Adult Mouse Spinal Cord Research Articles

High-resolution gene expression atlases for adult and developing mouse brain and spinal cord

Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).

Reining in pain

UCSF researchers have shown that neuronal precursor cells from fetal mouse brains can be transplanted into the spinal cord of adult mice to treat neuropathic pain. The embryonic brain cells are being developed by Neurona.

Localization of neurones expressing the gap junction protein Connexin45 within the adult spinal dorsal horn: a study using Cx45-eGFP reporter mice

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

Forebrain GABAergic Neuron Precursors Integrate into Adult Spinal Cord and Reduce Injury-Induced Neuropathic Pain

Neuropathic pain is a chronic debilitating disease characterized by mechanical allodynia and spontaneous pain. Because symptoms are often unresponsive to conventional methods of pain treatment, new therapeutic approaches are essential. Here, we describe a strategy that not only ameliorates symptoms of neuropathic pain but is also potentially disease modifying. We show that transplantation of immature telencephalic GABAergic interneurons from the mouse medial ganglionic eminence (MGE) into the adult mouse spinal cord completely reverses the mechanical hypersensitivity produced by peripheral nerve injury. Underlying this improvement is a remarkable integration of the MGE transplants into the host spinal cord circuitry, in which the transplanted cells make functional connections with both primary afferent and spinal cord neurons. By contrast, MGE transplants were not effective against inflammatory pain. Our findings suggest that MGE-derived GABAergic interneurons overcome the spinal cord hyperexcitability that is a hallmark of nerve injury-induced neuropathic pain.

Localization of endogenous morphine‐like compounds in the mouse spinal cord

Morphine, codeine, morphine-6-glucuronide, and morphine-3-glucuronide are synthesized de novo in mammalian cells and in the central nervous system. Knowledge on endogenous morphine-like compound distribution in the adult mouse brain has been recently improved, and new hypotheses have been suggested about the potential implications in brain physiology. Endogenous morphine-like compounds have been shown to be synthesized in the spinal cord, but their localization is unknown. Here we describe the distribution of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the adult mouse spinal cord using a well-validated antibody. By using different microscopy approaches, we found the presence of morphine, codeine, or morphine glucuronides in γ-aminobutyric acid (GABA)-ergic neurons and astrocytes of the spinal cord. Whereas GABAergic neurons containing endogenous morphine-like compounds were located primarily in the ventral horn, astrocytes that were labeled for morphine-like compounds were found throughout the gray matter and the white matter. Our study demonstrates the possibility that endogenous morphine-like compounds in the central nervous system have other functions beyond their analgesic functions.

Epigenetic Regulation of Motor Neuron Cell Death through DNA Methylation

DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene-regulatory regions. It is unknown whether aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased fivefold and twofold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.

Inhibition of the Ca2+-Dependent K+Channel,KCNN4/KCa3.1, Improves Tissue Protection and Locomotor Recovery after Spinal Cord Injury

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²⁺ signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.

Novel molecular targets of the neuroprotective/neurorescue multimodal iron chelating drug M30 in the mouse brain

The novel multifunctional brain permeable iron, chelator M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline] was shown to possess neuroprotective activities in vitro and in vivo, against several insults applicable to various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present study, we demonstrate that systemic chronic administration of M30 resulted in up-regulation of hypoxia-inducible factor (HIF)-1α protein levels in various brain regions (e.g. cortex, striatum, and hippocampus) and spinal cord of adult mice. Real-time RT-PCR revealed that M30 differentially induced HIF-1α-dependent target genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), enolase-1, transferrin receptor (TfR), heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), and glucose transporter (GLUT)-1. In addition, mRNA expression levels of the growth factors, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) and three antioxidant enzymes (catalase, superoxide dismutase (SOD)-1, and glutathione peroxidase (GPx)) were up-regulated by M30 treatment in a brain-region-dependent manner. Signal transduction immunoblotting studies revealed that M30 induced a differential enhanced phosphorylation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), protein kinase B (PKB/Akt), and glycogen synthase kinase-3β (GSK-3β). Together, these results suggest that the multifunctional iron chelator M30 can up-regulate a number of neuroprotective-adaptive mechanisms and pro-survival signaling pathways in the brain that might function as important therapeutic targets for the drug in the context of neurodegenerative disease therapy.

