Touch gives new life: mechanosensation modulates spinal cord adult neurogenesis

  • A Corrigendum to this article was published on 26 March 2012

Abstract

The ability to respond to a wide range of novel touch sensations and to habituate upon repeated exposures is fundamental for effective sensation. In this study we identified adult spinal cord neurogenesis as a potential novel player in the mechanism of tactile sensation. We demonstrate that a single exposure to a novel mechanosensory stimulus induced immediate proliferation of progenitor cells in the spinal dorsal horn, whereas repeated exposures to the same stimulus induced neuronal differentiation and survival. Most of the newly formed neurons differentiated toward a GABAergic fate. This touch-induced neurogenesis reflected the novelty of the stimuli, its diversity, as well as stimulus duration. Introducing adult neurogenesis as a potential mechanism of response to a novel stimulus and for habituation to repeated sensory exposures opens up potential new directions in treating hypersensitivity, pain and other mechanosensory disorders.

Introduction

Our world is surrounded by various mechanosensory inputs, which are captured by our largest sensory organ—the skin. We are constantly exposed to both novel and familiar mechanosensory inputs such as shaking hands, putting on a piece of clothing or walking barefoot on the grass. These stimuli vary in their duration, novelty and their functional significance; some are persistent stimuli, like the touch of the grass under ones feet, whereas others are more acute, like a bug suddenly crawling on ones leg. Obviously, these signals require different levels of excitability or habituation by the nervous system.

Habituation in sensory systems is a key to effective perception; among other features, it allows better ‘signal to noise’ detection. Many of the habituation mechanisms involve desensitization at the level of the single sensory cell, as in the case of the eye's rod cells, along with higher-level processes and system plasticity. In the mechanosensory system, a large body of evidence supports the existence of plasticity and habituation process at the single cell level, mostly in the form of receptor sensitivity.1 In this study we introduce a new level of plasticity in the mechanosensory modality in the form of adult neurogenesis, manifested in the dorsal horn of the spinal cord.

Recent studies have identified neurogenesis in the adult spinal cord, both in healthy and in diseased tissues.2, 3, 4, 5, 6, 7, 8 Although the origin of the newly formed cells and their potential functional role is still unknown, adult neurogenesis of the spinal cord is reported to be mostly confined to the dorsal horn.2, 4 As mechanosensory information travels from the skin to the sensory cortex through specialized interneurons in this region,9, 10, 11 and based on the induction of neurogenesis in another sensory system, the olfactory bulb,12, 13, 14, 15, 16 we hypothesized that adult spinal cord neurogenesis may be involved in the response to touch and to mechanosensory inputs.

By exposing mice to novel mechanosensory stimuli for varying periods of time and of different diversity, we found that novel sensory stimuli induce progenitor cell proliferation in the sensory dorsal pathway of the adult spinal cord, and that the duration of the stimulation and its diversity affect their subsequent γ-aminobutyric acid (GABA)ergic neuronal differentiation and survival. Thus, our results are of potential functional relevance to neurogenesis in the dorsal pathway of adult spinal cord, with likely implications to plasticity and habituation in mechanosensation and touch.

Materials and methods

Animals

C57BL/6J mice were supplied by the Animal Breeding Center of the Weizmann Institute of Science. Animals were housed from birth under standard housing conditions. For all experiments, 8-week-old male mice were used. Mice were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee.

Sensory-enriched environment (SEE)

Mice were kept in cages covered with standard bedding. For the SEE sessions, mice were moved into new cages, the bottoms of which were covered with different types of textures: sandpapers comprising different sizes of embedded particles (coarse (p20), medium (p60) and fine (p150)), gravel and sponge. Each group was exposed to a different combination of these textures: cages in the group defined as ‘multiple’ comprised sections with each of these different textures, whereas cages in the ‘uniform’ group were covered with a single sensory texture, p60 sandpaper (Figure 1a). Mice in the ‘altered’ group were placed each day in a cage covered with a different single textured material, either the sandpapers or gravel or sponge. The duration of exposure to the SEE is indicated in text. In general, for single exposure, mice were placed in the SEE for 2 h; for repeated exposures, mice were exposed to SEE for 2 h, twice a day with an 8-h interval for 7 consecutive days; and in the group of continuous exposure, animals were permanently housed in the SEE cages. As social behavior and animal handling has been previously shown to affect neurogenesis, at least in the hippocampus,17 in all the described experiments, the animals were maintained in fixed groups of five animals and housed in routine housing conditions (cage size: 32 × 16 × 12.5 cm). Water and food were freely available in their standard positions. The control group was housed under similar conditions as the SEE group, and for the training session mice were placed in a new cage, covered with standard bedding.

