There is a growing body of evidence that cytokines contribute to both induction and maintenance of neuropathic pain derived from changes in dorsal root ganglia (DRG), including the activity of the primary sensory neurons and their satellite glial cells (SGC). We used immunofluorescence and in situ hybridization methods to provide evidence that chronic constriction injury (CCI) of the sciatic nerve induces synthesis of interleukin-6 (IL-6) in SGC, elevation of IL-6 receptor (IL-6R) and activation of signal transducer and activator of transcription 3 (STAT3) signalling. Unilateral CCI of the rat sciatic nerve induced mechanoallodynia and thermal hyperalgesia in ipsilateral hind paws, but contralateral paws exhibited only temporal changes of sensitivity. We demonstrated that IL-6 mRNA and protein, which were expressed at very low levels in naïve DRG, were bilaterally increased not only in L4-L5 DRG neurons but also in SGC activated by unilateral CCI. Besides IL-6, substantial increase of IL-6R and pSTAT3 expression occurred in SGC following CCI, however, IL-6R associated protein, gp130 levels did not change. The results may suggest that unilateral CCI of the sciatic nerve induces bilateral activation of SGC in L4-L5 DRG to transduce IL-6 signalling during neuroinflammation.

Epilepsy is characterized by both neuronal and astroglial dysfunction. The endogenous anticonvulsant adenosine, the level of which is largely controlled by astrocytes, might provide a crucial link between astrocyte and neuron dysfunction in epilepsy. Here we have studied astrogliosis, a hallmark of the epileptic brain, adenosine dysfunction and the emergence of spontaneous seizures in a comprehensive approach that includes a new mouse model of focal epileptogenesis, mutant mice with altered brain levels of adenosine, and mice lacking adenosine A1 receptors. In wild-type mice, following a focal epileptogenesis-precipitating injury, astrogliosis, upregulation of the adenosine-removing astrocytic enzyme adenosine kinase (ADK), and spontaneous seizures coincide in a spatio-temporally restricted manner. Importantly, these spontaneous seizures are mimicked by untreated transgenic mice that either overexpress ADK in brain or lack A1 receptors. Conversely, mice with reduced ADK in the forebrain do not develop either astrogliosis or spontaneous seizures. Our studies define ADK as a crucial upstream regulator of A1 receptor-mediated modulation of neuronal excitability, and support the ADK hypothesis of epileptogenesis in which upregulation of ADK during astrogliosis provides a crucial link between astrocyte and neuron dysfunction in epilepsy. These findings define ADK as rational target for therapeutic intervention.

A key neurotrophin responsible for the survival and function of basal forebrain (BF) cholinergic neurons is brain-derived neurotrophic factor (BDNF). A number of studies now indicate that a source of this factor may be BF astrocytes. This study was designed to define the role of BF-astrocyte-derived BDNF on cholinergic neurons. Moreover, it investigated regulatory events that modulate BDNF content and release. In initial work BDNF derived from BF-astrocyte-conditioned medium (ACM) was found to increase both numbers of BF acetylcholinesterase (AChE+) cholinergic neurons and the cholinergic synthetic enzyme choline acetyltransferase (ChAT). Western blots, immunocytochemistry and pharmacological inhibition studies revealed that glutamate, through group I metabotropic glutamate receptors (mGluR), increases the intracellular levels of BDNF in BF astrocytes in culture, as well as its release. Furthermore, the release of BDNF is mediated by the actions of PLC, IP3 and internal stores of Ca2+. These results suggest that BF astrocytes serve as local sources of BDNF for cholinergic neurons, and that they may be regulated as such by the neuronal signal, glutamate, through the mediation of group I metabotropic receptors and the PLC pathway.

Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol¯). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.

The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU2M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.

Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy.

Extracellular matrix (ECM) in the brain is composed of molecules synthesized and secreted by neurons and glial cells in a cell-type-specific and activity-dependent manner. During development, ECM plays crucial roles in proliferation, migration and differentiation of neural cells. In the mature brain, ECM undergoes a slow turnover and supports multiple physiological processes, while restraining structural plasticity. In the first part of this review, we discuss the contribution of ECM molecules to different forms of plasticity, including developmental plasticity in the cortex, long-term potentiation and depression in the hippocampus, homeostatic scaling of synaptic transmission and metaplasticity. In the second part, we focus on pathological changes associated with epileptogenic mutations in ECM-related molecules or caused by seizure-induced remodeling of ECM. The available data suggest that ECM components regulating physiological plasticity are also engaged in different aspects of epileptogenesis, such as dysregulation of excitatory and inhibitory neurotransmission, sprouting of mossy fibers, granule cell dispersion and gliosis. At the end, we discuss combinatorial approaches that might be used to counteract seizure-induced dysregulation of both ECM molecules and extracellular proteases. By restraining ECM modification and preserving the status quo in the brain, these treatments might prove to be valid therapeutic interventions to antagonize the progression of epileptogenesis.