Human neural progenitors from different foetal forebrain regions remyelinate the adult mouse spinal cord

Improving oligodendroglial differentiation from human foetal neural progenitor cells remains a primordial issue to accomplish successful cell-based therapies in myelin diseases. Here, we combined in situ, in vitro and in vivo approaches to assess the oligodendrogenic potential of different human foetal forebrain regions during the first trimester of gestation. We show for the first time that the initial wave of oligodendrocyte progenitor emergence in the ventral telencephalon onsets as early as 7.5 weeks into gestation. Interestingly, in vitro, isolation of ganglionic eminences yielded oligodendrocyte progenitor-enriched cultures, as compared with cortex and thalamus. Most importantly, single injection of human neural progenitors into rodent models of focal gliotoxic demyelination revealed the great capacity of these cells to survive, extensively migrate and successfully remyelinate the spinal cord, irrespective of their origin. Thus, our study brings novel insights into the biology of early human foetal neural progenitor cells and offers new support for the development of cellular therapeutics for myelin disorders.

SDF1 in the dorsal corticospinal tract promotes CXCR4+ cell migration after spinal cord injury

BackgroundStromal cell-derived factor-1 (SDF1) and its major signaling receptor, CXCR4, were initially described in the immune system; however, they are also expressed in the nervous system, including the spinal cord. After spinal cord injury, the blood brain barrier is compromised, opening the way for chemokine signaling between these two systems. These experiments clarified prior contradictory findings on normal expression of SDF1 and CXCR4 as well as examined the resulting spinal cord responses resulting from this signaling.MethodsThese experiments examined the expression and function of SDF1 and CXCR4 in the normal and injured adult mouse spinal cord primarily using CXCR4-EGFP and SDF1-EGFP transgenic reporter mice.ResultsIn the uninjured spinal cord, SDF1 was expressed in the dorsal corticospinal tract (dCST) as well as the meninges, whereas CXCR4 was found only in ependymal cells surrounding the central canal. After spinal cord injury (SCI), the pattern of SDF1 expression did not change rostral to the lesion but it disappeared from the degenerating dCST caudally. By contrast, CXCR4 expression changed dramatically after SCI. In addition to the CXCR4+ cells in the ependymal layer, numerous CXCR4+ cells appeared in the peripheral white matter and in the dorsal white matter localized between the dorsal corticospinal tract and the gray matter rostral to the lesion site. The non-ependymal CXCR4+ cells were found to be NG2+ and CD11b+ macrophages that presumably infiltrated through the broken blood-brain barrier. One population of macrophages appeared to be migrating towards the dCST that contains SDF1 rostral to the injury but not towards the caudal dCST in which SDF1 is no longer present. A second population of the CXCR4+ macrophages was present near the SDF1-expressing meningeal cells.ConclusionsThese observations suggest that attraction of CXCR4+ macrophages is part of a programmed response to injury and that modulation of the SDF1 signaling system may be important for regulating the inflammatory response after SCI.

SDF1 in the dorsal corticospinal tract promotes CXCR4+ cell migration after spinal cord injury

Stromal cell-derived factor-1 (SDF1) and its major signaling receptor, CXCR4, were initially described in the immune system; however, they are also expressed in the nervous system, including the spinal cord. After spinal cord injury, the blood brain barrier is compromised, opening the way for chemokine signaling between these two systems. These experiments clarified prior contradictory findings on normal expression of SDF1 and CXCR4 as well as examined the resulting spinal cord responses resulting from this signaling. These experiments examined the expression and function of SDF1 and CXCR4 in the normal and injured adult mouse spinal cord primarily using CXCR4-EGFP and SDF1-EGFP transgenic reporter mice. In the uninjured spinal cord, SDF1 was expressed in the dorsal corticospinal tract (dCST) as well as the meninges, whereas CXCR4 was found only in ependymal cells surrounding the central canal. After spinal cord injury (SCI), the pattern of SDF1 expression did not change rostral to the lesion but it disappeared from the degenerating dCST caudally. By contrast, CXCR4 expression changed dramatically after SCI. In addition to the CXCR4+ cells in the ependymal layer, numerous CXCR4+ cells appeared in the peripheral white matter and in the dorsal white matter localized between the dorsal corticospinal tract and the gray matter rostral to the lesion site. The non-ependymal CXCR4+ cells were found to be NG2+ and CD11b+ macrophages that presumably infiltrated through the broken blood-brain barrier. One population of macrophages appeared to be migrating towards the dCST that contains SDF1 rostral to the injury but not towards the caudal dCST in which SDF1 is no longer present. A second population of the CXCR4+ macrophages was present near the SDF1-expressing meningeal cells. These observations suggest that attraction of CXCR4+ macrophages is part of a programmed response to injury and that modulation of the SDF1 signaling system may be important for regulating the inflammatory response after SCI.

An Approach to Experimental Synaptic Pathology Using Green Fluorescent Protein-Transgenic Mice and Gene Knockout Mice to Show Mitochondrial Permeability Transition Pore-Driven Excitotoxicity in Interneurons and Motoneurons

Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.