Figure 1
figure1

Single exposure to mechanosensory stimulation induces proliferation in the dorsal horn of the adult spinal cord. (a) Illustration of the sensory-enriched environment (SEE)—‘multiple’ and ‘uniform’ paradigms. (b) 5-Bromo-2-deoxyuridine-positive (BrdU+) cells in the dorsal horn of spinal cords taken from adult control mice (left panel), or mice exposed once to a single SEE environment for 2 h (right panel, scale: 100 μm; magnification × 20); BrdU (75 mg kg–1; twice a day) was administered for 7 consecutive days. (c) Quantification of BrdU-labeled cells in the gray matter of the spinal cord of control and sensory-enriched mice exposed to either single texture (‘uniform’) or to a combination of textures (‘multiple’); BrdU (75 mg kg–1; twice a day) was administered for 7 consecutive days (n=3–5 per group; factorial analysis of variance (ANOVA); F=42.63; P=0.0001). (d, left panel) Experimental design; single exposure to combination of textures SEEmultiple followed by a single injection of BrdU (300 mg kg–1) at various time points relative to the SEE. The mice were killed 8 h after the BrdU injection. (d, right panel) Quantification of BrdU-labeled cells in the dorsal part of the spinal cord (n=3–5 per group; factorial ANOVA; F=4.29; P=0.028). (e, f) Double labeling for BrdU (green) and (e) Sox-2 (both in the central canal (cc) and in the dorsal horn (dh)), and (f) NG2 in mice that received BrdU parallel to the SEE and were killed 8 h later (magnification × 40; scale: (e) 10 μm; (f) 100μm). Confocal Z-axis sectioning imagines are provided for each marker (scale 10 μm). (g) Co-labeling for NG2 (green), doublecortin (DCX; red) and Hoechst labeling for nuclear localization in the dorsal horn. DCX+/NG2+ cells are marked by a full arrow and DCX/NG2+ is marked by an arrow head (magnification × 40; scale: 10 μm). ANOVA followed by Fisher's test; *P<0.05. The specific region analyzed in each panel is shown in the attached scheme.

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BrdU administration

5-Bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, St Louis, MO, USA) was dissolved by sonication in phosphate-buffered saline and injected intraperitoneally (75 mg kg–1 body weight) twice daily for 7 days immediately following the SEE exposure, or 14 days before the SEE exposures (as indicated in the text); animals were killed 14 or 28 days after the first SEE exposure. For identification of the stage following exposure in which the new cells formed, the mice were treated with a single administration of BrdU (300 mg kg–1 body weight) at different time points following the exposure, and analyzed 8 h after the injection. BrdU dose was chosen based on previous studies that suggested that 300 mg kg–1 is necessary to obtain efficient labeling of most of the S-phase cells;18, 19 such labeling is crucial for comparing between two different groups. This dose was previously shown to be nontoxic,4, 20 and was previously used to measure cell proliferation in mice. Indeed, in our experiments, no behavioral or physiological side effects were noticed.

Immunohistochemistry

Following phosphate-buffered saline perfusion, spinal cords were postfixed with Bouin's fixative (Sigma-Aldrich) for 48 h and then embedded in paraffin. Paraffin sections, 6-μm thick, were used throughout the study. The paraffin was removed by successive rinsing of slides with a gradient of xylene and ethanol. Following antigen retrieval by heating, the slides were blocked with blocking solution (20% horse serum with 0.3% Triton) and incubated for 48 h with specified combinations of the following primary antibodies. For BrdU staining, the slides were incubated in 2N HCl at 37 °C for 30 min following microwave treatment. The following primary antibodies were used: rat anti-BrdU (1:100; Oxford Biotechnology, Oxford, UK), goat anti-DCX (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-GABA (1:500; Sigma-Aldrich), rabbit anti-GAD65/67 (1:100; Abcam, Cambridge, MA, USA), rabbit anti-calretinin (1:1000; Chemicon, Temecula, CA, USA), rabbit anti-calbindin (1:200; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Sox-2 (1:150; Abcam), rabbit anti-NSE (1:50; Millipore, Billerica, MA, USA), mouse anti-HuC/D (1:50; Molecular Probes, Eugene, OR, USA) and rabbit anti-NG2 (1:150; Millipore). After rinsing in phosphate-buffered saline, sections were incubated for 1 h with the appropriate secondary antibodies (1:200; Jackson Immunoresearch Laboratories, Cambridgeshire, UK). For nuclear labeling, Hoechst 33 342 staining (1:2000; Molecular Probes) was done before mounting and covering.