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.

Gene expression changes during cell differentiation are thought to be coordinated by histone modifications, but still little is known about the role of specific histone deacetylases (HDACs) in cell fate decisions in vivo. Here we demonstrate that the catalytic function of HDAC2 is required in adult, but not embryonic neurogenesis. While brain development and adult stem cell fate were normal upon conditional deletion of HDAC2 or in mice lacking the catalytic activity of HDAC2, neurons derived from both zones of adult neurogenesis die at a specific maturation stage. This phenotype is correlated with an increase in proliferation and the aberrant maintenance of proteins normally expressed only in progenitors, such as Sox2, also into some differentiating neurons, suggesting that HDAC2 is critically required to silence progenitor transcripts during neuronal differentiation of adult generated neurons. This cell-autonomous function of HDAC2 exclusively in adult neurogenesis reveals clear differences in the molecular mechanisms regulating neurogenesis during development and in adulthood.

It has been known for some time that the somata of neurons in sensory ganglia respond to electrical or chemical stimulation and release transmitters in a Ca2+-dependent manner. The function of the somatic release has not been well delineated. A unique characteristic of the ganglia is that each neuronal soma is tightly enwrapped by satellite glial cells (SGCs). The somatic membrane of a sensory neuron rarely makes synaptic contact with another neuron. As a result, the influence of somatic release on the activity of adjacent neurons is likely to be indirect and/or slow. Recent studies of neuron–SGC interactions have demonstrated that ATP released from the somata of dorsal root ganglion neurons activates SGCs. They in turn exert complex excitatory and inhibitory modulation of neuronal activity. Thus, SGCs are actively involved in the processing of afferent information. In this review, we summarize our understanding of bidirectional communication between neuronal somata and SGCs in sensory ganglia and its possible role in afferent signaling under normal and injurious conditions. The participation of purinergic receptors is emphasized because of their dominant roles in the communication.

The high level of intercellular communication mediated by gap junctions between astrocytes indicates that, besides individual astrocytic domains, a second level of organization might exist for these glial cells as they form communicating networks. Therefore, the contribution of astrocytes to brain function should also be considered to result from coordinated groups of cells. To evaluate the shape and extent of these networks we have studied the expression of connexin 43, a major gap junction protein in astrocytes, and the intercellular diffusion of gap junction tracers in two structures of the developing brain, the hippocampus and the cerebral cortex. We report that the shape of astrocytic networks depends on their location within neuronal compartments in a defined brain structure. Interestingly, not all astrocytes are coupled, which indicates that connections within these networks are restricted. As gap junctional communication in astrocytes is reported to contribute to several glial functions, differences in the shape of astrocytic networks might have consequences on neuronal activity and survival.

Astrocytes were classically considered as simple supportive cells for neurons without a significant role in information processing by the nervous system. However, considerable amounts of evidence obtained by several groups during the past years demonstrated the existence of a bidirectional communication between astrocytes and neurons, which prompted a re-examination of the role of astrocytes in the physiology of the nervous system. While neurons base their excitability on electrical signals generated across the membrane, astrocytes base their cellular excitability on variations of the Ca2+ concentration in the cytosol. This article discusses our current knowledge of the properties of the synaptically evoked astrocyte Ca2+ signal, which reveals that astrocytes display integrative properties for synaptic information processing. Astrocytes respond selectively to different axon pathways, discriminate between the activity of different synapses and their Ca2+ signal is non-linearly modulated by the simultaneous activity of different synaptic inputs. Furthermore, this Ca2+ signal modulation depends on astrocyte cellular intrinsic properties and is bidirectionally regulated by the level of synaptic activity. Finally, astrocyte Ca2+ elevations can trigger the release of gliotransmitters, which modulate neuronal activity as well as synaptic transmission and plasticity, hence granting the bidirectional communication with neurons. Consequently, astrocytes can be considered as cellular elements involved in information processing by the nervous system.

Activity-dependent neuroprotective protein (ADNP, ∼123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using α-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by ∼1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Like neurons and astrocytes, oligodendrocytes have a variety of neurotransmitter receptors and ion channels. However, except for facilitating the rapid conduction of action potentials by forming myelin and buffering extracellular K+, little is known about the direct involvement of oligodendrocytes in neuronal activities. To investigate their physiological roles, we focused on oligodendrocytes in the alveus of the rat hippocampal CA1 region. These cells were found to respond to exogenously applied glutamate by depolarization through N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Electrical stimulation of the border between the alveus and stratum oriens evoked inward currents through several routes involving glutamate receptors and inward rectifier K+ channels. Moreover, electrical stimulation resembling in vivo activity evoked long-lasting depolarization. To examine the modulatory effects of oligodendrocytes on neuronal activities, we performed dual, whole-cell recording on CA1 pyramidal neurons and oligodendrocytes. Direct depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that oligodendrocytes increase the conduction velocity of action potentials by a mechanism additional to saltatory conduction, and that they have active roles in information processing in the brain.

