Cholera Toxin Beta Research Articles

Computational study to investigate Proteus mirabilis proteomes for multi-epitope vaccine construct design

Proteus mirabilis is a gram-negative bacterium particularly known for its unique swarming ability. The swarming gives the bacteria ability to enhance adherence to the catheter surface and epithelium cells of the urethra to cause catheter associated urinary tract infections. P. mirabilis has evolved resistant to antibiotics. Additionally, there is an approved vaccine against P. mirabilis, thus demanding for identification of new vaccine targets. This gram-negative bacterium consists of 19,502 core proteins, out of which 19,063 are redundant proteins and remaining 439 are non-redundant proteins. The non-redundant proteins have 21 proteins present on the cell surface out of which 11 proteins are virulent. Antigenicity analysis predicted only 2 proteins as antigenic (fimbrial biogenesis outer membrane usher protein and ligand-gated channel protein). Four and seven B-cells epitopes were predicted from the former and later proteins, respectively. The predicted B-cells epitopes were used for T- cells epitopes prediction. The predicted epitopes were linked to each other through GPGPG linkers and joined with cholera toxin beta subunit adjuvant. A multi-epitopes vaccine construct consisting of 226 residues was docked with MHC-I, MHC-II and TLR-4. The best docked complex in each case has binding energy of −714.6, −744.6 and −829.5 kcal/mol, respectively. Moreover, the docking results were validated through molecular dynamics simulation and binding free energies estimation. The net energy of −137.2 kcal/mol was calculated for vaccine-MHC-I complex, −133.39 kcal/mol for vaccine-MHC-II and −158.68 kcal/mol for vaccine-TLR-4 complex. The designed vaccine construct could provoke immune responses against targeted pathogen and may be used in experimental testing. Communicated by Ramaswamy H. Sarma

Spinal trigeminal subnucleus pars muralis: An interface for apneic reflex

Noxious stimulation of the nasal cavity elicits protective reflexes for the upper respiratory tract, including sneezing, mucus secretion, apnea and cardiovascular reflexes. Yet, no studies have shown the full circuit responsible for the apneic response upon noxious stimulation of the nasal mucosa. Here, we show that injection of cholera toxin beta subunit into the nasal mucosa leads to terminal labeling in the transition zone between the interpolaris and caudalis subnuclei of the trigeminal sensory complex, i.e. subnucleus muralis (SpVm). We also provide physiological evidence that ammonia vapor delivered into the nasal cavity induces an apneic reaction and activates some SpVm cells. Subnucleus muralis cells also respond to mechanical stimulation of the narial pad and the nasal mucosa. Yet, none of the muralis cells responded to vibrissa deflection. Electrolytic lesion of SpVm or unilateral transection of the anterior ethmoidal nerve prevent apnea induced by ammonia delivery to the nasal epithelium, demonstrating their crucial role in the apneic reflex. We also found direct projections from SpVm to the respiratory centers, particularly the preBötzinger complex (preBötC). Inhibition of inspiratory preBötC cells and increased firing of expiratory preBötC cells are associated with ammonia‐induced apnea.Anatomical and physiological data presented here support the hypothesis that nasal induced apnea may be mediated by direct projections from nasal innervated Subnucleus muralis neurons to the PreBötC complex.

Towards A Microbead Occlusion Model of Glaucoma for a Non-Human Primate

Glaucoma is a group of optic neuropathies associated with aging and sensitivity to intraocular pressure (IOP). The disease causes vision loss through the degeneration of retinal ganglion cell neurons and their axons in the optic nerve. Using an inducible model of glaucoma, we elevated IOP in the squirrel monkey (Saimiri boliviensis) using intracameral injection of 35 μm polystyrene microbeads and measured common pathogenic outcomes in the optic projection. A 42% elevation in IOP over 28 weeks reduced anterograde transport of fluorescently-labeled cholera toxin beta from retina to the lateral geniculate nucleus (60% decrease), and to the superior colliculus (49% decrease). Pressure also reduced survival of ganglion cellaxons in the optic nerve by 22%. The same elevation caused upregulation of proteins associated with glaucomatous neurodegeneration in the retina and optic nerve, including complement 1q, interleukin 6, and brain-derived neurotrophic factor. That axon degeneration in the nerve lagged deficits in anterograde transport is consistent with progression in rodent models, while the observed protein changes also occur in tissue from human glaucoma patients. Thus, microbead occlusion in a non-human primate with a visual system similar to our own represents an attractive model to investigate neurodegenerative mechanisms and therapeutic interventions for glaucoma.