Quantification

For microscopic analysis, a fluorescence microscope (E800; Nikon, Tokyo, Japan) or laser-scanning confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) was used. An observer, blind to the identity of the samples, counted the number of labeled cells from a total of 18 coronal spinal cord sections per mouse, taken from six different locations separated by 0.5 cm along the spinal cord. 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. Hoechst staining was routinely used for nuclear labeling, which served to verify quantification of the cells. Cell counting was performed using high-magnification imaging (objective × 40). In order to avoid overestimation because of counting fragments of cells that spanned several sections, only cells that had an intact morphology and a nucleus that was >5 μm in diameter were counted. Confocal Z-sectioning was performed in order to verify double labeling. The fluorescence microscope was equipped with a digital camera (DXM 1200F; Nikon) and with either a 20 × NA 0.50 or 40 × NA 0.75 objective lens (Plan Fluor; Nikon). The confocal microscope was equipped with LSM 510 laser scanning (three lasers: Ar 488, HeNe 543 and HeNe 633) and with a 40 × oil-immersion NA 1.3 Plan Neofluor objective lens. Recordings were made on postfixed tissues at 24 °C using acquisition software (ACT-1 (Nikon); or LSM (Carl Zeiss)).

Statistical analysis

Data were analyzed using Student's t-test or factorial analysis of variance followed by Fisher's least significant difference procedure. Error bars represent s.e.m. Statistical data that are given in the text represent average±s.e.m.

Results

Single exposure to novel tactile stimuli induces immediate cell proliferation, which depends on the diversity and intensity of the sensory stimuli

Based on our hypothesis that neurogenesis may be involved in mechanosensation, we first examined whether a novel tactile stimulation would induce changes in adult spinal cord neurogenesis. We therefore placed adult mice in a SEE consisting of cages covered with novel textures (sandpaper, gravel or sponge; Figure 1a) for 2 h. Immediately before the SEE exposure and for an additional 6 subsequent days, the mice were injected with BrdU (75 mg kg–1) twice a day to label proliferating cells. Mice were analyzed 14 days after exposure to SEE. Using immunohistochemical analysis, we found that a 2-h exposure to SEE resulted in a 2.5-fold increase in the number of cells labeled with BrdU in the dorsal horn (n=3–5 per group; Figures 1b and c), indicating cellular proliferation. To determine whether the degree of proliferation reflects the intensity and diversity of the novel stimuli, we compared two different SEE paradigms; ‘uniform’, composed of the single novel sensory texture (sandpaper), and ‘multiple’, comprising several different novel textures (Figure 1a). The exposure to multiple sensory inputs induced more intense cell proliferation (1.4-fold increase, multiple vs uniform; Figure 1c), suggesting that cell proliferation not only reflected the novelty of the sensory stimuli, but also its diversity.

In order to gain a better insight into the nature of this cell-renewal process, we aimed to identify the specific time frame in which proliferation took place in response to the SEE. As in the above-described experiment, the mice were analyzed only at a single time point, 14 days after the SEE. To this end, we repeated the experiment and administered a single dose of BrdU (300 mg kg–1), immediately, 24 h or 7 days after the exposure to a single touch stimulus. In each case, the animals were analyzed for cell renewal 8 h following the BrdU injection. We found that the exposure to a novel touch sensation elicited an immediate proliferative response, which was detected as early as 2 h after the exposure, and dramatically declined thereafter (n=3–5 per group; Figure 1d).

To determine whether these proliferating cells expressed cellular markers associated with neural progenitor cells, we co-labeled the cells with Sox-2, a transcription factor required for stem-cell maintenance in the central nervous system (Figure 1e).13 A small fraction (8±1%) of the BrdU+ cells was also found to be positive for this progenitor cell marker. Although the exposure to SEE did not affect the percentage of this population, the numbers of BrdU+/Sox2+ cells were increased following the sensory stimulus (155±32 versus 279±32, control and SEE, respectively; P<0.05, Student's t-test). Such double-labeled cells were found both in the central canal and in the gray matter of the dorsal horn.

