Ketogenic Diet Alleviates Mechanical Allodynia in the Models of Inflammatory and Neuropathic Pain in Male Mice.

Inflammation is involved in the induction of chronic inflammatory and neuropathic pain. Moreover, the ketogenic diet, a high-fat, low-carbohydrate, and adequate protein diet, has an anti-inflammatory effect. Thus, we hypothesized that a ketogenic diet has a therapeutic effect on both types of chronic pain. In the present study, we investigated the effect of a ketogenic diet on mechanical allodynia, a chronic pain symptom, in formalin-induced chronic inflammatory pain and nerve injury-induced neuropathic pain models using adult male mice. Formalin injection into the hind paw induced mechanical allodynia in both the injected and intact hind paws, and the ketogenic diet alleviated mechanical allodynia in both hind paws. In addition, the ketogenic diet prevented formalin-induced edema. Furthermore, the diet alleviated mechanical allodynia induced by peripheral nerve injury. Thus, these findings indicate that a ketogenic diet has a therapeutic effect on chronic pain induced by inflammation and nerve injury.

KCC2 downregulation after sciatic nerve injury enhances motor function recovery.

Injury to mature neurons induces downregulated KCC2 expression and activity, resulting in elevated intracellular [Cl-] and depolarized GABAergic signaling. This phenotype mirrors immature neurons wherein GABA-evoked depolarizations facilitate neuronal circuit maturation. Thus, injury-induced KCC2 downregulation is broadly speculated to similarly facilitate neuronal circuit repair. We test this hypothesis in spinal cord motoneurons injured by sciatic nerve crush, using transgenic (CaMKII-KCC2) mice wherein conditional CaMKIIα promoter-KCC2 expression coupling selectively prevents injury-induced KCC2 downregulation. We demonstrate, via an accelerating rotarod assay, impaired motor function recovery in CaMKII-KCC2 mice relative to wild-type mice. Across both cohorts, we observe similar motoneuron survival and re-innervation rates, but differing post-injury reorganization patterns of synaptic input to motoneuron somas-for wild-type, both VGLUT1-positive (excitatory) and GAD67-positive (inhibitory) terminal counts decrease; for CaMKII-KCC2, only VGLUT1-positive terminal counts decrease. Finally, we recapitulate the impaired motor function recovery of CaMKII-KCC2 mice in wild-type mice by administering local spinal cord injections of bicuculline (GABAA receptor blockade) or bumetanide (lowers intracellular [Cl-] by NKCC1 blockade) during the early post-injury period. Thus, our results provide direct evidence that injury-induced KCC2 downregulation enhances motor function recovery and suggest an underlying mechanism of depolarizing GABAergic signaling driving adaptive reconfiguration of presynaptic GABAergic input.

Controlled activation of cortical astrocytes modulates neuropathic pain-like behaviour.

Chronic pain is a major public health problem that currently lacks effective treatment options. Here, a method that can modulate chronic pain-like behaviour induced by nerve injury in mice is described. By combining a transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with concurrent activation of astrocytes in the somatosensory cortex (S1) by either low intensity transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia-like behaviour previously established by partial sciatic nerve ligation (PSL). Such activation of astrocytes initiated spine plasticity to reduce those synapses formed shortly after PSL. This reversal from allodynia-like behaviour persisted well beyond the active treatment period. Thus, our study demonstrates a robust and potentially translational approach for modulating pain, that capitalizes on the interplay between noxious afferents, sensitized central neuronal circuits, and astrocyte-activation induced synaptic plasticity.

The KCNQ channel inhibitor XE991 suppresses nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglion neurons.

