Neonatal bilateral whisker trimming in male mice age-dependently alters brain neurotransmitter levels and causes adolescent onsets of social behavior abnormalities.

Tactile perception via whiskers is important in rodent behavior. Whisker trimming during the neonatal period affects mouse behaviors related to both whisker-based tactile cognition and social performance. However, the molecular basis of these phenomena is not completely understood. To solve this issue, we investigated developmental changes in transmitters and metabolites in various brain regions of male mice subjected to bilateral whisker trimming during the neonatal period (10 days after birth [BWT10 mice]). We discovered significantly lower levels of 3-methoxy-4-hydroxyphenyl glycol (MHPG), the major noradrenaline metabolite, in various brain regions of male BWT10 mice at both early/late adolescent stages (at P4W and P8W). However, reduced levels of dopamine (DA) and their metabolites were more significantly identified at P8W in the nuclear origins of monoamine (midbrain and medulla oblongata) and the limbic system (frontal cortex, amygdala, and hippocampus) than at P4W. Furthermore, the onset of social behavior deficits (P6W) was observed later to the impairment of whisker-based tactile cognitive behaviors (P4W). Taken together, these findings suggest that whisker-mediated tactile cognition may contribute toprogressive abnormalities in social behaviors in BWT10 mice accompanied by impaired development of dopaminergic systems.

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities.

Rett syndrome (RTT) is a neurodevelopmental disorder with X-linked dominant inheritance caused mainly by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. The effects of various Mecp2 mutations have been extensively assessed in mouse models, but none adequately mimic the symptoms and pathological changes of RTT. In this study, we assessed the effects of Mecp2 gene deletion on female rats (Mecp2+/-) and found severe impairments in social behavior [at 8 weeks (w), 12 w, and 23 w of age], motor function [at 16 w and 26 w], and spatial cognition [at 29 w] as well as lower plasma insulin-like growth factor (but not brain-derived neurotrophic factor) and markedly reduced acetylcholine (30%-50%) in multiple brain regions compared to female Mecp2+/+ rats [at 29 w]. Alternatively, changes in brain monoamine levels were relatively small, in contrast to reports on mouse Mecp2 mutants. Female Mecp2-deficient rats express phenotypes resembling RTT and so may provide a robust model for future research on RTT pathobiology and treatment.

Exposure to valproic acid during middle to late-stage corticogenesis induces learning and social behavioral abnormalities with attention deficit/hyperactivity in adult mice.

Single prenatal exposure to valproic acid (VPA) in rodents is a widely used preclinical model of autism spectrum disorder (ASD). Continuous prenatal VPA exposure has been recently proposed as a new ASD model that closely captures the neuropathological features of ASD, including increases in cerebral cortex volume and the number of cortical upper layer neurons. We investigated the influence of prenatal VPA exposure on the behavior of adult offspring of pregnant dams that received intraperitoneal injections of VPA twice on one day during the genesis of cortical upper layer neurons. Mice exposed to VPA at E14 (E14-VPA) showed typical behavior abnormalities including reduced social interaction, hyperactivity, and poor maze learning due to attention deficit/impulsivity relative to healthy controls. Histological analysis revealed that E14-VPA mice had significantly increased neuronal density and impaired neural activity in the prefrontal cortex, but not the somatosensory area, which is likely linked to the observed abnormalities in social behavior. These results suggest that this VPA exposure method is a good model for gaining new insights into the underlying neuropathology of ASD.

Pyrroloquinoline quinone improves abnormal functional development of whisker-mediated tactile perception and social behaviors caused by neonatal whisker trimming.