Interestingly, 41±7% of the immediately proliferating cells expressed NG2 (Figure 1f). These cells were recently suggested to serve not only as oligodendrocyte precursors but also as precursors of other cells, including a potential source of new GABAergic interneurons.21 As these pluripotent NG2-positive cells were reported to coexpress doublecortin (DCX),22 we performed parallel analysis of NG2 together with DCX. Both the total numbers of NG2+ cells and of those that were double positive for DCX increased following the single SEE exposure (NG2+: 3459±521 vs 4869±480, P<0.05, Student's t-test; NG2+/DCX+: 916±323 vs 1718±171,P<0.05, Student's t-test; control vs SEE singlemultiple, respectively) (Figure 1g).

Altogether, these findings suggest that exposure to a novel mechanosensory stimulus for 2 h induces an immediate proliferation of progenitor cells, some of which can potentially differentiate into a neuronal cell fate.

Repeated exposure to the same stimuli inhibits cell proliferation and induces neuronal differentiation of newly formed cells

To examine whether repeated exposures to the SEE would induce a form of habituation, we introduced mice repeatedly to the same touch stimulus. Mice were exposed to the SEE in four different regimes: single exposure (as in the previous experiments), repeated exposures (two SEE sessions a day separated by an 8-h interval, and repeated for 7 consecutive days), continuous exposure to a uniform texture (animals were permanently housed in the SEE cages), and continuous exposure to a stimulus that was altered on a daily basis (the SEE housing cages were altered, with daily introduction of novel stimuli; ‘altered’; Figure 2a, left panel). We found that in contrast to the increased proliferative response achieved by increasing the number of concurrent signals (‘uniform’ vs ‘multiple’ sensory stimuli; Figure 1c), repeated exposures to a single texture had no effect on proliferation, and continuous exposure to the same SEE even inhibited it by 50% (n=3–5 per group; Figure 2a, right panel). However, by altering the sensory stimuli daily for the mice housed in the SEE cages, the inhibitory effect on the proliferation was attenuated (n=3–5 per group; Figure 2a, right panel).

Figure 2
figure2

Repeated exposures to sensory stimulation induce neuronal differentiation. (a, left panel) The experimental paradigm. (a, right panel) The quantification of 5-bromo-2-deoxyuridine (BrdU)-labeled cells in the gray matter of the spinal cord as a function of the duration/frequency of exposure to sensory-enriched environment (SEE); SEEsingle is single exposure to a uniform texture; SEERepeated is repeated exposures, with equal intervals between exposures, to a uniform texture; SEEContinuous is housing of mice in SEE cases of uniform texture; or a texture that was rotated daily (‘Continuousaltered’). BrdU (75 mg kg–1) was administrated twice a day for 7 consecutive days, starting from the first SEE (n=3–5 per group; factorial analysis of variance (ANOVA); F=11.75; P=0.0001). (b) Quantification of doublecortin (DCX)-labeled cells in the dorsal gray matter of the spinal cord of the control and sensory-enriched mice (n=4–8 per group; factorial ANOVA; F=12.15; P=0.0009 followed by Fisher's test; *P<0.05). (c) DCX labeling in adult control mice or mice exposed to SEERepeated (scale: 100 μm; magnification × 20). Arrows indicate positive cells. Enlargement of the boxed area in the low magnification panel is presented (scale: 10 μm; magnification × 40). (d, e) Co-labeling for both BrdU and DCX without (d), and with (e) Hoechst labeling for nuclear localization (scale: 10 μm; magnification × 40). (f) Experimental design; repeated exposures to SEE for 2 h twice a day, with an interval of 8 h, was performed using the following paradigms: SEEMultiple group was exposed to the same combination of textures every day; SEEUniform group was exposed to the same single novel texture (sand paper); and SEEaltered group was exposed to a single texture rotated on a daily basis. (g) Quantification of DCX+ cells in the dorsal gray matter in each of the treatment groups (n=5 per group; factorial ANOVA; F=11.0; P=0.009 followed by Fisher's test; *P<0.05).

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In other neurogenic niches, it was shown that a stimulus can also induce differentiation, especially if it is presented after the proliferation; therefore, we considered the possibility that the lack of proliferation might be because of increased differentiation. To examine if differentiation indeed took place, we examined the expression of the immature neuronal marker DCX in the dorsal horn of the spinal cord. Repeated exposures to SEE increased the number of cells that expressed DCX (n=4–8 per group; Figures 2b–e). Combined analysis with NG2 showed an increase in the numbers of DCX+/NG2 cells (1143±129 vs 4267±532, control vs SEE repeated, respectively, P=0.002). DCX+/NG2 cells were suggested to be committed to a neuronal lineage derived from the NG2+/DCX+ cells,22 a population that also increased following single SEE. Moreover, the fraction of this population (DCX+/NG2) out of the DCX+ cells increased following the repeated exposures (43±3 vs 72±3%, control vs SEE repeated, respectively, P=0.001).

