1:100; AbD Serotec, Raleigh, NC, http://www.ab-direct.com), goat anti-doublecortin (DCX) (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit anti-glial fibrillary acidic protein (GFAP) (1:200; Dako, Glostrup, Denmark, http://www.dako.com), rabbit anti-glutamic acid decarboxylase (GAD) 65/67 (1:200; Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti--aminobutyric acid (GABA) (1:500; Sigma), rabbit anti-proliferating cell nuclear antigen (PCNA) (1:100; Santa Cruz Biotechnology), mouse anti-stromal-derived factor-1 (SDF-1) (1:50; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and rabbit anti-CXC-chemokine receptor-4 (CXCR4) (1:50; eBioscience Inc., San Diego, http://www.ebioscience.com). After rinsing in PBS, sections were incubated for 1 hour at room temperature with the following secondary antibodies: Cy-3-conjugated donkey anti-goat, Cy-3- or Cy-2-conjugated donkey anti-rabbit, Cy-3-conjugated donkey anti-mouse, or Cy-2-conjugated donkey anti-rat (1:200; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). For nuclear staining, we used Hoechst 33342 (1:2,000; Molecular Probes, Eugene, OR, http://probes.invitrogen.com).

Quantification
For microscopic analysis, we used a Zeiss LSM 510 confocal laser scanning microscope (40x magnification; Carl Zeiss, Jena, Germany, http://www.zeiss.com) or a Nikon E800. To verify double labeling, confocal Z-sectioning was performed (supplemental online Fig. 2). We counted the number of labeled cells in a total of 18 axial spinal cord sections per mouse. The sections were taken from six different 0.5-cm-distanced locations along the spinal cord (three consecutive sections from each area). To obtain an estimate of the number of labeled cells per mm³ volume, the average number of cells counted in the selected sections (average surface area = 1 mm², thickness = 0.006 mm) was multiplied by 166.66. In order to avoid overestimation due to counting fractions of cells that appeared within the section, we took special care to count only cells that had an intact morphology and a nucleus that was larger than 5 µm in diameter.

Spinal Cord Injury
Mice were anesthetized, their spinal cords were exposed by laminectomy at T12, and a force of 200 kdyn was placed for 1 second on the laminectomized cord using an Infinite Horizon spinal cord impactor (Precision Systems and Instrumentation, Lexington, KY, http://www.presysin.com); this device was previously shown to inflict a well calibrated contusive injury of the spinal cord [10].

	RESULTS

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To investigate the possibility that neurogenesis occurs in the adult spinal cord, we searched for young neurons in the healthy spinal cord by immunohistochemical staining for DCX, a marker of early neuronal differentiation. We detected the presence of DCX+ cells in intact spinal cords (Fig. 1A, 1B). Many DCX+ cells were localized adjacent to the ependymal cell layer of the central canal (Fig. 1C). Quantitative analysis revealed significantly more DCX+ cells in the gray matter than in the white matter and in the dorsal (especially laminae I-III) versus the ventral parts of the spinal cord (Fig. 1D). Although expression of DCX is considered a good indicator of neurogenesis [11], DCX is reportedly also expressed by neurons in CNS areas where local neurogenesis does not occur [12]. To verify that the DCX+ cells detected in the intact spinal cord in our study were indeed newly formed, we injected the cell-proliferation marker BrdU twice daily for 5 days before killing the mice and analyzing their spinal cords. All BrdU+/DCX+ cells had small nuclei (5–10 µm in diameter) and short processes (Fig. 1E). To further confirm the presence of newly formed young neurons, we double-stained for DCX and PCNA, which is expressed during the cell cycle by actively dividing cells. PCNA+/DCX+ cells were detected in the dorsal part of the spinal cord, suggesting that the DCX+ cells were indeed newly formed ones (Fig. 1F).