Afferent neuropeptide Y projections to the ventral tegmental area in normal-weight male Wistar rats.

The hypothalamic neuropeptide Y (NPY) circuitry is a key regulator of feeding behavior. NPY also acts in the mesolimbic dopaminergic circuitry, where it can increase motivational aspects of feeding behavior through effects on dopamine output in the nucleus accumbens (NAc) and on neurotransmission in the ventral tegmental area (VTA). Endogenous NPY in the NAc originates from local interneurons and afferent projections from the hypothalamic arcuate nucleus (Arc). However, the origin of endogenous NPY in the VTA is unknown. We determined, in normal‐weight male Wistar rats, if the source of VTA NPY is local, and/or whether it is derived from VTA‐projecting neurons. Immunocytochemistry, in situ hybridization and RT‐qPCR were utilized, when appropriate in combination with colchicine treatment or 24 hr fasting, to assess NPY/Npy expression locally in the VTA. Retrograde tracing using cholera toxin beta (CTB) in the VTA, fluorescent immunocytochemistry and confocal microscopy were used to determine NPY‐immunoreactive afferents to the VTA. NPY in the VTA was observed in fibers, but not following colchicine pretreatment. No NPY‐ or Npy‐expressing cell bodies were observed in the VTA. Fasting for 24 hr, which increased Npy expression in the Arc, failed to induce Npy expression in the VTA. Double‐labeling with CTB and NPY was observed in the Arc and in the ventrolateral medulla. Thus, VTA NPY originates from the hypothalamic Arc and the ventrolateral medulla of the brainstem in normal‐weight male Wistar rats. These afferent connections link hypothalamic and brainstem processing of physiologic state to VTA‐driven motivational behavior.

Inhibitory Projections in the Mouse Auditory Tectothalamic System.

The medial geniculate body (MGB) is the target of excitatory and inhibitory inputs from several neural sources. Among these, the inferior colliculus (IC) is an important nucleus in the midbrain that acts as a nexus for auditory projections, ascending and descending, throughout the rest of the central auditory system and provides both excitatory and inhibitory inputs to the MGB. In our study, we assessed the relative contribution from presumed excitatory and inhibitory IC neurons to the MGB in mice. Using retrograde tract tracing with cholera toxin beta subunit (CTβ)-Alexa Fluor 594 injected into the MGB of transgenic, vesicular GABA transporter (VGAT)-Venus mice, we quantitatively analyzed the projections from both the ipsilateral and contralateral IC to the MGB. Our results demonstrate inhibitory projections from both ICs to the MGB that likely play a significant role in shaping auditory processing. These results complement prior studies in other species, which suggest that the inhibitory tectothalamic pathway is important in the regulation of neuronal activity in the auditory forebrain.

Hydrogen postconditioning promotes survival of rat retinal ganglion cells against ischemia/reperfusion injury through the PI3K/Akt pathway

Retinal ischemia/reperfusion injury (IRI) plays a crucial role in the pathophysiology of various ocular diseases. Our previous study have shown that postconditioning with inhaled hydrogen (H2) (HPC) can protect retinal ganglion cells (RGCs) in a rat model of retinal IRI. Our further study aims to investigate potential mechanisms underlying HPC-induced protection. Retinal IRI was performed on the right eyes of rats and was followed by inhalation of 67% H2 mixed with 33% oxygen immediately after ischemia for 1 h daily for one week. RGC density was counted using haematoxylin and eosin (HE) staining, retrograde labelling with cholera toxin beta (CTB) and TUNEL staining, respectively. Visual function was assessed using flash visual evoked potentials (FVEP) and pupillary light reflex (PLR). The phosphorylated Akt was analysed by RT-PCR and western blot. The results showed that administration of HPC significantly inhibited the apoptosis of RGCs and protected the visual function. Simultaneously, HPC treatment markedly increased the phosphorylations of Akt. Blockade of PI3K activity by inhibitors (LY294002) dramatically abolished its anti-apoptotic effect and lowered both visual function and Akt phosphorylation levels.Taken together, our results demonstrate that HPC appears to confer neuroprotection against retinal IRI via the PI3K/Akt pathway.