BACKGROUND: XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is reportedly a potent and selective Kv7 (KCNQ) channel inhibitor. This study aimed to evaluate how XE991 affects nicotinic responses in intracardiac ganglion neurons. METHODS: We studied how the KCNQ channel inhibitor XE991 could affect nicotinic responses in acutely isolated rat intracardiac ganglion neurons using a perforated patch-clamp recording configuration and Ca2+ imaging. RESULTS: XE991 reversibly and concentration-dependently inhibited the nicotine (10 µM)-induced current with an IC50 of 14.4 µM. The EC50 values for nicotine-induced currents in the absence and presence of 10 µM XE991 were 8.7 and 12.0 µM, respectively. Because XE991 suppressed the maximum response of the nicotine concentration-response curve, the inhibitory effect of this drug appears to be noncompetitive. In addition, linopirdine reduced the amplitude of 10 µM nicotine-induced currents with an IC50 value of 16.9 µM. The inorganic KCNQ channel inhibitor Ba2+ affected neither the nicotine-induced current nor the inhibitory effect of XE991 on the nicotinic response. The KCNQ activator flupirtine at a concentration of 10 µM slightly but markedly inhibited the nicotine-induced current. Finally, XE991 inhibited the nicotine-induced elevation of intracellular calcium concentration and the nicotine-induced firing of action potentials. CONCLUSION: We propose that XE991 inhibits nicotinic acetylcholine receptors in intracardiac ganglion neurons, which in turn attenuate nicotine-induced neuronal excitation.

Downregulation of ten-eleven translocation-2 triggers epithelial differentiation during organogenesis.

DNA methylation of cytosine bases is a major epigenetic modification that regulates gene expression and vertebrate development. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and active DNA demethylation influences gene expression specific to each developmental stage, although recent reports have shown that TET also has a non-catalytic function. In fetal mice, the epithelium in the submandibular gland (SMG) buds as a derivative of the oral cavity at embryonic day 11 (E11) and, by E15, it begins to differentiate into the salivary epithelium, which expresses water-channel aquaporin 5 (AQP5). The functional differentiation of the SMG epithelium can be regulated epigenetically, but how TET enzymes contribute is largely unknown. Here, we used several techniques, including hydroxymethylated DNA immunoprecipitation qPCR and histological analysis, to examine the changes in 5hmC levels and AQP5 and TET expression during SMG development. We found that 5hmC levels and AQP5 expression increased in the E15 SMG epithelium, while TET2 expression in the terminal buds decreased at E15. In agreement with the in vivo observations, Tet2 inhibition ex vivo led to the upregulation of AQP5 expression in terminal buds of the SMG epithelium. These results suggest that the downregulation of TET2 expression at E15 is a critical epigenetic event that establishes the epithelial fate for functional SMGs during development.

Thermosensitive TRPV4 channels mediate temperature-dependent microglia movement.

Microglia maintain central nervous system homeostasis by monitoring changes in their environment (resting state) and by taking protective actions to equilibrate such changes (activated state). These surveillance and protective roles both require constant movement of microglia. Interestingly, induced hypothermia can reduce microglia migration caused by ischemia, suggesting that microglia movement can be modulated by temperature. Although several ion channels and transporters are known to support microglia movement, the precise molecular mechanism that regulates temperature-dependent movement of microglia remains unclear. Some members of the transient receptor potential (TRP) channel superfamily exhibit thermosensitivity and thus are strong candidates for mediation of this phenomenon. Here, we demonstrate that mouse microglia exhibit temperature-dependent movement in vitro and in vivo that is mediated by TRPV4 channels within the physiological range of body temperature. Our findings may provide a basis for future research into the potential clinical application of temperature regulation to preserve cell function via manipulation of ion channel activity.

Photoactivatable CaMKII induces synaptic plasticity in single synapses.

Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.

Histamine depolarizes rat intracardiac ganglion neurons through the activation of TRPC non-selective cation channels.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 µM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

Overexpression of neuronal K+-Cl- co-transporter enhances dendritic spine plasticity and motor learning.

