Phase-specific plasticity of synaptic structures in the somatosensory cortex of living mice during neuropathic pain
© Kim et al; licensee BioMed Central Ltd. 2011
Received: 29 July 2011
Accepted: 9 November 2011
Published: 9 November 2011
Postsynaptic dendritic spines in the cortex are highly dynamic, showing rapid morphological changes including elongation/retraction and formation/elimination in response to altered sensory input or neuronal activity, which achieves experience/activity-dependent cortical circuit rewiring. Our previous long-term in vivo two-photon imaging study revealed that spine turnover in the mouse primary somatosensory (S1) cortex markedly increased in an early development phase of neuropathic pain, but was restored in a late maintenance phase of neuropathic pain. However, it remains unknown how spine morphology is altered preceding turnover change and whether gain and loss of presynaptic boutons are changed during neuropathic pain.
Here we used short-term (2-hour) and long-term (2-week) time-lapse in vivo two-photon imaging of individual spines and boutons in the S1 cortical layer 1 of the transgenic mice expressing GFP in pyramidal neurons following partial sciatic nerve ligation (PSL). We found in the short-term imaging that spine motility (Δ length per 30 min) significantly increased in the development phase of neuropathic pain, but returned to the baseline in the maintenance phase. Moreover, the proportion of immature (thin) and mature (mushroom) spines increased and decreased, respectively, only in the development phase. Long-term imaging data showed that formation and elimination of boutons moderately increased and decreased, respectively, during the first 3 days following PSL and was subsequently restored.
Our results indicate that the S1 synaptic structures are rapidly destabilized and rearranged following PSL and subsequently stabilized in the maintenance phase of neuropathic pain, suggesting a novel therapeutic target in intractable chronic pain.
Neuropathic pain, the effective treatment of which is still lacking, is caused by a lesion along the somatosensory system and lasts for prolonged periods once it developed. Earlier findings from macroscopic brain imaging studies have suggested that maladaptive plastic changes, such as hyperexcitability and reorganization, in the primary somatosensory (S1) cortex play active roles in the chronification of neuropathic pain [1, 2]. Recently, we further proposed at the synaptic level the rapid and phase-specific remodeling of neuronal connections in the S1 cortex during neuropathic pain , because turnover of dendritic spines in the S1 cortex of living mice markedly increased during the early development phase of neuropathic pain and was restored during the subsequent maintenance phase of neuropathic pain. However, it is still unknown how spine is morphologically changed preceding the occurrence of gain and loss in the differential phases of neuropathic pain. Do presyaptic axonal boutons change their morphology and turnover rate correlated with dendritic spine remodeling during neuropathic pain development? To address these questions, we conducted a short- and long-term in vivo two-photon imaging of layer 1 spines and boutons in the S1 cortex of M-line mice, which express GFP in a small subset of layer 5 pyramidal neurons , before and after partial sciatic nerve ligation (PSL) . Layer 5 pyramidal neurons are the major output cells in the S1cortex and their distal tuft dendrites in layer 1 that are innervated by thalamocortical and corticocortical long-range projections as well as local circuit inputs, encode information about hind limb stimuli .
In summary, spine morphology and bouton turnover in the S1 cortex are phase-specifically changed following PSL injury, correlating with long-term spine remodeling data in our previous study . Thus, it can be collectively suggested that synaptic structures in the S1 cortex are destabilized and rearranged in the early development phase of neuropathic pain and subsequently stabilized in the late maintenance phase. Such stabilization of new circuits might be a structural correlate of long-lasting nature of neuropathic pain, as represented by the sustained head enlargement of new persistent spines that were generated in the development phase during the late phase of neuropathic pain . Investigations to clarify whether and how the S1 synapse remodeling directly contribute to neuropathic pain behaviors are currently in progress.
Materials and methods
All animal experiments were approved by the Animal Research Committee of the National Institutes of Natural Sciences. For the short-term imaging, GFP-M-line mice (male, 3-month old) were implanted with a small open-skull glass window over the left S1 cortex under urethane anesthesia (1.9 g/kg) as described previously . High-resolution imaging of dendrites (0.08 μm/pixel, 15-30 optical planes, 0.5 μm z-step) or axons (0.14 μm/pixel, 10-30 optical planes, 1.0 μm z-step) within 100 μm below the cortical surface (i.e. layer 1) were performed every 30 min for 2 hours with two-photon laser scanning microscopy (Olympus FV1000MPE microscope, Spectra-Physics Mai Tai Ti:sapphire laser at 950 nm). The location of imaged dendrites/axons was then confirmed inside the S1 hind paw area by using intrinsic optical signal imaging . For the long-term imaging, 2-month old M-line mice were implanted with a glass window and repeated two-photon imaging of the same axons (3-day intervals for 2 weeks) within the S1 hind paw area under isoflurane anesthesia (~1.5%) was started at 5 weeks after the window implantation . Metamorph (Molecular Devices) and ImageJ (http://rsbweb.nih.gov/ij/) were used to analyze individual spines and boutons from three-dimensional image stacks, blind to the experimental conditions. Detailed criteria for scoring, classifying and analyzing the spines and boutons are described elsewhere [3, 9, 16, 18]. Briefly, only spines that were clearly protruded from the shaft (> 0.4 μm laterally) and boutons that were three times brighter than the shaft were included in analysis. Spine length was measured from the dendritic shaft to the tip of spines and the motility was calculated as average absolute Δ length per 30 min. Spines were classified into three different types: mushroom (the diameter of spine head is two times larger than neck diameter and the length of spine head is longer than neck length), stubby (no head & total spine length is less than 1 μm) and thin (the length of spine head is shorter than neck length & total spine length is longer than 1 μm). Changes in the gain and loss rates of boutons were determined as the percentages of bouton formation and elimination between two successive imaging sessions, relative to the total bouton number in the former session. Only boutons that clearly separated from other bouton-like swellings over the 2-week imaging period were included in the analysis of integrated brightness, which was calculated by summing the intensity of all pixels comprising a bouton after background subtraction, divided by mean intensity of the adjacent axonal shaft. Each integrated brightness value was normalized to the value at PSL 0 d. For PSL injury, the right sciatic nerve was exposed at high-thigh level and 1/3-1/2 the diameter of the nerve was ligated with 9-0 suture. For sham-operation, the nerve was exposed, but left intact. The behavioral sign of tactile allodynia was assessed by using von Frey hair test. Data are presented as mean ± s.e.m. P values were calculated using a paired t-test or one-way ANOVA followed by a Dunnet's multiple comparison test unless otherwise stated.
We thank H. Ishibashi, T. Nemoto, H. Wake, Y. Takatsuru, K. Eto and H. Inada for excellent technical advice and critical discussions on the experiments and manuscript; M. Yoshitomo and T. Ohba for expert technical assistance; S. Park for providing a mouse illustration. This work was supported by the Grant-in-Aid for Scientific Research (A)(22240042) from the Japan Society for the Promotion of Science (JSPS) to J.N.; and the JSPS foreign researcher fellowship (P08456), Grant-in-Aid for Scientific Research (20.08456) from JSPS and Basic Science Research Program through the National Research Foundation funded by the Ministry of Education, Science and Technology, Korea (R11-2005-014) to S.K.K.
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