Tau accumulation in the retina promotes early neuronal dysfunction and precedes brain pathology in a mouse model of Alzheimer\u2019s disease

BackgroundTau is an axon-enriched protein that binds to and stabilizes microtubules, and hence plays a crucial role in neuronal function. In Alzheimer’s disease (AD), pathological tau accumulation correlates with cognitive decline. Substantial visual deficits are found in individuals affected by AD including a preferential loss of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. At present, however, the mechanisms that underlie vision changes in these patients are poorly understood. Here, we asked whether tau plays a role in early retinal pathology and neuronal dysfunction in AD.MethodsAlterations in tau protein and gene expression, phosphorylation, and localization were investigated by western blots, qPCR, and immunohistochemistry in the retina and visual pathways of triple transgenic mice (3xTg) harboring mutations in the genes encoding presenilin 1 (PS1M146 V), amyloid precursor protein (APPSwe), and tau (MAPTP301L). Anterograde axonal transport was assessed by intraocular injection of the cholera toxin beta subunit followed by quantification of tracer accumulation in the contralateral superior colliculus. RGC survival was analyzed on whole-mounted retinas using cell-specific markers. Reduction of tau expression was achieved following intravitreal injection of targeted siRNA.ResultsOur data demonstrate an age-related increase in endogenous retinal tau characterized by epitope-specific hypo- and hyper-phosphorylation in 3xTg mice. Retinal tau accumulation was observed as early as three months of age, prior to the reported onset of behavioral deficits, and preceded tau aggregation in the brain. Intriguingly, tau build up occurred in RGC soma and dendrites, while tau in RGC axons in the optic nerve was depleted. Tau phosphorylation changes and missorting correlated with substantial defects in anterograde axonal transport that preceded RGC death. Importantly, targeted siRNA-mediated knockdown of endogenous tau improved anterograde transport along RGC axons.ConclusionsOur study reveals profound tau pathology in the visual system leading to early retinal neuron damage in a mouse model of AD. Importantly, we show that tau accumulation promotes anterograde axonal transport impairment in vivo, and identify this response as an early feature of neuronal dysfunction that precedes cell death in the AD retina. These findings provide the first proof-of-concept that a global strategy to reduce tau accumulation is beneficial to improve axonal transport and mitigate functional deficits in AD and tauopathies.

MMP-2 as a modulator of dendritic shrinkage and axonal regeneration: neuroinflammation as the driving force