To characterize the nature of the signal that induces this differentiation, we used the same paradigm as above and introduced mice to repeated exposures of different novelties (Figure 2f) including cages covered with: (1) multiple tactile textures (sandpapers of different roughness, gravel and sponge; ‘multiple’); (2) a single tactile texture (medium roughness sandpaper, ‘uniform’); and (3) alternating textures on a daily basis (one of the sandpapers, gravel or sponge; ‘altered’). Although the number of exposures and the time spent in the SEE in all groups were similar, significant differences were found in the numbers of newly formed neurons under the different conditions. Thus, mice that were exposed repeatedly to either a single stimulus (uniform) or to a cage containing a multiple but fixed set of textures (multiple) showed a similar increase in differentiation relative to mice maintained in standard housing (n=5 per group; Figure 2g). Interestingly, the group that was exposed to repeated stimuli, the nature of which was changed on a daily basis (‘altered’), exhibited a significantly greater increase in neuronal differentiation (n=5 per group; Figure 2g). Thus, this finding suggested that the novelty of the mechanosensory stimulus is an important factor in determining the number of differentiating newly formed neurons.

Taking into consideration the dynamic nature of this process, we aimed to determine whether prolonged exposure induced both proliferation and subsequent differentiation of the same proliferating cells, or whether it induced differentiation of the pre-existing pool of proliferating cells. To this end, we first created a new labeled pool of cells by injection of BrdU (75 mg kg–1; for 7 consecutive days, twice a day) to naive animals. The mice were exposed to repeated SEE 14 days later. We found a greater than twofold increase in the BrdU-labeled neurons (DCX+/BrdU+ cells) in the group prelabeled with BrdU compared with the group injected with BrdU concomitantly with the SEE exposure (DCX+/BrdU+ cells out of DCX+; 10.3±2.1 vs 4.5±1.0%, respectively; n=4 to 5 per group). These findings thus indicated that repeated sensory stimuli induced neuronal differentiation from both newly proliferated progenitors and pre-existing ones.

The newly formed cells are mostly localized in the gray matter of the lumbar segments of the dorsal horn

Taken together, our results identified spinal cord adult neurogenesis as a novel aspect of touch sensation plasticity, highly sensitive to the nature and duration of the stimuli. Brief exposure to a novel stimulus induced immediate cell proliferation in the dorsal horn of the spinal cord, whereas prolonged exposure affected the differentiation of the newly formed cells into immature (DCX+) neurons. To determine whether these newly formed immature neurons were preferentially formed at specific locations, we spatially characterized these proliferation and differentiation events. Analyzing the cells 8 h after exposure to a single novel stimulus revealed that the majority of the proliferating (BrdU+) cells were located in the gray matter (n=5; Figure 3a). Similarly, analyzing the cells 14 days after sensory stimuli revealed that the immature DCX+ neurons produced following repeated SEE exposures were also mainly located in the gray matter (n=5; Figure 3b) of the dorsal horn (n=5; Figure 3c). Repeated SEE exposures specifically increased the numbers of immature neurons in this area (n=5 per group; Figures 3d and e) as well as in the central canal (n=5 per group; Figures 3f and g), which is known to maintain neural progenitor cells.2, 5 Analysis of consecutive sections along the anterior–posterior axis revealed that the increase in neurogenesis was most strongly evident in sections derived from the lumbar segments (n=5; Figure 3h).