View larger version (47K): [in this window] [in a new window]   Figure 1. Distribution and characterization of nascent neurons in the adult spinal cord. (A): Axial section of the adult mouse spinal cord showing cells expressing the early neuronal differentiation marker DCX. (B): Higher magnification of the boxed area in (A). DCX+ cells appear in both gray and white matter and around the central canal (arrow indicates the location of the central canal). (C): Representative images showing DCX+ cells localized at the ependymal layer of the central canal of the spinal cord. (D): Quantitative analysis of the distribution of DCX+ cells in the white and gray matter along the dorsal and ventral parts of the spinal cord (*** p < .001, Student's t test). Early neuronal proliferation: DCX+ cells incorporate BrdU (E) or express the endogenous cell proliferation marker PCNA (F). (G–I): Quantitative analysis of neurogenesis over time. (G): Mice were killed 1, 7, or 28 days after BrdU pulse, and axial sections of their spinal cord were analyzed for BrdU+ and DCX+ cells (Fisher's test, F = 9.77, p = .003 and F = 5.4, p = .002 for BrdU+ cells and BrdU+/DCX+ cells, respectively). (H): Relative distribution of newly formed neurons in different regions of the spinal cord expressed as the ratio between the number of BrdU+/DCX+ cells within the gray versus the white matter and between the dorsal versus the ventral sector (* Fisher's test, F = 5.018, p = .031 and F = 5.215, p = .025, respectively). (I): Relative degree of neuronal differentiation in different regions of the spinal cord expressed as the ratio between the percentage of BrdU+/DCX+ cells of the total BrdU+ cells within the gray versus the white matter and between the dorsal versus the ventral sector (* Fisher test, F = 14.79, p = .002 and F = 23.17, p = .0002, respectively). Scale bar, 50 µm in (A, C) and 10 µm in (B, E, F). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; d, day; DCX, doublecortin; PCNA, proliferating cell nuclear antigen.

Newly formed DCX+ neurons with similar morphology and size to those we found in the spinal cord were described in neocortex and striatum [9], brain regions outside the classic neurogenic regions. Those newly formed cortical neurons exhibit the characteristics of inhibitory interneurons. To determine whether the new neurons that we identified in the spinal cord displayed similar characteristics, we double-stained spinal cord sections for BrdU and GAD-65/67, a key enzyme in the biosynthetic pathway of the neurotransmitter GABA. At day 1 after BrdU injection, none of the BrdU+ cells were labeled with GAD-65/67. However, at day 28 after BrdU injection, we were able to detect BrdU+ cells that were labeled with GAD-65/67 (Fig. 2A). Double staining for DCX and GAD-65/67 demonstrated that new neurons in the spinal cord expressed GAD-65/67 (Fig. 2B). Double staining for GABA and BrdU further confirmed that these newly formed cells were GABAergic (Fig. 2C). Quantitative analysis revealed that 46% of the BrdU+ cells were also positive for GAD. Notably, the newly formed neurons in the spinal cord did not express the neuronal marker NeuN, in line with the finding by Horner et al. [2] that no BrdU+/NeuN+ cells are present in the intact spinal cord.

We postulated that the uneven distribution of newly formed neurons throughout the different spinal cord sectors could result from the presence of a gradient of specific guidance molecules. In a murine model of amyotrophic lateral sclerosis, progenitor cells expressing LacZ under the nestin promoter in spinal cords of diseased mice are localized mainly in the dorsal part of the spinal cord, although the damaged motor neurons reside ventrally [5]. Those progenitor cells express the chemokine receptor CXCR4, a receptor for the chemokine SDF-1, suggesting that SDF-1 signaling regulates migration of the progenitors. To examine this possibility in normal mice, we stained spinal cord sections for DCX and CXCR4. Some of the DCX+ cells in the dorsal part of the spinal cord also expressed CXCR4 (Fig. 3A), suggesting that SDF-1 plays a role in directing the dorsal migration of the newly formed neurons. Analysis of adult spinal cord revealed that SDF-1 expression was limited to the dorsal white matter (medial to the dorsal horn) and the central canal. Further analysis revealed that, in the dorsal part of the spinal cord, SDF-1 expression was associated with cells that were labeled with the astroglial marker GFAP, whereas in the central canal, SDF-1 expression was associated with the ependymal cell population (Fig. 3B–3D). The association between SDF-1 expression in the superficial dorsal horn and the relative abundance of neurogenesis in the dorsal part of the spinal cord suggest that SDF-1/CXCR4 signaling plays a role in adult spinal cord neurogenesis.

View larger version (15K): [in this window] [in a new window]   Figure 2. GABAergic phenotype of newly formed neurons in the adult spinal cord. Axial section of the adult mouse spinal cord showing expression of GAD-65/67 by BrdU+ cells (A) and by DCX+ cells (B); production of GABA by BrdU+ cells (C). Scale bar, 10 µm. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; DCX, doublecortin; GABA, -aminobutyric acid; GAD, glutamic acid decarboxylase.

View larger version (34K): [in this window] [in a new window]   Figure 3. SDF-1/CXCR4 signaling plays a role in adult spinal cord neurogenesis. (A): Expression of the chemokine receptor CXCR4 by DCX+ cells. (B): Double staining for SDF-1 and the astroglial marker GFAP. (C): Higher magnification of the boxed area in (B) showing astroglial expression of SDF-1 in the medial dorsal white matter. (D): SDF-1 is expressed in the central canal on GFAP– cells in the ependymal layer. Scale bar, 50 µm in (B) and 10 µm in (A, C, D). Abbreviations: CXCR4, CXC-chemokine receptor-4; DCX, doublecortin; GFAP, glial fibrillary acidic protein; SDF-1, stromal-derived factor-1.

Our group recently discovered that immune cells contribute to the maintenance of adult neurogenesis in the subventricular zone and dentate gyrus [6]. These findings, and the fact that adverse conditions in the CNS are usually associated with local immune activity, prompted us to examine whether immune activity also contributes to spinal cord neurogenesis under physiological conditions. We found significantly fewer DCX+ cells in the intact spinal cords of mice with SCID than in genetically matched wild-type controls (Fig. 4A, 4B). We further found, in the spinal cords of transgenic mice overexpressing a T-cell receptor for myelin basic protein, significantly more DCX+ cells than in the spinal cords of their matched wild-type controls (Fig. 4C). This is consistent with results we recently obtained in the dentate gyrus [6]. Thus, T cells, especially those of anti-myelin specificity, positively regulate spinal cord neurogenesis.

View larger version (24K): [in this window] [in a new window]   Figure 4. Contribution of myelin-specific T cells to adult spinal cord neurogenesis. (A): Representative images showing DCX+ cells in the dorsal part of the spinal cord of wild-type versus SCID mice (scale bar, 50 µm). (B): Quantification of DCX+ cells in spinal cord sections from wild-type versus SCID mice and (C) in spinal cord sections from wild-type versus TMBP transgenic mice. (* p < .05, *** p < .001, Student's t test). Abbreviations: DCX, doublecortin; SCID, severe combined immune deficiency; TMBP, T-cell receptor for myelin basic protein; WT, wild-type.

	DISCUSSION

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A previous study by Horner, Gage, and colleagues [2] revealed proliferating cells in the adult intact spinal cord that give rise to glial cells (oligodendrocytes and astrocytes). In that study, the authors used the neuronal markers NeuN (for postmitotic neurons) and Tuj-1 (ß-III tubulin, for immature neurons) and did not detect any cells double-labeled with BrdU and these neuronal markers. In line with the results of that study, our analysis also did not reveal any new proliferating cells that were stained for NeuN or ß-III tubulin. However, utilizing two markers of proliferation (BrdU and PCNA) together with the neuronal marker DCX and the GABAergic markers GAD-65/67 and GABA, we demonstrated that the DCX+ cells were indeed newly formed cells with neuronal properties. Previous studies that used transgenic mice that express LacZ reporter under the promoter of the neuronal progenitor marker nestin demonstrated the presence of proliferating neural progenitor cells in the ependymal zone surrounding the central canal under both normal and pathological conditions [4, 5]. Although this finding is consistent with our data, in the current study we are not only showing proliferation of progenitor cells but are also providing evidence for neurogenesis.

Our quantitative analysis of spinal cord neurogenesis over three different time points following BrdU uptake showed that although proliferating cells were found in similar numbers throughout the different sectors of the spinal cord (dorsal/ventral and white/gray matter), the number of new neurons and their fraction out of the total proliferating population significantly increased in the dorsal gray matter by day 28. These changes in distribution of the newly formed neurons likely reflect three developmental processes: migration of cells toward the dorsal gray matter, preferential survival of new neurons at the dorsal sector relative to non-neuronal proliferating cells, and continued differentiation that took place in the dorsal sector by the 28-day time point. The latter possibility is also consistent with the finding that cells expressing a GABAergic phenotype (i.e., BrdU+GAD-65/67+ and BrdU+GABA+ cells) were only found at day 28 after BrdU injection.

The presence of proliferating cells (BrdU+ or PCNA+) within the dorsal part of the spinal cord suggests that the newly formed GABAergic neurons in the adult spinal cord arise from a different progenitor-cell population than those of the central canal, in accordance with the model proposed by Horner et al. [2]. Although we do not exclude the possibility that early precursor cells migrate to the dorsal part of the spinal cord from other locations, such as the central canal, our observations suggest that the final proliferative stages during the process of adult spinal cord neurogenesis occur in situ, within the dorsal part of the spinal cord. The expression of SDF-1 in this region might indicate the relevance of CXCR4/SDF-1 signaling in attracting progenitor cells or restricting their position to the dorsal region. This notion is supported by studies showing that SDF-1 is expressed at the dorsal neural tube during development and acts as an attractant for migrating sensory neurons [13]. In the adult brain, astrocyte-derived SDF-1 was found to attract neural progenitor cells to injured sites after ischemic injury [14].

In agreement with our previous findings [6, 15], the results of the present study suggest that specific T-cell populations that recognize CNS-specific proteins contribute to the maintenance of neurogenesis in the adult spinal cord. The fivefold stable increase in neurogenesis following spinal cord injury, relative to noninjured control mice (supplemental online Fig. 1), might be linked to local immune-cell activity. Our group has recently discovered that injury-induced neurogenesis from endogenous precursors in the spinal cord is associated with local immunity and can be augmented by immunization with myelin-related antigens, provided that the evoked local immune response is carefully regulated [10]. In contrast, an uncontrolled immune response can be detrimental to neurogenesis [16, 17]. Other studies have suggested a relationship between adult neural stem cells and immune activity in the spinal cord [3, 18].

A key question that arises from these findings relates to the functional relevance of the newly formed immature GABAergic neurons in the adult intact spinal cord. Recent studies have demonstrated that newly formed neurons in the hippocampus are functional even before they express a mature phenotype [19, 20]; these results suggest that the new spinal-cord neurons described here might also have a physiological role while still in their immature state. The number of new neurons formed in the adult spinal cord falls within the same order of magnitude as those formed in the hippocampus, further suggesting that such a small cell population could have a functional role in the spinal cord. The location and morphology of the newly formed cells together with their GABAergic expression profile suggest that they function as inhibitory interneurons [9]. GABAergic interneurons in the dorsal horn of the spinal cord are thought to participate in modulation of sensory input, including pain [21, 22]. A link between neurogenesis and sensation of pain was recently found in a study showing that transplantation of neural stem cells into the injured spinal cord, while improving motor recovery, can also induce allodynia [23]. However, if the differentiation of the transplanted stem cells is restricted to the neuronal or oligodendroglial lineages, allodynia is alleviated [23]. The possibility that the new GABAergic neurons in the adult spinal cord function in the modulation of sensory input remains to be tested. Another intriguing possibility is that GABA released through a paracrine, nonsynaptic manner from the nascent GABAergic neurons provides trophic support to neural circuits in the dorsal part of the spinal cord. Such a noncanonical release of GABA was shown to be important for neuronal development [24]. In conclusion, this study demonstrates the presence of newly formed immature neurons expressing GABAergic characteristics as a novel cell population in the adult spinal cord and introduces a possible role for adult stem/progenitor cells in spinal cord plasticity in adulthood.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank S. Schwarzbaum and R. Halper for editing the manuscript. M.S. is the incumbent of the Maurice and Ilse Katz Professorial Chair in Neuroimmunology. The work was partially supported by the IsrALS Research Fund, by a research grant from the Julius and Ray Charlestein Foundation, and by the Mario Negri-Weizmann Collaboration Fund. R.S. and Y.Z. contributed equally to this work.

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This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 2006-0705v1 25/9/2277    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shechter, R. Articles by Schwartz, M. Search for Related Content PubMed PubMed Citation Articles by Shechter, R. Articles by Schwartz, M.