Event Abstract Back to Event MMP-2 as a modulator of dendritic shrinkage and axonal regeneration: neuroinflammation as the driving force Lien Andries1*, Manuel Salinas Navarro1, Kim Lemmens1, Lies De Groef1 and Lieve Moons1 1 KU Leuven, Biology, Belgium A limited number of experiments performed across the animal kingdom hints to improved axonal regeneration upon stimulation of dendritic shrinkage in adult injured neurons. In this study, we use the visual system as a model to investigate dendrite remodeling and axonal regeneration following injury. Damage to the adult mammalian optic nerve, along which the retinal ganglion cells (RGCs) project their axons, is characterized by axonal degeneration, shrinkage of the dendrites in the inner plexiform layer (IPL) of the retina and eventually RGC death. Notably, inflammatory stimulation (IS) within the retina has been suggested to transform the RGCs into an active regenerative state. Several players essential to this regeneration-promoting effect of IS have been identified. However, our understanding of the multiple glial- or macrophage-derived factors that may synergistically contribute or potentiate the beneficial effects of IS, is still incomplete. Here, we propose MMP-2 as one of the modulators of dendritic and axonal responses after optic nerve crush (ONC) (degeneration model) or ONC combined with IS (regeneration model). Besides the well-known axonal regeneration promoting effect of IS, our data now suggest that IS also affects dendritic shrinkage. This was assessed by measuring the thickness of the IPL using optical coherence tomography (OCT) and (immuno)histological stainings on retinal sections. Wild type (WT) mice subjected to ONC+IS showed increased IPL thinning as compared to mice subjected to ONC solely. Furthermore, MMP-2 seems to exert a role in both dendritic and axonal processes. On the one hand, Mmp-2-/- mice were characterized by a delayed IPL thinning: at 4 and 7dpi, wild-type (WT) mice already show significant IPL thinning, while this is not the case for Mmp-2-/- mice. On the other hand, axonal regeneration was assessed by quantifying fluorescently labelled cholera toxin beta (CTB-alexa 488)-positive axons in optic nerve sections. Mmp-2-/- mice show less axonal regeneration in the optic nerve. A significant lower number of CTB+ regenerating axons was found in Mmp-2-/- mice versus WT mice. Notably, after ONC+IS, Müller glia and infiltrating immune cells were identified as the major producers of MMP-2. Bone marrow transplantations, FACS and cytokine profiling are currently ongoing to unravel the downstream cellular and molecular players. Taken together, MMP-2 plays a beneficial role during axonal regeneration and affects both dendritic and axonal compartments. Furthermore, MMP-2 expression by invading immune cells puts MMP-2 forward as a molecule linking IS to enhanced axonal regeneration. Our results suggest that the underlying mechanism of neuroinflammatory-induced axonal regeneration might partially reside in an increased potential to promote dendritic retraction, yet, more research is needed to investigate the link between dendritic shrinkage and axonal regeneration. Keywords: MMP-2, Neuroinflammation, Optic Nerve Injuries, axonal regeneration, dendrite remodeling Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Oral Presentation Topic: Disorders of the Nervous System Citation: Andries L, Salinas Navarro M, Lemmens K, De Groef L and Moons L (2019). MMP-2 as a modulator of dendritic shrinkage and axonal regeneration: neuroinflammation as the driving force. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00091 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Miss. Lien Andries, KU Leuven, Biology, Leuven, 3000, Belgium, lien.andries@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lien Andries Manuel Salinas Navarro Kim Lemmens Lies De Groef Lieve Moons Google Lien Andries Manuel Salinas Navarro Kim Lemmens Lies De Groef Lieve Moons Google Scholar Lien Andries Manuel Salinas Navarro Kim Lemmens Lies De Groef Lieve Moons PubMed Lien Andries Manuel Salinas Navarro Kim Lemmens Lies De Groef Lieve Moons Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Postconditioning with inhaled hydrogen promotes survival of retinal ganglion cells in a rat model of retinal ischemia/reperfusion injury

Retinal ischemia/reperfusion (I/R) injury plays a crucial role in the pathophysiology of various ocular diseases. Intraperitoneal injection or ocular instillation with hydrogen (H2)-rich saline was recently shown to be neuroprotective in the retina due to its anti-oxidative and anti-inflammatory effects. Our study aims to explore whether postconditioning with inhaled H2 can protect retinal ganglion cells (RGCs) in a rat model of retinal I/R injury. Retinal I/R injury was performed on the right eyes of rats and was followed by inhalation of 67% H2 mixed with 33% oxygen immediately after ischemia for 1h daily for one week. RGC density was counted using haematoxylin and eosin (HE) staining and retrograde labeling with cholera toxin beta (CTB). Visual function was assessed using flash visual evoked potentials (FVEP) and pupillary light reflex (PLR). Potential biomarkers of retinal oxidative stress and inflammatory responses were measured, including the expression of 4-Hydroxynonenalv (4-HNE), interleukin-1 beta (IL1-β) and tumor necrosis factor alpha (TNF-α). HE and CTB tracing showed that the survival rate of RGCs in the H2-treated group was significantly higher than the rate in the I/R group. Rats with H2 inhalation showed better visual function in assessments of FVEP and PLR. Moreover, H2 treatment significantly decreased the number of 4-HNE-stained cells in the ganglion cell layer and inhibited the retinal overexpression of IL1-β and TNF-α that was induced by retinal I/R injury. Our results demonstrate that postconditioning with inhaled high-dose H2 appears to confer neuroprotection against retinal I/R injury via anti-oxidative, anti-inflammatory and anti-apoptosis pathways.