The neuronal K+-Cl- cotransporter KCC2 maintains a low intracellular Cl- concentration and facilitates hyperpolarizing GABAA receptor responses. KCC2 also plays a separate role in stabilizing and enhancing dendritic spines in the developing nervous system. Using a conditional transgenic mouse strategy, we examined whether overexpression of KCC2 enhances dendritic spines in the adult nervous system and characterized the effects on spine dynamics in the motor cortex in vivo during rotarod training. Mice overexpressing KCC2 showed significantly increased spine density in the apical dendrites of layer V pyramidal neurons, measured in vivo using two-photon imaging. During modest accelerated rotarod training, mice overexpressing KCC2 displayed enhanced spine formation rates, greater balancing skill at higher rotarod speeds and a faster rate of learning in this ability. Our results demonstrate that KCC2 enhances spine density and dynamics in the adult nervous system and suggest that KCC2 may play a role in experience-dependent synaptic plasticity.

Microglia mediate non-cell-autonomous cell death of retinal ganglion cells.

Excitotoxicity is well known in the neuronal death in the brain and is also linked to neuronal damages in the retina. Recent accumulating evidence show that microglia greatly affect excitotoxicity in the brain, but their roles in retina have received only limited attention. Here, we report that retinal excitotoxicity is mediated by microglia. To this end, we employed three discrete methods, that is, pharmacological inhibition of microglia by minocycline, pharmacological ablation by an antagonist for colony stimulating factor 1 receptor (PLX5622), and genetic ablation of microglia using Iba1-tTA::DTAtetO/tetO mice. Intravitreal injection of NMDA increased the number of apoptotic retinal ganglion cells (RGCs) followed by reduction in the number of RGCs. Although microglia did not respond to NMDA directly, they became reactive earlier than RGC damages. Inhibition or ablation of microglia protected RGCs against NMDA. We found up-regulation of proinflammatory cytokine genes including Il1b, Il6 and Tnfa, among which Tnfa was selectively blocked by minocycline. PLX5622 also suppressed Tnfa expression. Tumor necrosis factor α (TNFα) signals were restricted in microglia at very early followed by spreading into other cell types. TNFα up-regulation in microglia and other cells were significantly attenuated by minocycline and PLX5622, suggesting a central role of microglia for TNFα induction. Both inhibition of TNFα and knockdown of TNF receptor type 1 by siRNA protected RGCs against NMDA. Taken together, our data demonstrate that a phenotypic change of microglia into a neurotoxic one is a critical event for the NMDA-induced degeneration of RGCs, suggesting an importance of non-cell-autonomous mechanism in the retinal neuronal excitotoxicity.

Cortical astrocytes prime the induction of spine plasticity and mirror image pain.

Peripheral nerve injury causes maladaptive plasticity in the central nervous system and induces chronic pain. In addition to the injured limb, abnormal pain sensation can appear in the limb contralateral to the injury, called mirror image pain. Because synaptic remodeling in the primary somatosensory cortex (S1) has critical roles in the induction of chronic pain, cortical reorganization in the S1 ipsilateral to the injured limb may also accompany mirror image pain. To elucidate this, we conducted in vivo 2-photon calcium imaging of neuron and astrocyte activity in the ipsilateral S1 after a peripheral nerve injury. We found that cross-callosal inputs enhanced the activity of both S1 astrocytes and inhibitory neurons, whereas activity of excitatory neurons decreased. When local inhibitory circuits were blocked, astrocyte-dependent spine plasticity and allodynia were revealed. Thus, we propose that cortical astrocytes prime the induction of spine plasticity and mirror image pain after peripheral nerve injury. Moreover, this result suggests that cortical synaptic rewiring could be sufficient to cause allodynia on the uninjured periphery.

Activity-dependent functions of non-electrical glial cells.

Electrical activity is essential for brain function. However, neurons, the electrically active cells, are less numerous than the non-electrical glial cells in the central nervous system. The non-electrical components modify the function of neural circuits, depending on the electrical neuronal activity, by wrapping synapses, myelinating axons and phagocytozing the neuronal components. Moreover, recent evidence has suggested that they contribute to neurological and psychiatric disease by regulating neuronal circuits, ultimately affecting their behaviour. In this review, we highlight the physiological functions of glial cells, particularly the electrical activity-dependent processes, to provide further insight into their role in brain function.