Abnormalities in tactile perception and response, such as sensory defensiveness, are core features of autism spectrum disorder (ASD) and may be associated with impaired communication skills. However, the influences of tactile perception deficits on the development of social behaviors and neuronal circuits related to emotional regulation of social interactions remain unclear. Whiskers are the most important tactile apparatus in rodents. We previously reported that adult mice receiving bilateral whisker trimming for 10 days after birth (BWT10) exhibited deficits in whisker-mediated tactile discrimination, abnormal social behaviors, and hyperactivity of brain emotional systems under psychological stress. Pyrroloquinoline quinone (PQQ) is an essential nutrient with important roles in central nervous system development and function through modulation of glutamatergic N-methyl-d-aspartate receptor (NMDAR) activity. Here we examined the effect of neonatal PQQ administration on the behavioral abnormalities of BWT10 mice. PQQ treatment significantly reversed abnormal social behavior in adult BWT10 mice as detected by three-chamber social interaction and social dominance tube tests, and improved whisker perception as revealed by the gap-crossing test. In addition, PQQ reversed hyperactivity in emotional systems as evidenced by c-Fos expression pattern following elevated-platform stress. These data suggest that PQQ may be a promising candidate therapeutic drug for neurodevelopmental disorders such as ASD.

Priming with FGF2 stimulates human dental pulp cells to promote axonal regeneration and locomotor function recovery after spinal cord injury.

Human dental pulp cells (DPCs), adherent cells derived from dental pulp tissues, are potential tools for cell transplantation therapy. However, little work has been done to optimize such transplantation. In this study, DPCs were treated with fibroblast growth factor-2 (FGF2) for 5-6 consecutive serial passages and were transplanted into the injury site immediately after complete transection of the rat spinal cord. FGF2 priming facilitated the DPCs to promote axonal regeneration and to improve locomotor function in the rat with spinal cord injury (SCI). Additional analyses revealed that FGF2 priming protected cultured DPCs from hydrogen-peroxide-induced cell death and increased the number of DPCs in the SCI rat spinal cord even 7 weeks after transplantation. The production of major neurotrophic factors was equivalent in FGF2-treated and untreated DPCs. These observations suggest that FGF2 priming might protect DPCs from the post-trauma microenvironment in which DPCs infiltrate and resident immune cells generate cytotoxic reactive oxygen species. Surviving DPCs could increase the availability of neurotrophic factors in the lesion site, thereby promoting axonal regeneration and locomotor function recovery.

Neonatal Whisker Trimming Impairs Fear/Anxiety-Related Emotional Systems of the Amygdala and Social Behaviors in Adult Mice.

Abnormalities in tactile perception, such as sensory defensiveness, are common features in autism spectrum disorder (ASD). While not a diagnostic criterion for ASD, deficits in tactile perception contribute to the observed lack of social communication skills. However, the influence of tactile perception deficits on the development of social behaviors remains uncertain, as do the effects on neuronal circuits related to the emotional regulation of social interactions. In neonatal rodents, whiskers are the most important tactile apparatus, so bilateral whisker trimming is used as a model of early tactile deprivation. To address the influence of tactile deprivation on adult behavior, we performed bilateral whisker trimming in mice for 10 days after birth (BWT10 mice) and examined social behaviors, tactile discrimination, and c-Fos expression, a marker of neural activation, in adults after full whisker regrowth. Adult BWT10 mice exhibited significantly shorter crossable distances in the gap-crossing test than age-matched controls, indicating persistent deficits in whisker-dependent tactile perception. In contrast to controls, BWT10 mice exhibited no preference for the social compartment containing a conspecific in the three-chamber test. Furthermore, the development of amygdala circuitry was severely affected in BWT10 mice. Based on the c-Fos expression pattern, hyperactivity was found in BWT10 amygdala circuits for processing fear/anxiety-related responses to height stress but not in circuits for processing reward stimuli during whisker-dependent cued learning. These results demonstrate that neonatal whisker trimming and concomitant whisker-dependent tactile discrimination impairment severely disturbs the development of amygdala-dependent emotional regulation.

A novel 2-decenoic acid thioester ameliorates corticosterone-induced depression- and anxiety-like behaviors and normalizes reduced hippocampal signal transduction in treated mice.