Trigeminal nerve activation in response to inflammatory stimuli has been shown to increase neuron–glia communication via gap junctions in trigeminal ganglion. The goal of this study was to identify changes in the expression of gap junction proteins, connexins (Cxs), in trigeminal ganglia in response to acute or chronic joint inflammation. Although mRNA for Cxs 26, 36, 40 and 43 was detected under basal conditions, protein expression of only Cxs 26, 36 and 40 increased following capsaicin or complete Freund's adjuvant (CFA) injection into the temporomandibular joint (TMJ). While Cx26 plaque formation between neurons and satellite glia was transiently increased following capsaicin injections, Cx26 plaque formation between neurons and satellite glia was sustained in response to CFA. Interestingly, levels of Cx36 and Cx40 were only elevated in neurons following capsaicin or CFA injections, but the temporal response was similar to that observed for Cx26. In contrast, Cx43 expression was not increased in neurons or satellite glial cells in response to CFA or capsaicin. Thus, trigeminal ganglion neurons and satellite glia can differentially regulate Cx expression in response to the type and duration of inflammatory stimuli, which likely facilitates increased neuron–glia communication during acute and chronic inflammation and pain in the TMJ.

Little is known about the functional connectivity between astrocytes in the CNS. To explore this issue we photo-released glutamate onto a single astrocyte in murine hippocampal slices and imaged calcium responses. Photo-release of glutamate causes a metabotropic glutamate receptor (mGluR)-dependent increase in internal calcium in the stimulated astrocyte and delayed calcium elevations in neighboring cells. The delayed elevation in calcium was not caused by either neuronal activity following synaptic transmission or by glutamate released from astrocytes. However, it was reduced by flufenamic acid (FFA), which is consistent with a role for adenosine triphosphate (ATP) release from astrocytes as an intercellular messenger. Exogenous ligands such as ATP (1 µM) increased the number of astrocytes that were recruited into coupled astrocytic networks, indicating that extracellular accumulation of neurotransmitters modulates neuronal excitability, synaptic transmission and functional coupling between astrocytes.

Action potential conduction velocity increases dramatically during early development as axons become myelinated. Integral to this process is the clustering of voltage-gated Na+ (Nav) channels at regularly spaced gaps in the myelin sheath called nodes of Ranvier. We show here that some aspects of peripheral node of Ranvier formation are distinct from node formation in the CNS. For example, at CNS nodes, Nav1.2 channels are detected first, but are then replaced by Nav1.6. Similarly, during remyelination in the CNS, Nav1.2 channels are detected at newly forming nodes. By contrast, the earliest Nav-channel clusters detected during developmental myelination in the PNS have Nav1.6. Further, during PNS remyelination, Nav1.6 is detected at new nodes. Finally, we show that accumulation of the cell adhesion molecule neurofascin always precedes Nav channel clustering in the PNS. In most cases axonal neurofascin (NF-186) accumulates first, but occasionally paranodal neurofascin is detected first. We suggest there is heterogeneity in the events leading to Nav channel clustering, indicating that multiple mechanisms might contribute to node of Ranvier formation in the PNS.

Adhesive and repellent molecular cues guide migrating cells and growing neurites during development. They also contribute to synaptic function, learning and memory in adulthood. Here, we review the roles of cell adhesion molecules of the immunoglobulin superfamily (Ig-CAMs) and semaphorins (some of which also contain Ig-like domains) in regulation of synaptic transmission and plasticity. Interestingly, among the seven studied Ig-CAMs, the neuronal cell adhesion molecule proved to be important for all tested forms of hippocampal plasticity, while its associated unusual glycan polysialic acid is necessary and sufficient part for synaptic plasticity only at CA3-CA1 synapses. In contrast, Thy-1 and L1 specifically regulate long-term potentiation (LTP) at synapses formed by entorhinal axons in the dentate gyrus and cornu ammonis, respectively. Contactin-1 is important for long-term depression but not for LTP at CA3-CA1 synapses. Analysis of CHL1-deficient mice illustrates that at intermediate stages of development a deficit in a cell adhesion molecule is compensated but appears as impaired LTP during early and late postnatal development. The emerging mechanisms by which adhesive Ig-CAMs contribute to synaptic plasticity involve regulation of activities of NMDA receptors and L-type Ca2+ channels, signaling via mitogen-activated protein kinase p38, changes in GABAergic inhibition and motility of synaptic elements. Regarding repellent molecules, available data for semaphorins demonstrate their activity-dependent regulation in normal and pathological conditions, synaptic localization of their receptors and their potential to elevate or inhibit synaptic transmission either directly or indirectly.

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon–SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry, including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels. Regrowth is ‘staggered’ such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow. ‘Naked’ axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of β III tubulin, PGP 9.5 and GAP43/B50 were complex, implying a pause in their regrowth, and were most prominent at the proximal stump–regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges. Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.