Retina-on-a-chip: a microfluidic platform for point access signaling studies.

We report on a microfluidic platform for culture of whole organs or tissue slices with the capability of point access reagent delivery to probe the transport of signaling events. Whole mice retina were maintained for multiple days with negative pressure applied to tightly but gently bind the bottom of the retina to a thin poly-(dimethylsiloxane) membrane, through which twelve 100μm diameter through-holes served as fluidic access points. Staining with toluidine blue, transport of locally applied cholera toxin beta, and transient response to lipopolysaccharide in the retina demonstrated the capability of the microfluidic platform. The point access fluidic delivery capability could enable new assays in the study of various kinds of excised tissues, including retina.

Hydrogen-rich saline promotes survival of retinal ganglion cells in a rat model of optic nerve crush.

ObjectiveTo investigate the effect of molecular hydrogen (H2) in a rat model subjected to optic nerve crush (ONC).MethodsWe tested the hypothesis that after optic nerve crush (ONC), retinal ganglion cell (RGC) could be protected by H2. Rats in different groups received saline or hydrogen-rich saline every day for 14 days after ONC. Retinas from animals in each group underwent measurements of hematoxylin and eosin (H&E) staining, cholera toxin beta (CTB) tracing, gamma synuclein staining, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining 2 weeks post operation. Flash visual evoked potentials (FVEP) and pupillary light reflex (PLR) were then tested to evaluate the function of optic nerve. The malondialdehyde (MDA) level in retina was evaluated.ResultsH&E, gamma synuclein staining and CTB tracing showed that the survival rate of RGCs in hydrogen saline-treated group was significantly higher than that in saline-treated group. Apoptosis of RGCs assessed by TUNEL staining were less observed in hydrogen saline-treated group. The MDA level in retina of H2 group was much lower than that in placebo group. Furthermore, animals treated with hydrogen saline showed better function of optic nerve in assessments of FVEP and PLR.ConclusionThese results demonstrated that H2 protects RGCs and helps preserve the visual function after ONC and had a neuroprotective effect in a rat model subjected to ONC.

Immunopanning of Retrograde-Labeled Corticospinal Motor Neurons from Early Postnatal Rodents

Here we describe a method to purify corticospinal motor neurons (CSMNs). It combines the versatility of retrograde labeling with the advantages of immunopanning. Rat cortices with CSMNs that have been labeled with cholera toxin beta (CTB) are dissected and dissociated, and the CSMNs are specifically purified via immunopanning with an anti-CTB antibody. We show the efficacy of this method in early rat pups by purifying CSMNs to >99% purity. The method can be used to highly purify any neuronal cell type whose projections can be selectively labeled via retrograde tracing.

Astrocyte Reactivity: A Biomarker for Retinal Ganglion Cell Health in Retinal Neurodegeneration.

Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity (migration/proliferation, hypertrophy and GFAP expression) in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application.

Pharmacokinetics of GM1 Ganglioside Following Parenteral Administration

The pharmacokinetic parameters of monosialotetrahexosylganglioside (GM1) have been determined in healthy volunteers at 3 dose levels: 100, 200, 300 mg. Each dose was administered to separate groups of 12 volunteers. GM1 levels were determined in plasma, urine, and faeces by a method based on the property of the cholera toxin beta subunit to react specifically with GM1 ganglioside. A non-compartmental model was applied to determine standard pharmacokinetic parameters. The average AUC increased with dose (1002 +/- 121.2, 1306 +/- 146.1, 3155 +/- 121.6 micrograms mL-1 h after 100, 200, 300 mg, respectively). Plasma clearance was less than 3 mL min-1 and the distribution volume was close to the plasma volume (on average between 4.3 and 7.2 L). Mean residence time was about 43 h for all doses. GM1 was not detected in urine, while in faeces the amount of GM1 determined was similar to the baseline values obtained before dosing.