Possible involvement of syntaxin 1A downregulation in the late phase of allodynia induced by peripheral nerve injury

Syntaxin 1A is a membrane protein playing an integral role in exocytosis and membrane trafficking. The superficial dorsal horn (SDH) of the spinal cord, where nociceptive synaptic transmission is modulated, is rich in this protein. We recently reported that peripheral nerve ligation-induced nociceptive responses are considerably enhanced in syntaxin 1A-knockout mice [Takasusuki T, Fujiwara T, Yamaguchi S, Fukushima T, Akagawa K, Hori Y (2007) Eur J Neurosci 26:2179–2187]. On the basis of this earlier finding, we hypothesized that syntaxin 1A is involved in peripheral nerve injury-induced nociceptive plasticity. In this study, we examined this hypothesis by using nociceptive behavioral studies and tight-seal whole-cell recordings from neurons in the SDH of adult mouse spinal cord slices. Partial sciatic nerve ligation (PSNL) in adult male Institute of Cancer Research (ICR) mice increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). The amplitude of the mEPSCs did not exhibit any changes, suggesting that peripheral nerve injury is associated with increased synaptic release of excitatory neurotransmitters. Western blot and real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that PSNL gradually decreased the expression level of syntaxin 1A in the spinal SDH. This downregulation of syntaxin 1A took several days to develop, whereas behavioral allodynia developed within one day after PSNL. Syntaxin 1A knockdown by intrathecal injection of an antisense oligodeoxynucleotide against the syntaxin 1A gene led to the gradual development of allodynia. These results indicate a possible involvement of syntaxin 1A downregulation in the late maintenance phase of peripheral nerve injury-induced allodynia. In addition, syntaxin 1A knockdown by ribonucleic acid interference enhanced the axonal elongation and sprouting of spinal dorsal horn neurons in culture, suggesting that PSNL-induced syntaxin 1A downregulation may result in the rearrangement of the synaptic connections between neurons in the spinal dorsal horn. Taken together, it is possible to conclude that syntaxin 1A might be involved in spinal nociceptive plasticity induced by peripheral nerve injury.

Absence of IFNγ expression induces neuronal degeneration in the spinal cord of adult mice.

BackgroundInterferon gamma (IFNγ) is a pro-inflammatory cytokine, which may be up-regulated after trauma to the peripheral or central nervous system. Such changes include reactive gliosis and synaptic plasticity that are considered important responses to the proper regenerative response after injury. Also, IFNγ is involved in the upregulation of the major histocompatibility complex class I (MHC class I), which has recently been shown to play an important role in the synaptic plasticity process following axotomy. There is also evidence that IFNγ may interfere in the differentiation and survival of neuronal cells. However, little is known about the effects of IFNγ absence on spinal cord neurons after injury.MethodsWe performed a unilateral sciatic nerve transection injury in C57BL/6J (wild type) and IFNγ-KO (mutant) mice and studied motoneuron morphology using light and electron microscopy. One week after the lesion, mice from both strains were sacrificed and had their lumbar spinal cords processed for histochemistry (n = 5 each group) and transmission electron microscopy (TEM, n = 5 each group). Spinal cord sections from non-lesioned animals were also used to investigate neuronal survival and the presence of apoptosis with TUNEL and immunohistochemistry.ResultsWe find that presumed motoneurons in the lower lumbar ventral horn exhibited a smaller soma size in the IFNγ-KO series, regardless of nerve lesion. In plastic embedded sections stained with toluidine blue, the IFNγ-KO mice demonstrated a greater proportion of degenerating neurons in the ventral horn when compared to the control series (p < 0.05). Apoptotic death is suggested based on TUNEL and caspase 3 immunostaining. A sciatic nerve axotomy did not further aggravate the neuronal loss. The cellular changes were supported by electron microscopy, which demonstrated ventral horn neurons exhibiting intracellular vacuoles as well as degenerating nuclei and cytoplasm in the IFNγ-KO mice. Adjacent glial cells showed features suggestive of phagocytosis. Additional ultrastructural studies showed a decreased number of pre-synaptic terminals apposing to motoneurons in mutant mice. Nevertheless, no statistical difference regarding the input covering could be detected among the studied strains.ConclusionAltogether, these results suggest that IFNγ may be neuroprotective and its absence results in neuronal death, which is not further increased by peripheral axotomy.

VGLUT2-Dependent Glutamate Release from Nociceptors Is Required to Sense Pain and Suppress Itch

Itch can be suppressed by painful stimuli, but the underlying neural basis is unknown. We generated conditional null mice in which vesicular glutamate transporter type 2 (VGLUT2)-dependent synaptic glutamate release from mainly Nav1.8-expressing nociceptors was abolished. These mice showed deficits in pain behaviors, including mechanical pain, heat pain, capsaicin-evoked pain, inflammatory pain, and neuropathic pain. The pain deficits were accompanied by greatly enhanced itching, as suggested by (1) sensitization of both histamine-dependent and histamine-independent itch pathways and (2) development of spontaneous scratching and skin lesions. Strikingly, intradermal capsaicin injection promotes itch responses in these mutant mice,as opposed to pain responses in control littermates. Consequently, coinjection of capsaicin was no longer able to mask itch evoked by pruritogenic compounds. Our studies suggest that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Elimination of VGLUT2 in these nociceptors creates a mouse model of chronic neurogenic itch.