Figure 3
figure3

The newly formed cells are mostly localized in the gray matter of the dorsal horn of the lumbar segments. (a) Quantification of the number of 5-bromo-2-deoxyuridine (BrdU)-labeled cells in the dorsal region (gray matter (black bars) and white matter (white bars)) of the spinal cord of mice exposed to a single session of SEEmultiple and injected with a single injection of BrdU (300 mg kg–1) immediately before the sensory-enriched environment (SEE; ‘immediate’). No changes were seen in the number of proliferating cells in the white matter. Enhanced proliferation was attributed to proliferating cells in the gray matter. (be) Numbers of newly formed neurons (b) in the gray vs white matter, and (c) in the dorsal vs ventral horns in the SEERepeated mice (n=5; Student's t-test; (b) ***P=0.007; (c) **P=0.01). Relative distribution of doublecortin-positive (DCX+) cells (d) in the gray vs white matter, and (e) in the dorsal vs ventral horns in mice that were exposed to SEERepeated, and in control mice (n=5 per group; Student's t-test; (d) *P=0.01; (e) **P=0.003). (f) DCX-positive (red; marked by full arrow) and BrdU-positive (green; marked by arrow head) cells in the central canal of naive mice, and mice that were exposed to SEERepeated (scale=10 μm; magnification × 40). (g) Number of BrdU- and DCX-positive cells in the central canal of both groups (mean numbers of cells per 1 mm cord length; Student's t-test; n=5 per group; BrdU: P=0.7; DCX: *P=0.01). (h) Quantitative analysis of DCX+-labeled cells in the dorsal gray matter in coronal sections taken from the cervical, thoracic and lumbar areas of the spinal cords (n=5; factorial analysis of variance (ANOVA); F=3.802; P=0.01; followed by Fisher's test, *P<0.05).

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Most of the newly formed cells differentiate into GABAergic immature neurons

Although the role of adult neurogenesis in the central nervous system is still not clear, identifying its specific neuronal differentiation might shed some light on their potential role in the central nervous system. We hypothesized that at least some portion of the newly formed cells described above eventually differentiate into a GABAergic fate. This was supported by several lines of evidence. First, in the olfactory system, where neurogenesis continuously takes place, most of the immature neurons that reach the olfactory bulb differentiate into neurons expressing GABAergic granules.23 Second, recent reports suggested that progenitor cells expressing NG2 and differentiating into neurons appear to differentiate into GABAergic interneurons.24, 25 Finally, a previous report suggested the presence of immature GABAergic neurons in the dorsal horn of the spinal cord.4

To test our hypothesis and examine the cellular fate of the newly formed cells, we first verified that these cells indeed show markers of fully committed neurons. We used human neuronal protein (HuC/D) and neuronal-specific enolase (NSE), the markers that appear only in neurons. Analyzing cells 28 days after the first BrdU injection (75 mg kg–1, administrated twice a day for 7 consecutive days), which corresponds to 22 days after the last exposure to the novel sensory stimuli, enabled us to detect 55 co-labeled cells in 1 mm3 that were positive for both BrdU and HuC/D or NSE (Figures 4a and b). Further analysis revealed BrdU+ cells that coexpressed GABA and the enzyme responsible for production of this neurotransmitter, glutamic acid decarboxylase (GAD65/67) (Figures 4c and d). Among the BrdU+ cells, 53±4.5% expressed GABA. Some of these newly formed cells were also co-labeled with the calcium-binding protein, calretinin (Figure 4e), but we could not detect any cell that was co-labeled with calbindin (not shown), potentially indicating a specialized GABAergic differentiation state, or specific maturation phase.

Figure 4
figure4

The newly formed cells differentiate into γ-aminobutyric acid (GABA)ergic neurons. Animals were subjected to repeated exposures to sensory-enriched environment (SEE), and analyzed 28 days after first exposure. 5-Bromo-2-deoxyuridine (BrdU) was administered at the time of SEE exposure. (ae) Double labeling for BrdU and (a) human neuronal protein (HuC/D); (b) neuronal-specific enolase (NSE); (c) GAD65/67; (d) GABA; and (e) calretinin (CR) (scale: (a, b) 10 μm; (ce) 50 μm; boxed: 10 μm; all at magnification × 40). Double-positive cells are marked by full arrows, whereas arrowheads indicate BrdU+ cells that do not express the other tested neuronal marker. Confocal Z-axis scan of single cells positive for the indicated markers are provided. (f) Number of BrdU+ cells in the dorsal horn (following single injection of 300 mg kg–1 BrdU, administrated once parallel to the first sensory stimuli; n=6 per group). (g) Quantification of BrdU+/GABA+ cells in the dorsal horn 28 days following the first SEE exposure (BrdU was administrated parallel to the SEE sessions at a dose of 75 mg kg–1), (n=6 per group; Student's t-test; *P<0.05).