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

Cholera is an acute infection of the intestine caused by theVibrio cholerae bacterium. This bacterium, a member of Vibrionaceae family, is a facultatively anaerobic, Gram-negative, non-spore-forming curved rod, about 1.4–2.6 µm long, and capable of both respiratory and fermentative metabolism. Cholera, characterized by numerous voluminous watery stools, is often accompanied by vomiting (leading to bicarbonate loss). One of the most important virulence factors of cholera is enterotoxin (ctxAB). The cholera toxin beta subunit (CTB) is a pentameric non-toxic portion of V. cholerae toxin, responsible for holotoxin binding to the GM1-ganglioside receptor that is present on most nucleated cells. When conjugated to autoantigens, CTB dramatically increases its tolerogenic potential after oral administration. In the present study, the CTB gene from a hypertoxigenic V. cholerae strain was amplified and cloned. We first amplified the CTB gene with specific primers and the Prime STAR enzyme. The amplified CTB gene was then cloned in pET-28 vector and introduced into Escherichia coli DH5α. pET plasmids containing the CTB gene plasmids were then extracted from the bacteria and used to transform an expression host (BL21). After culturing and induction of positive bacteria, SDS-PAGE and Western blotting for protein determination and verification were performed. We also quantified the level of recombinant CTB by ELISA. Our results show that, due to its rapid growth, an expression host such as E. coli can be useful for production of CTB protein. The ELISA results showed yields of recombinant protein of up to 1 mg/L culture. However, optimal conditions for expression in our study included choice of host strain, temperature used for culture, and concentration of antibiotic and inducer. Beta subunit has many important scientific applications. There is a great deal of interest in the use of CTB as an adjuvant for vaccines targeted for delivery to the mucosa-associated lymphatic tissues. The importance and multifunctional nature of Beta subunit of V. cholerae enterotoxin, e.g., in oral vaccine preparation, will make the use of prokaryotic systems (such as Escherichia coli) for production of this protein most advantageous.

SOCS3 Deletion Promotes Optic Nerve Regeneration In Vivo

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

TRPV1 channels control synaptic plasticity in the developing superior colliculus

Long-term depression (LTD) in the rodent superior colliculus (SC) is regarded as a model of synaptic refinement because it can be induced during development but not in adults. We investigated the role of transient receptor potential vanilloid type-1 (TRPV1) channels in this type of synaptic plasticity. Experiments were carried out in pigmented mice aged between postnatal day 8 (P8) and 42 (P42) and in adult mice. Retinal axons to the SC were labelled by injection of cholera toxin-beta (CTbeta) into the eye. Immunohistochemical staining for CTbeta, TRPV1 and markers of glutamatergic and GABAergic cells and fibres (VGLUT1 and VGAT or GAD65, respectively) was performed by using multiple immunofluorescence. This showed that both glutamatergic retinal afferents to, and some GABAergic neurones in, the superficial SC are TRPV1 positive in juvenile but not adult mice. Field potential recordings were made from the superficial grey layer in parasagittal SC slices, and LTD (76 +/- 8% of control responses) was induced with a 50 Hz, 20 s tetanus. Activation of TRPV1 with resiniferatoxin also reduced field potential amplitude to 84 +/- 8% of control values. Blockade of TRPV1 with the selective antagonist 5'-iodo-resiniferatoxin prevented the induction of LTD (98 +/- 4% of control values), but did not cause its reversal if LTD was already established. N-acylphosphatidylethanolamine-specific phospholipase D and 12-lipoxygenase, two proposed endovanilloid biosynthesizing enzymes, were co-expressed with TRPV1 in the SC at P14 and P28. These results suggest that TRPV1 modulates retinocollicular responses in the developing SC and is activated during tetanic stimulation by endovanilloid ligands to participate in the induction of LTD.

