A GABAergic inhibitory component within the hippocampal commissural pathway.

Previous results from immunocytochemical and retrograde transport studies indicated that some GABAergic neurons in the hilus of the dentate gyrus may have axonal projections through the hippocampal commissure. This study has utilized a combined immunocytochemical and retrograde transport method as well as 2 anterograde electron-microscopic methods to determine the existence of this projection in rats. Combined tracer and immunofluorescence studies showed several double-labeled GABAergic neurons in the hilus contralateral to the injection site. The electron-microscopic studies revealed both degenerating and HRP-labeled commissural axons that formed symmetric synapses, the type shown to be formed by GABAergic terminals in the hippocampus. These data demonstrate a GABAergic component within the hippocampal commissural pathway and add further evidence that cortical GABAergic terminals are not derived exclusively from intrinsic neurons

Transcranial Low-Level Laser Therapy Improves Neurological Performance in Traumatic Brain Injury in Mice: Effect of Treatment Repetition Regimen

Low-level laser (light) therapy (LLLT) has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI). We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a) sham-TBI sham-treatment, (b) real-TBI sham-treatment, and (c) real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm[superscript 2], 18 J/cm[superscript 2], spot diameter 1 cm) while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU) at days 21–27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx) had significant improvements in neurological severity score (NSS), and wire-grip and motion test (WGMT). However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups) and less Fluoro-Jade staining for degenerating neurons (at 14 days) than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.National Institutes of Health (U.S.) (Grant R01AI050875)Center for Integration of Medicine and Innovative Technology (DAMD17-02-2-0006)United States. Dept. of Defense. Congressionally Directed Medical Research Programs (W81XWH-09-1-0514)United States. Air Force Office of Scientific Research. Military Photomedicine Program (FA9550-11-1-0331

Motor Unit Abnormalities in Dystonia musculorum Mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt27J mice. Phenotypic dt27J mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt27J mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt27J mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt27J muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt27J mice. This study reveals neuromuscular defects that likely contribute to the dt27J pathology and identifies a critical role for dystonin outside of sensory neurons

NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration

<p>Abstract</p> <p>Background</p> <p>Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.</p> <p>Methods</p> <p>Male C57BL/6 and NF-κB enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-κB were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91^phoximmunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain.</p> <p>Results</p> <p>Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-κB transcription and increased NOX gp91^phoxat 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91^phox+ immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91^phoxexpression coincided with increased production of O₂^-and O₂^-- derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation.</p> <p>Conclusions</p> <p>Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-κB signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.</p

Convulsant Doses of a Dopamine D1 Receptor Agonist Result in Erk-Dependent Increases in Zif268 and Arc/Arg3.1 Expression in Mouse Dentate Gyrus

Activation of dopamine D1 receptors (D1Rs) has been shown to induce epileptiform activity. We studied the molecular changes occurring in the hippocampus in response to the administration of the D1-type receptor agonist, SKF 81297. SKF 81297 at 2.5 and 5.0 mg/kg induced behavioural seizures. Electrophysiological recordings in the dentate gyrus revealed the presence of epileptiform discharges peaking at 30–45 min post-injection and declining by 60 min. Seizures were prevented by the D1-type receptor antagonist, SCH 23390, or the cannabinoid CB1 receptor agonist, CP 55,940. The effect of SKF 81297 was accompanied by increased phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK), in the granule cells of the dentate gyrus. This effect was also observed in response to administration of other D1-type receptor agonists, such as SKF83822 and SKF83959. In addition, SKF 81297 increased the phosphorylation of the ribosomal protein S6 and histone H3, two downstream targets of ERK. These effects were prevented by genetic inactivation of D1Rs, or by pharmacological inhibition of ERK. SKF 81297 was also able to enhance the levels of Zif268 and Arc/Arg3.1, two immediate early genes involved in transcriptional regulation and synaptic plasticity. These changes may be involved in forms of activity-dependent plasticity linked to the manifestation of seizures and to the ability of dopamine to affect learning and memory

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN

The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms

TGF-β induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid β plaques in Alzheimer's disease

The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo

Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing

We have mapped the motor neurons (MNs) supplying the major hindlimb muscles of transgenic (C57/BL6J-ChAT-EGFP) and wild-type (C57/BL6J) mice. The fluorescent retrograde tracer Fluoro-Gold was injected into 19 hindlimb muscles. Consecutive transverse spinal cord sections were harvested, the MNs counted, and the MN columns reconstructed in 3D. Three longitudinal MN columns were identified. The dorsolateral column extends from L4 to L6 and consists of MNs innervating the crural muscles and the foot. The ventrolateral column extends from L1 to L6 and accommodates MNs supplying the iliopsoas, gluteal, and quadriceps femoris muscles. The middle part of the ventral horn hosts the central MN column, which extends between L2–L6 and consists of MNs for the thigh adductor, hamstring, and quadratus femoris muscles. Within these longitudinal columns, the arrangement of the different MN groups reflects their somatotopic organization. MNs innervating muscles developing from the dorsal (e.g., quadriceps) and ventral muscle mass (e.g., hamstring) are situated in the lateral and medial part of the ventral gray, respectively.MN pools belonging to proximal muscles (e.g., quadratus femoris and iliopsoas) are situatedventral to those supplying more distal ones (e.g., plantar muscles). Finally, MNs innervatingflexors (e.g., posterior crural muscles) are more medial than those belonging to extensors ofthe same joint (e.g., anterior crural muscles). These data extend and modify the MN maps in the recently published atlas of the mouse spinal cord and may help when assessing neuronal loss associated with MN diseases