Origin of New Glial Cells in Intact and Injured Adult Spinal Cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.

Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain

Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) have large influences upon neuronal excitability. However, the participation of spinal HCN channels in chronic pain states, where pathological conditions are related to altered neuronal excitability, has not been clarified. Intraperitoneally (i.p.) or intrathecally (i.t.) administered ZD7288, a selective blocker of Ih channels, reduced thermal and mechanical hypersensitivity in mice under neuropathic conditions induced by the partial ligation of the sciatic nerve, while no analgesic effect was observed in naïve animals. Moreover, in the mouse formalin test, ZD7288 (i.p. and i.t.) reduced the licking/biting behavior observed during the second phase without affecting the first phase. To further explore the pain-modulatory action of spinal HCN channels, whole-cell patch clamp recordings were made from the visually identified substantia gelatinosa neurons in adult mouse spinal cord slices with an attached dorsal root, and A-fiber- and/or C-fiber-mediated monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the L4 or L5 dorsal root using a suction electrode. Bath-applied ZD7288 reduced A-fiber- and C-fiber-mediated monosynaptic EPSCs more preferentially in slices prepared from mice after peripheral nerve injury. In addition, ZD7288 reduced the frequency of miniature EPSCs without affecting their amplitude in cells receiving monosynaptic afferent inputs, indicating that it inhibits EPSCs via presynaptic mechanisms. The present behavioral and electrophysiological data suggest that spinal HCN channels, most likely at the primary afferent terminals, contribute to the maintenance of chronic pain.

Quantifying axon diameter and intra-cellular volume fraction in excised mouse spinal cord with q-space imaging

Q-space magnetic resonance imaging (QSI) can quantify white matter (WM) axonal architecture at the cellular level non-destructively, unlike histology, but currently has several limitations. First, current methodology does not differentiate between diffusing molecules occupying extra- or intra-cellular spaces (ECS and ICS, respectively). Second, accurate assessment of axonal architecture requires high-gradient amplitudes not clinically available. Third, the only direct QSI marker of axonal architecture has been mean axon diameter (MAD), even though other direct markers would be valuable as well. The objective was to investigate three QSI-based methods that address the above limitations. Method 1 employs a two-compartment model to account for signal from ECS and ICS. Method 2 uses data only from low q-values thereby obviating the need for high-gradient amplitudes. Method 3 empirically estimates ICS volume fraction and provides an additional metric of axonal architecture. We implemented each method on data from excised healthy adult mouse spinal cords collected previously using a home-built 50T/m z-gradient yielding sub-micron displacement resolution. Through comparison with histology, each method was evaluated for accuracy in assessing axonal architecture. MAD measured with Methods 1 and 2 showed good correlation with histology (R2=0.99 (p<0.0001), and 0.77 (p<0.01), respectively) and Bland–Altman analysis indicates that measurements from the two methods are not significantly different from histology. The third method measured ICS volume fractions (0.64±0.07) that were highly correlated (R2=0.92, p<0.05) with measurements from histology (0.68±0.07). These methods may provide insight into axonal architecture in normal and abnormal WM tissue but additional validation with more samples will be needed.

Polysialic Acid Glycomimetic Promotes Functional Recovery and Plasticity After Spinal Cord Injury in Mice

Regeneration after injury of the central nervous system is poor due to the abundance of molecules inhibiting axonal growth. Here we pursued to promote regeneration after thoracic spinal cord injury in young adult C57BL/6J mice using peptides which functionally mimic polysialic acid (PSA) and human natural killer cell-1 (HNK-1) glycan, carbohydrate epitopes known to promote neurite outgrowth in vitro. Subdural infusions were performed with an osmotic pump, over 2 weeks. When applied immediately after injury, the PSA mimetic and the combination of PSA and HNK-1 mimetics, but not the HNK-1 mimetic alone, improved functional recovery as assessed by locomotor rating and video-based motion analysis over a 6-week observation period. Better outcome in PSA mimetic-treated mice was associated with higher, as compared with control mice, numbers of cholinergic and glutamatergic terminals and monaminergic axons in the lumbar spinal cord, and better axonal myelination proximal to the injury site. In contrast to immediate post-traumatic application, the PSA mimetic treatment was ineffective when initiated 3 weeks after spinal cord injury. Our data suggest that PSA mimetic peptides can be efficient therapeutic tools improving, by augmenting plasticity, functional recovery when applied during the acute phase of spinal cord injury.

Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.