We characterized mice administered corticosterone (CORT) at a dose of 20 mg/kg for 3 weeks to determine their suitability as a model of mood disorders and found that the time immobilized in the tail suspension test was longer and the time spent in the open arms of the elevated plus-maze test was shorter than those of the vehicle-treated group, findings demonstrating that chronic CORT induced both depression-like and anxiety-like behaviors. Furthermore, the levels of phosphorylated extracellular signal-regulated kinase (pERK) 1/2 in the hippocampus and cerebral cortex were reduced in the CORT-treated group. Using this model, we investigated the protective effect of the ester, thioester, and amide compounds of 2-decenoic acid derivatives (termed compounds A, B, and C, respectively). The potency of the protective activity against the CORT-induced depression-like or anxiety-like behaviors and the reduction in pERK1/2 level were found to be in the following order: compound B > compound C > compound A. Therefore, we further investigated the therapeutic activity of only compound B, and its effect on depression-like behavior was observed after oral administration for 1 or 2 weeks, and its effect on anxiety-like behavior was observed after oral administration for 3 weeks. The ratios of phosphorylated ERK1/2, Akt, and cAMP-response element-binding protein to their respective nonphosphorylated forms were smaller in the CORT-treated group than in the vehicle-treated group; however, subsequent treatment with compound B at either 0.3 or 1.5 mg/kg significantly ameliorated this reduction. Compound B appeared to elicit intracellular signaling, similar to that elicited by brain-derived neurotrophic factor, and its mode of action was shown to be novel and different from that of fluvoxamine, a currently prescribed drug for mood disorders.

Neurotrophin-3 influences the number and the laminar fate of cortical progenitors in the developing cerebral cortex of mice through the MEK/ERK1/2 signaling pathway.

The laminar formation in the developing cerebral cortex requires precisely regulated generation of phenotype-specific neurons. To determine whether neurotrophin-3 (NT3) is involved in this formation, we investigated the effects of NT3 administration in the telencephalic ventricular space on 13.5-day-old mouse embryos. NT3 increased the number of newly generated neurons and altered the neuronal phenotypes in the position and the transcription factors-expression profiles; the neuronal phenotypes originally committed for layer IV neurons were altered toward for layers II/III neurons. The former effects were observed when the parent progenitor cells were exposed to NT3 in the G1- to S-phase, whereas the latter effects were observed with exposure in the G1-phase. In addition, in vitro experiments revealed that the laminar fate alteration by NT3 was observed in the dissociated primary culture of cortical progenitors and the NT3 actions were suppressed by cotreatment with the MEK/ERK inhibitor. These observations suggest that NT3 is involved in the laminar formation of the developing cerebral cortex through the intercellular MEK/ERK pathway.

Anxiolytic-like effect of trans-2-decenoic acid ethyl ester in stress-induced anxiety-like model mice.

Brain-derived neurotrophic factor (BDNF) is considered to be involved in the etiology and treatment of stress-related mood disorders including anxiety. However, application of BDNF itself has many technical and pharmacological problems such as instability and/or a lack of appropriate delivery systems. To overcome these, we developed trans-2-decenoic acid ethyl ester (DAEE) as a stable and small molecule with BDNF-like activities. In the present study, we tested the activities of DAEE on a stress-induced anxiety-like mouse model. Mice were kept in cages and subjected to 3 sets of sequential leaning, drenching, and rotation as chronic mild stresses applied for 1-2 days over a 3-week period; and the anxiety-like symptom (reduced time spent in open arm of the maze) was evaluated by use of the elevated plus-maze test. A daily intraperitoneal administration of DAEE competed against the expression of the anxiety-like symptom when administered during the stress-loading period, and became therapeutic when administered after the stress-loading. This activity was accompanied by amelioration of the stress-induced reduction in the levels of BDNF and neurotrophin-3 mRNAs and phosphorylated extracellular signal-regulated kinases (ERK) 1/2 in the hippocampus. These results demonstrated that DAEE behaved like an anxiolytic and ameliorated this characteristic anxiety-like symptom, suggesting that DAEE may be a promising candidate for a novel anxiolytic with a new mechanism of action.

