RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation.

BACKGROUND: Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. METHODS: A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. RESULTS: Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1ß. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. CONCLUSIONS: These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

Acute stress induces severe neural inflammation and overactivation of glucocorticoid signaling in interleukin-18-deficient mice.

Interleukin-18 (IL18) is an inflammatory cytokine that is related to psychiatric disorders such as depression and cognitive impairment. We previously found that IL18 deficiency may cause hippocampal impairment, resulting in depression-like behavioral changes. However, the potential role of IL18 in stressful conditions remains uncertain. In the present study, we examined the effect of IL18 on neural inflammation and stress tolerance during acute stress. Littermate Il18+/+ and Il18-/- mice were exposed to a single restraint stress for 6 h, and all assessments were performed 18 h after the mice were released from the restraint. In Il18-/- mice exposed to acute stress, the immobility times in both the forced swim test and tail suspension test were decreased, although no difference was observed in Il18+/+ mice. Il1ß, Il6, and Tnfα expression levels in the hippocampus of stressed Il18-/- mice were significantly higher than those in the other groups. Moreover, the numbers of astrocytes and microglia, including those in the active form, were also increased compared with those in other groups. Regarding the molecular mechanism, the HSF5 and TTR genes were specifically expressed in stressed Il18-/- mice. As a potential treatment, intracerebral administration of IL18 to Il18-/- mice resulted in partial recovery of changes in behavioral assessments. Our results revealed that IL18-deficient mice were more sensitive and had a longer response to acute stress than that in normal mice. In addition, neural inflammation and augmentation of glucocorticoid signals caused by stress were more intense and remained longer in Il18-/- mice, resulting in behavioral changes. In conclusion, IL18 might be an indispensable factor that modulates the stress response and maintains balance between neural inflammation and glucocorticoid signaling.

A Bone Morphogenetic Protein Signaling Inhibitor, LDN193189, Converts Ischemia-Induced Multipotent Stem Cells into Neural Stem/Progenitor Cell-Like Cells.

Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner.

Morphologically dynamic dendritic spines are the major sites of neuronal plasticity in the brain; however, the molecular mechanisms underlying their morphological dynamics have not been fully elucidated. Phldb2 is a protein that contains two predicted coiled-coil domains and the pleckstrin homology domain, whose binding is highly sensitive to PIP3. We have previously demonstrated that Phldb2 regulates synaptic plasticity, glutamate receptor trafficking, and PSD-95 turnover. Drebrin is one of the most abundant neuron-specific F-actin-binding proteins that are pivotal for synaptic morphology and plasticity. We observed that Phldb2 bound to drebrin A (adult-type drebrin), but not to drebrin E (embryonic-type drebrin). In the absence of Phldb2, the subcellular localization of drebrin A in the hippocampal spines and its distribution in the hippocampus were altered. Immature spines, such as the filopodium type, increased relatively in the CA1 regions of the hippocampus, whereas mushroom spines, a typical mature type, decreased in Phldb2-/- mice. Phldb2 suppressed the formation of an abnormal filopodium structure induced by drebrin A overexpression. Taken together, these findings demonstrate that Phldb2 is pivotal for dendritic spine morphology and possibly for synaptic plasticity in mature animals by regulating drebrin A localization.

Morphology of Schwann Cell Processes Supports Renal Sympathetic Nerve Terminals With Local Distribution of Adrenoceptors.

Nerves in the renal parenchyma comprise sympathetic nerves that act on renal arteries and tubules to decrease blood flow and increase primary urine reabsorption, respectively. Synaptic vesicles release neurotransmitters that activate their effector tissues. However, the mechanisms by which neurotransmitters exert individual responses to renal effector cells remain unknown. Here, we investigated the spatial and molecular compositional associations of renal Schwann cells (SC) supporting the nerve terminals in male rats. The nerve terminals of vascular smooth muscle cells (SMCs) enclosed by renal SC processes were exposed through windows facing the effectors with presynaptic specializations. We found that the adrenergic receptors (ARs) α2A, α2C, and ß2 were localized in the SMC and the basal side of the tubules, where the nerve terminals were attached, whereas the other subtypes of ARs were distributed in the glomerular and luminal side, where the norepinephrine released from nerve endings may have indirect access to ARs. In addition, integrins α4 and ß1 were coexpressed in the nerve terminals. Thus, renal nerve terminals could contact their effectors via integrins and may have a structure, covered by SC processes, suitable for intensive and directional release of neurotransmitters into the blood, rather than specialized structures in the postsynaptic region.