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To test whether sensory exposure affects the survival rate of the newly formed cells, we evaluated the number of surviving BrdU-positive cells 28 days after the first SEE exposure (following a single 300 mg kg–1 injection of BrdU given immediately before the first exposure to SEE). Similar to the effect of exposure to novel odors on neurogenesis in the olfactory bulb, we found that repeated exposures to the same stimuli, unlike a single exposure, led to an overall twofold increase in the number of surviving newly formed cells (n=6 per group; Figure 4f). To quantify the number of newly formed GABAergic cells, we repeated the above-described experiment while using a protocol of repeated BrdU injections (75 mg kg–1; 7 consecutive days), which improves the detection of such cells. Repeated exposures resulted in an increased number of newly formed (BrdU+) GABAergic cells (n=6 per group; Figure 4g). These findings thus indicate that repeated exposure to sensory stimuli supports the survival of the newly formed GABAergic immature neurons.

Discussion

Our data suggest spinal cord adult neurogenesis as a novel player in touch sensation, responsive to different aspects of the mechanosensory input, including the novelty of the stimulus, its duration and its diversity. We showed that single exposure to novel mechanosensory stimulus resulted in an increase in the number of proliferating cells in the dorsal horn of the adult rodent spinal cord in correlation with the diversity of the new stimuli presented (higher diversity resulted in a greater number of proliferating cells). The proliferative response of the progenitor cells was found to be an immediate one, and was evident as early as 2 h following exposure to the novel stimuli (the effect dramatically declined thereafter). Furthermore, repeated exposures to the same stimulus induced neuronal differentiation and survival of the progenitor cells, possibly reflecting a mechanism of habituation (Figure 5).

Figure 5
figure5

Schematic representation of spinal cord adult neurogenesis in the sensory-enriched environment (SEE) model. Single exposure to a novel mechanosensory stimulus induces immediate proliferation from an apparent reservoir of progenitor cells (DCX+/NG2+; Sox2+) in the adult spinal cord. In contrast, repeated exposure to the same stimulus induces neuronal differentiation and survival of the new cells, mostly into immature (DCX+, NG2, NeuN, Calbindin) GABAergic (GABA+, GAD 65/67+) neurons (NSE+, HuC/D+, Calretinin+), and inhibits their additional proliferation.

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In contrast to the proliferation phase, which was found to be a reflection of both the novelty and diversity of the stimuli, the extent of neuronal differentiation depended mainly on the novelty of the stimuli (repeated exposures to various novel stimuli presented together (‘multiple’) had a weaker effect on the differentiation compared with presenting the same number of novel stimuli on a rotating basis (‘altered’). Notably, the cellular fate of these cells was mostly GABAergic. Our observation that prolonged exposures to the same stimuli increased the number of GABAergic cells might reflect a habituation or de-sensitization processes related to these stimuli. This possibility is especially attractive in light of the fact that in the superficial lamina of the dorsal spinal cord, many inhibitory interneurons express and respond to GABA.26

The origin of the newly formed cells is not clear. It was first suggested by Horner et al.2 that cells continuously proliferate at the outer circle of the adult spinal cord. Further characterization of this niche by others revealed that these cells routinely differentiate to immature neurons and are predominately found in the dorsal gray matter.4 The exposure to novel stimuli results in an immediate proliferation of cells, the majority of which are NG2 positive. As reviewed by Nishiyama et al.,27 the lineage progression of NG2+ cells, which are the largest pool of postnatal proliferative progenitors, is a generally controversial issue. NG2-expressing cells, originally suggested to serve only as oligodendrocyte precursors, are now considered to be a heterogeneous population. Several recent studies have suggested that at least some of these assumed oligodendrocyte precursors retain the ability to generate interneurons and, thus, are suggested to have properties of multipotential progenitors. Although the lineage progression of NG2+ cells toward a neuronal fate is still under debate, evidence from in vitro and in vivo experiments increasingly support such a possibility. It was demonstrated that a sizeable fraction of postnatal NG2 proteoglycan-expressing progenitor cells in the hippocampus are proliferative precursors, whose progeny appear to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs.24 Moreover, in adult nonconstitutively active neurogenic niches, such as the neocortex, it has been shown that a fraction of NG2-expressing cells in the adult brain express neuronal markers, suggesting that the source of the newly generated GABAergic cells are NG2+ progenitors.21, 24 However, under normal conditions, the vast majority of NG2-positive cells in the adult neocortex that incorporate BrdU remain NG2 positive many weeks after the BrdU injection, and their differentiation to immature neurons is a relatively rare event. Tamura et al.22 reported that such NG2-expressing multipotent progenitor cells are also DCX positive, and that in the rare process of their differentiation to neuronal committed cells, they downregulate NG2 and maintain the expression of DCX. In line with this suggested model of fate decision, in the current study we found that the proliferative population in the spinal cord in response to SEE stimuli is mostly NG2+/DCX+ and repeated stimulations increase the fraction of the neuronal committed DCX+/NG2 cells. Accordingly, the sensory-sensitive progenitor population in the spinal cord, described here, could represent a novel niche from which such neuronal differentiation can occur.