Injury-induced accumulation of glial cell line-derived neurotrophic factor in the rostral part of the injured rat spinal cord.

The spinal cord of a 7-week-old female Wistar rat was hemi-transected at thoracic position 10 with a razor blade, and changes in glial cell line-derived neurotrophic factor (GDNF) protein and mRNA expression levels in the spinal cord were examined. GDNF protein and mRNA expression levels were evaluated by enzyme immunoassay and reverse transcription polymerase chain reaction, respectively. Although GDNF is distributed in the healthy spinal cord from 150 to 400 pg/g tissue in a regionally dependent manner, hemi-transection (left side) of the spinal cord caused a rapid increase in GDNF content in the ipsilateral rostral but not in the caudal part of the spinal cord. On the other hand, injury-induced GDNF mRNA was distributed limitedly in both rostral and caudal stumps. These observations suggest the possibility that increased GDNF in the rostral part is responsible for the accumulation of GDNF that may be constitutively transported from the rostral to caudal side within the spinal cord. Although such local increase of endogenous GDNF protein may not be sufficient for nerve regeneration and locomotor improvement, it may play a physiological role in supporting spinal neurons including motoneurons.

Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth.

Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3'-untranslated region (3'-UTR), producing mRNAs with variable 3' ends. Because 3'-UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

2-decenoic acid ethyl ester, a compound that elicits neurotrophin-like intracellular signals, facilitating functional recovery from cerebral infarction in mice.

In our previous study, we found that trans-2-decenoic acid ethyl ester (DAEE), a derivative of a medium-chain fatty acid, elicits neurotrophin-like signals including the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in cultured mouse cortical neurons. Here, we examined the efficacy of intraperitoneal administration of DAEE on the treatment of a mouse model of the cerebral infarction caused by unilateral permanent middle cerebral artery occlusion (PMCAO). DAEE-treatment (100 µg/kg body weight injected at 0.5, 24, 48, 72 h after PMCAO) significantly restored the mice from PMCAO-induced neurological deficits including motor paralysis when evaluated 48, 72, and 96 h after the PMCAO. Furthermore, DAEE facilitated the phosphorylation of ERK1/2 on the infarction side of the brain when analyzed by Western immunoblot analysis, and it enhanced the number of phosphorylated ERK1/2-positive cells in the border areas between the infarction and non-infarction regions of the cerebral cortex, as estimated immunohistochemically. As the infarct volume remained unchanged after DAEE-treatment, it is more likely that DAEE improved the neurological condition through enhanced neuronal functions of the remaining neurons in the damaged areas rather than by maintaining neuronal survival. These results suggest that DAEE has a neuro-protective effect on cerebral infarction.

Antidepressant-like activity of 10-hydroxy-trans-2-decenoic Acid, a unique unsaturated Fatty Acid of royal jelly, in stress-inducible depression-like mouse model.

Symptoms of depression and anxiety appeared in mice after they had been subjected to a combination of forced swimming for 15 min followed by being kept in cages that were sequentially subjected to leaning, drenching, and rotation within 1-2 days for a total of 3 weeks. The animals were then evaluated by the tail-suspension test, elevated plus-maze test, and open-field test at 1 day after the end of stress exposure. Using these experimental systems, we found that 10-hydroxy-trans-2-decenoic acid (HDEA), an unsaturated fatty acid unique to royal jelly (RJ), protected against the depression and anxiety when intraperitoneally administered once a day for 3 weeks simultaneously with the stress loading. Intraperitoneally administered RJ, a rich source of HDEA, was also protective against the depression, but RJ given by the oral route was less effective. Our present results demonstrate that HDEA and RJ, a natural source of it, were effective in ameliorating the stress-inducible symptoms of depression and anxiety.

Prenatal immune challenge compromises the normal course of neurogenesis during development of the mouse cerebral cortex.