Exploring Molecular Mechanisms Involved in the Development of the Depression-Like Phenotype in Interleukin-18-Deficient Mice.

Interleukin-18 (IL-18) is an inflammatory cytokine that has been linked to energy homeostasis and psychiatric symptoms such as depression and cognitive impairment. We previously revealed that deficiency in IL-18 led to hippocampal abnormalities and resulted in depression-like symptoms. However, the impact of IL-18 deficiency on other brain regions remains to be clarified. In this study, we first sought to confirm that IL-18 expression in neural cells can be found in human brain tissue. Subsequently, we examined the expression of genes in the prefrontal cortex of Il18 -/- mice and compared it with gene expression in mice subjected to a chronic mild stress model of depression. Extracted genes were further analyzed using Ingenuity® Pathway Analysis, in which 18 genes common to both the chronic mild stressed model and Il18 -/- mice were identified. Of those, 16 were significantly differentially expressed between Il18+/+ and Il18 -/- mice. We additionally measured protein expression of α-2-HS-glycoprotein (AHSG) and transthyretin (TTR) in serum and the brain. In the prefrontal cortex of Il18 -/- mice, TTR but not AHSG was significantly decreased. Conversely, in the serum of Il18 -/- mice, AHSG was significantly increased but not TTR. Therefore, our results suggest that in IL-18-deficit conditions, TTR in the brain is one of the mediators causally related to depression, and AHSG in peripheral organs is one of the regulators inducing energy imbalance. Moreover, this study suggests a possible "signpost" to clarify the molecular mechanisms commonly underlying the immune system, energy metabolism, neural function, and depressive disorders.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Relationship between lamellar sensory corpuscles distributed along the upper arm's deep arteries and pulsating sensation of blood vessels.

Vibration is detected by mechanoreceptors, including Pacinian corpuscles (PCs), which are widely distributed in the human body including the adventitia of large blood vessels. Although the distribution of PCs around large limb vessels has been previously reported, there remains no consensus on their distribution in the adventitia of the human deep blood vessels in the upper arm. In addition, the physiological functions of PCs located around the deep limb blood vessels remain largely unknown. This study aimed to elucidate detailed anatomical features and physiological function of lamellar sensory corpuscles structurally identified as PCs using the immunohistochemical methods around the deep vessels in the upper arm. We identified PCs in the connective tissue adjacent to the deep vessels in the upper arm using histological analysis and confirmed that PCs are located in the vascular sheath of the artery and its accompanying vein as well as in the connective tissue surrounding the vascular sheath and nerves. PCs were densely distributed on the distal side of deep vessels near the elbow. We also examined the relationship between vascular sound and pulsating sensation to evaluate the PCs functions around deep arteries and veins and found that the vascular sound made by pressing the brachial arteries in the upper arm was associated with the pulsating sensation of the examinee. Our results suggest that PCs, around deep vessels, function as bathyesthesia sensors by detecting vibration from blood vessels.

Interleukin-18-deficient mice develop hippocampal abnormalities related to possible depressive-like behaviors.

Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

PIP3-Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover.

The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2-/- mice.

Platelet-derived growth factor receptor α gene is regulated by multiple first exons.

Transcription of the platelet-derived growth factor receptor α (PDGFRA/Pdgfra) gene is considered to be precisely regulated. We have previously reported that the PDGFRA/Pdgfra gene is regulated by a dual promoter system in human and mouse, in which a novel PDGFRA/Pdgfra transcript has a first exon (exon 1ß) different from that of the canonical PDGFRA/Pdgfra transcript (exon 1α). To elucidate the function of each transcript, we first investigated the contribution of different PDGFRA transcripts to final protein levels. Notably, knockdown experiments suggested the existence of other PDGFRA transcripts, and we identified five additional first exons (exons 1γ, 1δ, 1ε, 1ζ, and 1η) in intron 1 in both the human and mouse genes. The first exons of the mouse Pdgfra gene showed unique expression patterns: exon 1α was broadly expressed; exon 1ß was highly expressed in embryos; exon 1γ was observed at relatively high levels in the adult central nervous system (CNS); and exon 1δ was expressed at relatively high levels in the developing CNS. Furthermore, in silico analysis of common putative transcription factor binding sites in the upstream regions of the first exons of both human and mouse PDGFRA/Pdgfra genes predicted common (such as Sry, Mzf1, and Cdx) and unique (such as Sox5, Lmo2, and GATA) transcription factors. Our findings show the diversity of the transcriptional regulation of the PDGFRA/Pdgfra gene.