The roles of cortical astrocytes in chronic pain and other brain pathologies.

Astrocytes are the most abundant cell type in the brain. Several decades ago, they were considered to be only support cells in the central nervous system. Recent studies using advanced technologies have clarified that astrocytes play more active roles in regulating neuronal function and remodeling synaptic structures by releasing molecules called gliotransmitters. In addition to various physiological functions, astrocytes are activated under disease conditions, such as chronic pain, releasing molecules that in turn cause reorganization of the central nervous system microstructure and disrupt behavior in pathological conditions. In the present review, we summarize cortical astrocyte function in chronic pain and other neurological disorders and discuss the role of astrocytes in brain pathologies.

Astrocytes with previous chronic exposure to amyloid ß-peptide fragment 1-40 suppress excitatory synaptic transmission.

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long-term exposure of amyloid ß-peptide (Aß) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long-term Aß exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal-naïve neurons co-cultured with astrocytes that have previously experienced chronic Aß1-40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post-synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aß1-40 is attributable to a small number of synapses with higher release probability.

Activation-Dependent Rapid Postsynaptic Clustering of Glycine Receptors in Mature Spinal Cord Neurons.

Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal development in vivo. While presynaptic mechanisms of the shift of inhibitory neurotransmission are well investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the first postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in a depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of GlyRs in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for 1 h in the mature stage. Furthermore, live cell-imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca2+ nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission but also the inhibitory synaptic plasticity in mature nervous system.

Microglia contact induces synapse formation in developing somatosensory cortex.

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8-10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca(2+) transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS.

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

Noradrenergic refinement of glutamatergic neuronal circuits in the lateral superior olivary nucleus before hearing onset.

Neuronal circuit plasticity during development is fundamental for precise network formation. Pioneering studies of the developmental visual cortex indicated that noradrenaline (NA) is crucial for ocular dominance plasticity during the critical period in the visual cortex. Recent research demonstrated tonotopic map formation by NA during the critical period in the auditory system, indicating that NA also contributes to synaptic plasticity in this system. The lateral superior olive (LSO) in the auditory system receives glutamatergic input from the ventral cochlear nucleus (VCN) and undergoes circuit remodeling during postnatal development. LSO is innervated by noradrenergic afferents and is therefore a suitable model to study the function of NA in refinement of neuronal circuits. Chemical lesions of the noradrenergic system and chronic inhibition of α2-adrenoceptors in vivo during postnatal development in mice disrupted functional elimination and strengthening of VCN-LSO afferents. This was potentially mediated by activation of presynaptic α2-adrenoceptors and inhibition of glutamate release because NA presynaptically suppressed excitatory postsynaptic current (EPSC) through α2-adrenoceptors during the first two postnatal weeks in an in vitro study. Furthermore, NA and α2-adrenoceptor agonist induced long-term suppression of EPSCs and decreased glutamate release. These results suggest that NA has a critical role in synaptic refinement of the VCN-LSO glutamatergic pathway through failure of synaptic transmission. Because of the ubiquitous distribution of NA afferents and the extensive expression of α2-adrenoceptors throughout the immature brain, this phenomenon might be widespread in the developing central nervous system.

A double bond-conjugated dimethylnitrobenzene-type photolabile nitric oxide donor with improved two-photon cross section.

Photocontrollable NO donors enable precise spatiotemporal release of NO under physiological conditions. We designed and synthesized a novel dimethylnitrobenzene-type NO donor, Flu-DNB-DB, which contains a carbon-carbon double bond in place of the amide bond of previously reported Flu-DNB. Flu-DNB-DB releases NO in response to one-photon activation in the blue wavelength region, and shows a greatly increased two-photon cross-section (δu) at 720 nm (Flu-DNB: 0.12 GM, Flu-DNB-DB: 0.98 GM). We show that Flu-DNB-DB enables precisely controlled intracellular release of NO in response to 950 nm pulse laser irradiation for as little as 1s. This near-infrared-light-controllable NO source should be a valuable tool for studies on the biological roles of NO.