The Effects of Aging on Hypoglossal Motoneurons in Rats

Aging can result in a loss of neuronal cell bodies and a decrease in neuronal size in some regions of the brain and spinal cord. Motoneuron loss in the spinal cord is thought to contribute to the progressive decline in muscle mass and strength that occurs with age (sarcopenia). Swallowing disorders represent a large clinical problem in elderly persons; however, age-related alterations in cranial motoneurons that innervate muscles involved in swallowing have been understudied. We aimed to determine if age-related alterations occurred in the hypoglossal nucleus in the brainstem. If present, these changes might help explain alterations at the neuromuscular junction and changes in the contractile properties of tongue muscle that have been reported in older rats. We hypothesized that with increasing age there would be a loss of motoneurons and a reduction in neuronal size and the number of primary dendrites associated with each hypoglossal motoneuron. Neurons in the hypoglossal nucleus were visualized with the neuronal marker NeuN in young (9-10 months), middle-aged (24-25 months), and old (32-33 months) male F344/BN rats. Hypoglossal motoneurons were retrograde-labeled with injections of Cholera Toxin beta into the genioglossus muscle of the tongue and visualized using immunocytochemistry. Results indicated that the number of primary dendrites of hypoglossal motoneurons decreased significantly with age, while no age-associated changes were found in the number or size of hypoglossal motoneurons. Loss of primary dendrites could reduce the number of synaptic inputs and thereby impair function.

Connections of cat auditory cortex: II. Commissural system.

The commissural projections between 13 areas of cat auditory cortex (AC) were studied using retrograde tracers. Areal and laminar origins were characterized as part of a larger study of thalamic input and cortical origins of projections to each area. Cholera toxin beta subunit (CTbeta) and cholera toxin beta subunit gold-conjugate (CTbetaG) were injected separately within an area or in different areas in an experiment. The areas were identified independently with SMI-32, which revealed differences in neurofilament immunoreactivity in layers III, V, and VI. Each area received convergent AC input from 3 to 6 (mean, 5) contralateral areas. Most of the projections (>75%) were homotopic and from topographically organized loci in the corresponding area. Heterotopic projections (>1 mm beyond the main homotopic projection) constituted approximately 25% of the input. Layers III and V contained >95% of the commissural neurons. Commissural projection neurons were clustered in all areas. Commissural divergence, assessed by double labeling, was less than 3% in each area. This sparse axonal branching is consistent with the essentially homotopic connectivity of the commissural system. The many heterotopic origins represent unexpected commissural influences converging on an area. Areas more dorsal on the cortical convexity have commissural projections originating in layers III and V; more ventral areas favor layer III at the expense of layer V, to its near-total exclusion in some instances. Some areas have almost entirely layer III origins (temporal cortex and area AII), whereas others have a predominantly layer V input (anterior auditory field) or dual contributions from layers III and V (the dorsal auditory zone). A topographic distribution of commissural cells of origin is consistent with the order observed in thalamocortical and corticocortical projections, and which characterizes all extrinsic projection systems (commissural, corticocortical, and thalamocortical) in all AC areas. Thus, laminar as well as areal differences in projection origin distinguish the auditory cortical commissural system.

Activation of afferents to the ventral tegmental area in response to acute amphetamine: a double‐labelling study

The ventral tegmental area (VTA), primary source of the mesocorticolimbic dopaminergic system, is regarded as a critical site for initiation of behavioural sensitization to psychostimulants. The present study was undertaken to identify the neural pathways converging on the VTA that are potentially implicated in this process. Rats were sensitized by a single exposure to amphetamine (5 mg/kg, s.c.). The distribution of VTA-projecting neurons activated by amphetamine was examined by combining retrograde transport of the cholera toxin beta subunit (CTb), injected into the VTA, with immunodetection of Fos. The quantitative analysis of CTb-Fos double labelling demonstrates that amphetamine induced a rapid activation of Fos in a large number of brain areas projecting to the VTA. More than half of the CTb-Fos double-labelled neurons were located in the prefrontal cortex, lateral preoptic area-lateral hypothalamus, pontomesencephalic tegmentum, dorsal raphe nucleus, ventral pallidum and nucleus accumbens. In addition, scattered CTb-Fos double-labelled cells were observed in many other VTA afferent structures, such as claustrum, lateral septum, diagonal band-magnocellular preoptic nucleus, deep mesencephalic nucleus, oral part of pontine reticular nucleus and dorsomedial tegmental area. This suggests that systemic amphetamine activates a wide population of neurons projecting to the VTA that may be important for the modulation of neurobehavioural plasticity produced by this psychostimulant.