Distribution of Pacini-Like Lamellar Corpuscles in the Vascular Sheath of the Femoral Artery.

Pacinian corpuscles are vibration-sensing mechanoreceptors that are densely distributed in the dermis of the human hand. Although they are also known to occur in various other regions/structures throughout the human body, including the adventitia of large vessels, their precise distribution and function in arteries remain unclear. In the present study, we identified Pacini-like lamellar corpuscles (LCs) adjacent to the femoral artery, and investigated their distribution with respect to that structure via a histological analysis. We identified nine LCs that were localized in the connective tissue surrounding the femoral artery and vein. We showed that although their distribution was heterogeneous, they were predominantly concentrated on the dorsal side of the femoral artery. Immunohistochemical analyses revealed that the identified femoral artery LCs exhibited features characteristic of typical LCs located in the dermis of the index finger. Thus, the results of the present study contribute to an improved understanding of the function of femoral artery LCs. Anat Rec, 301:1809-1814, 2018. © 2018 Wiley Periodicals, Inc.

Negative Regulation of TRPA1 by AMPK in Primary Sensory Neurons as a Potential Mechanism of Painful Diabetic Neuropathy.

AMPK is a widely expressed intracellular energy sensor that monitors and modulates energy expenditure. Transient receptor potential ankyrin 1 (TRPA1) channel is a widely recognized chemical and thermal sensor that plays vital roles in pain transduction. In this study, we discovered a functional link between AMPK and TRPA1 in dorsal root ganglion (DRG) neurons, in which AMPK activation rapidly resulted in downregulation of membrane-associated TRPA1 and its channel activity within minutes. Treatment with two AMPK activators, metformin or AICAR, inhibited TRPA1 activity in DRG neurons by decreasing the amount of membrane-associated TRPA1. Metformin induced a dose-dependent inhibition of TRPA1-mediated calcium influx. Conversely, in diabetic db/db mice, AMPK activity was impaired in DRG neurons, and this was associated with a concomitant increase in membrane-associated TRPA1 and mechanical allodynia. Notably, these molecular and behavioral changes were normalized following treatment with AMPK activators. Moreover, high-glucose exposure decreased activated AMPK levels and increased agonist-evoked TRPA1 currents in cultured DRG neurons, and these effects were prevented by treatment with AMPK activators. Our results identify AMPK as a previously unknown regulator of TRPA1 channels. AMPK modulation of TRPA1 could thus serve as an underlying mechanism and potential therapeutic molecular target in painful diabetic neuropathy.

Differential Distribution of Renal Nerves in the Sympathetic Ganglia of the Rat.

The renal nerve plexus comprises efferent and afferent fibers. It controls urine production and bodily fluid homeostasis. Efferent fibers to the kidney include sympathetic nerve fibers from their main ganglia, the prevertebral suprarenal ganglia (SrG), and the paravertebral sympathetic chain ganglia (ChG). In the present study, we examined topological innervation from these ganglia to the renal parenchymal segments of the left kidney of the rat. Fluoro-Gold was injected into the rostral or caudal poles of the left kidney. Approximately 50% of the cells in the SrG of rats injected in the rostral pole were labeled, while 60% of the cells in the ChG T13 of rats injected in the caudal pole were labeled. In addition, we performed dual-probe retrograde tracing of the nerves using two kinds of fluorescent-conjugated cholera toxins (f-CTbs) injected into the rostral and caudal poles of the left kidney. The cells labeled with each f-CTb were distributed differently in the left SrG and the lower ChGs; no dual-labeled cells were found in these ganglia. Anterograde tracing with pCAGGS-tdTomato vector transfected into the left SrG showed that tdTomato-labeled nerve varicosities extended to the cortical arterioles and urinary tubules. Immunohistochemistry revealed that they were positive to tyrosine hydroxylase and synaptophysin, suggesting that they possessed sympathetic nerve endings. Our results show that renal efferent nerves in the SrG may control the rostral part of the kidney and innervate the multiple effectors in the cortex. Anat Rec, 300:2263-2272, 2017. © 2017 Wiley Periodicals, Inc.