Maternal infection during pregnancy is an environmental risk factor for the development of severe brain disorders in offspring, including schizophrenia and autism. However, little is known about the neurodevelopmental mechanisms underlying the association between prenatal exposure to infection and the emergence of cognitive and behavioral dysfunctions in later life. By injecting viral mimetic polyriboinosinic-polyribocytidylic acid (Poly I:C) into mice, we investigated the influence of maternal immune challenge during pregnancy on the development of the cerebral cortex of offspring. Our previous study showed that stimulation of the maternal immune system compromised the expression properties of transcription factors and the synaptogenesis of cortical neurons in upper layers but not those in deeper layers. The objective of the current study was to examine further whether maternal immune challenge has an influence on the cellular-biological features of the cortical progenitors that generate distinct cortical neuronal subtypes. We found the following abnormalities in the cortex of mice given the prenatal Poly I:C injection during later stages of cortical neurogenesis. First, proliferative activity and the expression of Pax6, which is a master regulator of the gene expression of transcription factors, were significantly decreased in the cortical progenitors. Second, the laminar allocation and gene expression were significantly altered in the daughter neurons generated at the same birth dates. These results demonstrate that specific abnormalities in the cortical progenitors preceded deficits in neuronal phenotypes. These changes may underlie the emergence of psychiatric brain and behavioral dysfunctions after in utero exposure to an infection.

Prenatal immune challenge compromises development of upper-layer but not deeper-layer neurons of the mouse cerebral cortex.

Maternal infection during pregnancy is an environmental risk factor for the offspring to develop severe brain disorders, including schizophrenia. However, little is known about the neurodevelopmental mechanisms underlying the association between prenatal exposure to infection and emergence of cognitive and behavioral dysfunctions later in life. By injecting the viral mimetic polyriboinosinic-polyribocytidylic acid (Poly I:C) into mice, we investigated the influence of maternal immune challenge during pregnancy on the development of the cerebral cortex, a responsive organ for cognition. Stimulation of the maternal immune system did not influence the cell number or density of the cortical neurons of postnatal 10-day-old and 8-week-old offspring, whereas gene expressions of upper-layer-specific transcription factors were significantly reduced, without affecting those of the deeper-layer ones. Moreover, the prenatal Poly I:C injection impaired synaptic development of the upper-layer neurons at a later stage, and there was a decrease in the synaptophysin- and glutamic acid decarboxylase-67-positive puncta surrounding the neuronal cell bodies and an increase in the dendritic spine density in postnatal 8-week-old offspring. Considering their importance for cognitive function, the specific abnormalities in the development of upper-layer neuronal phenotypes may underlie the development of psychiatric brain and behavioral dysfunctions emerging after in utero exposure to an infection.

Caffeic acid phenethyl ester reduces spinal cord injury-evoked locomotor dysfunction.

Caffeic acid phenethyl ester (CAPE) is a component of propolis, which is a substance taken from the hives of honeybees, and is known to exhibit an anti-inflammatory activity. Such activity has been thought to be partly based on its potential and specific inhibitory activities toward nuclear factor-κB, a transcription factor. Therefore, in the present study, we evaluated the effect of CAPE on functional locomotor recovery after spinal cord injury (SCI) caused by hemi-transection, because inflammatory responses are a major cause of the secondary injury observed following SCI and play a pivotal role in regulating the pathogenesis of acute and chronic SCI. When CAPE was i.p.-administered at a dosage of 10 µmol/kg, it enhanced the recovery of locomotor function and reduced the lesion size while suppressing the expression of the mRNAs for a pro-inflammatory cytokine interleukin-1ß and the inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase-2. These results suggest CAPE to be a promising therapeutic tool for reducing the secondary neuronal damage following primary physical injury to the spinal cord.

Ethanol extract of chinese propolis facilitates functional recovery of locomotor activity after spinal cord injury.

An ethanol extract of Chinese propolis (EECP) was given intraperitoneally to rats suffering from hemitransection of half of their spinal cord (left side) at the level of the 10th thoracic vertebra to examine the effects of the EECP on the functional recovery of locomotor activity and expression of mRNAs of inducible nitric oxide (NO) synthase (iNOS) and neurotrophic factors in the injury site. Daily administration of EECP after the spinal cord injury ameliorated the locomotor function, which effect was accompanied by a reduced lesion size. Furthermore, the EECP suppressed iNOS gene expression, thus reducing NO generation, and also increased the expression level of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the lesion site, suggesting that the EECP reduced the inflammatory and apoptotic circumstances through attenuation of iNOS mRNA expression and facilitation of mRNA expression of neurotrophins in the injured spinal cord. These results suggest that Chinese propolis may become a promising tool for wide use in the nervous system for reducing the secondary neuronal damage following primary physical injury.

2-Decenoic acid ethyl ester possesses neurotrophin-like activities to facilitate intracellular signals and increase synapse-specific proteins in neurons cultured from embryonic rat brain.

We presently found that medium-chain fatty acids (MCFAs) with 8-12 carbons and their esters facilitated activation (phosphorylation) of mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) 1/2 of cultured embryonic cortical/hippocampal neurons. In particular, trans-2-decenoic acid ethyl ester (DAEE) had the most potent activity. Additionally, DAEE activated phosphatidylinositol 3-kinase and cAMP-response element binding protein (CREB), suggesting that DAEE generates similar intracellular signal as neurotrophins. Therefore, details of the signal elicited by DAEE were examined in comparison with those of a neurotrophin, brain-derived neurotrophic factor (BDNF). We found that 1) DAEE phosphorylated MAPK/ERK1/2 via MEK activation without the involvement of tyrosine kinases of neurotrophin Trk receptors; 2) DAEE activated CREB predominantly through MAPK/ERK1/2 activation, not through other pathways such as cAMP/protein kinase A; and 3) DAEE increased the expression of RNAs of BDNF and neurotrophin-3 and the protein content of synapse-specific proteins such as synaptophysin, synapsin-1, and syntaxin. Based on these observations we propose that DAEE and some other derivatives of MCFAs having neurotrophin-like neurotrophic activities may become therapeutic tools for certain neurological or psychiatric disorders.

Stem cell factor induces heterotopic accumulation of cells (heterotopia) in the mouse cerebral cortex.

The stem cell factor (SCF)-c-kit signal transduction pathway plays an important role in the proliferation and migration of neural progenitor cells, but little is known about its function during the development of the cerebral cortex. We investigated the effects of SCF by directly administering it into the telencephalic ventricular space of 13.5-day-old mouse embryos. SCF produced the heterotopic accumulation of cortical cells in several distinct area of the cerebral cortex at the postnatal stage, including the subcortical periventricular area, marginal zone, and lateral ventricular space. Additional analysis revealed that the heterotopia included both neurons and astrocytes and that SCF initially increased the number of neural stem cells without affecting that of intermediate progenitors and also disturbed their organization. These results suggest that SCF alters the timing of the genesis and migration of neural stem/progenitor cells, which may lead to formation of the observed heterotopia.

Axonal regrowth downregulates the synthesis of glial cell line-derived neurotrophic factor in the lesioned rat sciatic nerve.

The effect of axonal regeneration on de novo synthesis of glial cell line-derived neurotrophic factor (GDNF) in rat sciatic nerves was examined. Transection of the sciatic nerve caused a prominent increase in the GDNF content in the distal segments within 1 week. The high level was sustained until 4 weeks in the animal model in which the nerve ends were ligated with thread (non-regeneration group); however, it was reduced to the original level within 2 or 4 weeks after the transection only in the segments invaded by regenerating axons in the models in which the nerve ends were coaptated (regeneration group). Expression of both GDNF protein and mRNA was decreased with a reciprocal increase in the density of neurofilaments, used as a marker of axonal ingrowth in distal segments of the regeneration group, suggesting that axonal contact turned off the GDNF-mediated nerve regeneration activity.