Most of these newly formed cells die within 28 days (in agreement with the cell fate in other neurogenic niches, especially the dentate gyrus of the hippocampus where most of the newly formed cells die within 4 weeks of proliferation28). However, of the remaining cells, 50% express markers associated with a GABAergic cell fate. Tamura et al.22 suggested GABAergic differentiation as the reasonable fate of the rare DCX+/NG2 sub-population, which were referred to as ‘immature neurons’, although such a maturation fate was not detected in the nonstimulated neocortex. Interestingly, Tamura et al.22 suggested that the low differentiation rate into GABAergic fate may be an outcome of an environment with minimal stimulation. In apparent correlation, our observations of increased survival of newly formed GABAergic cells following repeated sensory stimuli might constitute a response to a strongly stimulatory environment. Further support to this claim comes from our observation that sensory enrichment affects the relative proportions of this newly characterized sub-population, with an increased fraction of the DCX+/NG2 subset. Further studies are needed to test whether other enriched environmental conditions, such as social activities or exercise, can affect the fate of this rare population in other neurogenic niches.

GABAergic neuronal differentiation is one of the hallmarks that characterize adult neurogenesis in the olfactory system.12, 29 Interestingly, olfactory bulb GABAergic neurogenesis is enhanced by novel odors. In this study we suggest that in analogy to olfactory neurogenesis, which is associated with odor discrimination30, 31 and with mating behavior,32 neurogenesis in the dorsal horn of the adult spinal cord is related to the ability to discriminate and/or habituate different mechanosensory stimuli. In analogy to the effects of exposure to novel odors,30, 31 SEE prolonged the survival of the newly formed GABAergic cells in the spinal cord. The role of the newly formed GABAergic immature neurons is, as yet, unclear. Unlike their olfactory counterparts, which were shown to be capable of differentiating to mature circuit-integrated interneurons, we did not observe any mature neurons such as NeuN+/BrdU+ co-labeled cells after 4 weeks. It is possible that in the spinal cord neurogenic niche, the newly generated GABAergic immature neurons serve a transient neuromodulatory role, which supports a mechanism in which the niche plasticity undergoes fine tuning that is regulated both temporally and spatially.

The similarity between the neurogenic response to novel odors and the neurogenic response to the mechanosensory stimulation described in the present study might reflect a more general phenomenon of plasticity in sensory organs, although with different spatial and temporal dynamics. Such a mechanism may perhaps be of greater relevance to sensations that are subject to ‘background noise’ in the sensory input and those that involve continuous and uninterrupted input, such as hearing (background noise that is continually present), olfaction (a lingering smell in the room) and mechanosensation (the sensation derived from extended contact with everyday objects, such as clothes); these sensory organs are likely to utilize adult neurogenesis as part of their plasticity mechanism. This is in contrast to the fast dynamics of stimulus presentation in vision, which would benefit less from the use of neurogenesis in the encoding process.

Therapeutically, the formation of new, mostly GABAergic, neurons in the dorsal horn of the adult spinal cord, where nociceptive fibers terminate, may also be relevant for pain transduction, especially for allodynia, a syndrome characterized by sensations of pain in response to non-painful stimuli, and was previously shown to be ameliorated by transplantation of stem cells.33 Such pathology may potentially involve a maladaptive neurogenic process in this region of the spinal cord. Other conditions, which involve a mechanosensory component, are also potential subjects of intervention. Further understanding of this neurogenic process, responsive to mechanosensory inputs, may open new opportunities for noninvasive therapy.

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Acknowledgements

We thank Shelley Schwarzbaum for editing the manuscript. MS holds the Maurice and Ilse Katz Professorial Chair in Neuroimmunology. This work was supported in part by the High Q foundation and by IsrALS (to MS).

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Correspondence to M Schwartz.

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The authors declare no conflict of interest.

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Shechter, R., Baruch, K., Schwartz, M. et al. Touch gives new life: mechanosensation modulates spinal cord adult neurogenesis. Mol Psychiatry 16, 342–352 (2011). https://doi.org/10.1038/mp.2010.116

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Keywords

  • adult neurogenesis
  • enriched environment
  • habituation
  • mechanosensory
  • spinal cord
  • touch

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