Possible Role of the Myelinated Neural Network in the Parietal Peritoneum in Rats as a Mechanoreceptor.

A network of myelinated nerve fibers in the peritoneum covers the abdominal wall. We studied the topographic distribution of this network, explored the fibers' destination in the central nervous system, and examined the markers in these fibers in order to identify the nature of the sensation conveyed by the network of nerve fibers in rats. We used Sihler's method, which stains myelinated fibers in whole mount materials, and observed a dense nerve network and endings toward the peritoneal cavity in the peritoneum that covers the abdomen's lateral bulge. We studied the axonal transport of cholera toxin subunit B to investigate the central projections of this network in order to identify its function. After applying the tracer in the peritoneum, we observed many labeled terminals in the medial part of laminae 3-5 of the spinal cord. A small number of labeled terminals was observed in the dorsal nucleus of Clarke and gracile nucleus. Labeled somata were observed in the dorsal root ganglia (DRG). Most (96%) were larger than 35 µm. We performed immunohistochemistry of the abdominal wall, using antiserum against the 200-kD neurofilament (a marker for mechanosensory neurons). We observed many positive nerve fibers in the peritoneum. Because cell bodies in the DRG were large, their nerve terminals ended in the base of the dorsal horn, which is known to transmit proprioceptive information, and the network possesses the marker for mechanosensitive fibers; therefore, it appears that the myelinated nerve network conveys information about distension and/or contraction of the abdominal wall. Anat Rec, 300:1662-1669, 2017. © 2017 Wiley Periodicals, Inc.

Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling ß chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70.

The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.

Postnatal changes of interleukin-18 receptor immunoreactivity in neurons of the retrosplenial cortex in wild-type and interleukin-18 knock out mice.

Interleukin-18 (IL-18), which is involved in the inflammatory response, is also found in the cerebral cortex. IL-18 receptor-immunoreactive (IL-18R-ir) neurons are present in layer V of the retrosplenial cortex (RSC). In the adult IL-18 knock out (KO) mice, no IL-18R-ir neurons but many degenerated neurons are present in layer V of the RSC, suggesting that any changes in the neurons of layer V have occurred during postnatal development. We examined changes of IL-18R expression during postnatal development. In the wild-type mice, many IL-18R-ir neurons were present in layers II, III and VI of the RSC in 2-week-old mice, whereas they were sparsely observed in only layer III in 3-week-old mice. No IL-18R-ir neurons were present in 4- and 5-week-old mice. In older than 6-week-old mice, many IL-18R-ir neurons were present in layers V and VI. The IL-18KO mice showed IL-18R-ir neurons in layers II, III and VI at 2-weeks-old, and a few in layer III at 3-week-old mice, similar to that in the wild-type mice. No IL-18R-ir neurons were found in mice older than 4 weeks of age. Thus, IL-18 or IL-18R seem to be involved in the construction of neural circuits corresponding to events after 3-weeks of age.

Fine structure of interleukin 18 (IL-18) receptor-immunoreactive neurons in the retrosplenial cortex and its changes in IL18 knockout mice.

Interleukin 18 (IL-18) participates in the inflammatory immune response of lymphocytes. Delay in learning or memory are common in the IL-18 knockout mouse. Many IL-18-immunoreactive neurons are found in the retrosplenial cortex (RSC) and the subiculum. These neurons also contain the IL-18 receptor. We determined the location and the ultrastructure of the IL-18 receptor-immunoreactive neurons in the RSC and observed changes in the IL-18 receptor-immunoreactive neurons of the IL-18 knockout mouse. The IL-18 receptor-immunoreactive neurons were found specifically in layer V of the granular RSC. They were medium-sized neurons with a light oval nucleus and had little cytoplasm with many free ribosomes, rough endoplasmic reticulum and many mitochondria, but no Nissl bodies. The number of axosomatic terminals was about six per section. The IL-18 receptor-immunoreactive neurons were not found in the RSC in the IL-18 knockout mouse at 5 or 9 weeks of age. However, many small electron-dense neurons were found in layer V. Both the nucleus and cytoplasm were electron-dense, but not necrotic. The mitochondria and rough endoplasmic reticulum were swollen. The IL-18 receptor-immunoreactive neurons were presumed to be degenerating. The degeneration of the IL18-receptor-immunoreactive neurons in the RSC may cause the abnormal behaviors of the IL-18 knockout mice.

Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats.