Hair cell recovery in the vestibular sensory epithelia of mature guinea pigs

The progression of recovery of the vestibular sensory epithelia of guinea pigs after gentamicin-induced hair cell injury was assessed quantitatively and qualitatively. Evaluations were made of the number of cells bearing hair bundles by using scanning electron microscopy (SEM) and of identifiable hair cells in thin sections. Both assessment procedures showed that an initial loss of hair cells in utricular maculae is followed by significant recovery in the number of hair cells present. SEM also showed recovery in saccules comparable to that in utricles. During the recovery, progressive maturation of hair bundles, which exhibited features similar to those seen during normal ontogenetic development of hair cells, could be identified. The pattern and extent of hair cell loss and subsequent reappearance revealed by SEM corresponded with that derived from analysis of thin sections. This suggests that repair of nonlethally damaged hair cells is unlikely but, rather, that new hair cells are produced. An apparent decrease in supporting cell numbers was observed coincident with the increase in hair cell numbers. This complements previous morphological observations, which have suggested new hair cells arise from direct, nonmitotic transdifferentiation of supporting cells. The quantitative analyses indicate that more than half of the hair cells that are lost are replaced, but the recovery process does not result in complete restoration of the epithelium. Eight months after the end of drug treatment, the number of hair cells present was still significantly less than normal, and several other abnormalities persisted. There was also no evidence of any hair cell recovery in the organ of Corti. Thus, there appear to be limitations on the capacity for spontaneous replacement of lost hair cells in the mammalian inner ear. J. Comp. Neurol. 397:69–88, 1998. © 1998 Wiley-Liss, Inc.     

Orexin a phosphorylates the γ‐Aminobutyric acid type A receptor β2 subunit on a serine residue and changes the surface expression of the receptor in SH‐SY5Y cells exposed to propofol

Propofol activates the γ‐aminobutyric acid type A receptor (GABA_AR) and causes a reversible neurite retraction, leaving a thin, thread‐like structure behind; it also reverses the transport of vesicles in rat cortical neurons. The awakening peptide orexin A (OA) inhibits this retraction via phospholipase D (PLD) and protein kinase C? (PKC?). The human SH‐SY5Y cells express both GABA_ARs and orexin 1 and 2 receptors. These cells are used to examine the interaction between OA and the GABA_AR. The effects of OA are studied with flow cytometry and immunoblotting. This study shows that OA stimulates phosphorylation on the serine residues of the GABA_AR β₂ subunit and that the phosphorylation is caused by the activation of PLD and PKC?. OA administration followed by propofol reduces the cell surface expression of the GABA_AR, whereas propofol stimulation before OA increases the surface expression. The GABA_AR β₂ subunit is important for receptor recirculation, and the effect of OA on propofol‐stimulated cells may be due to a disturbed recirculation of the GABA_AR. © 2015 Wiley Periodicals, Inc.     

Pigment epithelium‐derived factor (PEDF) and PEDF‐receptor in the adult mouse brain: Differential spatial/temporal localization pattern

Pigment epithelium‐derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western‐blotting, and also by enzyme‐linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age‐dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.     

Transforming growth factor α with insulin stimulates cell proliferation in vivo in adult rat vestibular sensory epithelium

Hair cells, the sensory receptors of the mammalian inner ear, have long been thought to be produced only during embryogenesis, and postnatal hair cell loss is considered to be irreversible and is associated with permanent hearing and balance deficits. Little is known about the factors that regulate hair cell genesis and differentiation. The mitogenic effects of insulin and transforming growth factor α (TGFα) were assayed in vivo in normal and drug-damaged rat inner ear. Tritiated thymidine and autoradiographic techniques were used to identify cells synthesizing DNA. Simultaneous infusion of TGFα and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium. New supporting cells and putative new hair cells were produced. Infusion of insulin alone or TGFα alone failed to stimulate significant DNA synthesis. These results suggest that exogenous growth factors may have utility for therapeutic treatment of hearing and balance disorders in vivo. J. Comp. Neurol. 399:413–423, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of multiple bistratified retinal ganglion cells in a purkinje cell protein 2‐Cre transgenic mouse line

Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional‐selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, overexpression, and ectopic expression in a cell type‐specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type‐specific expression of Cre recombinase. In this study we characterized the Cre‐positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)‐cre mouse line. We found that all of the Cre‐positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On‐ and Off‐dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn‐3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On‐Off and On‐DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice. J. Comp. Neurol. 521:2165–2180, 2013. © 2012 Wiley Periodicals, Inc.     

A comparative study of the neural stem cell niche in the adult hypothalamus of human,mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non‐human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co‐expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.     

Specializations of intercellular junctions are associated with the presence and absence of hair cell regeneration in ears from six vertebrate classes

Sensory hair cell losses lead to hearing and balance deficits that are permanent for mammals, but temporary for nonmammals because supporting cells in their ears give rise to replacement hair cells. In mice and humans, vestibular supporting cells grow exceptionally large circumferential F‐actin belts and their junctions express E‐cadherin in patterns that strongly correlate with postnatal declines in regeneration capacity. In contrast, chicken supporting cells retain thin F‐actin belts throughout life and express little E‐cadherin. To determine whether the junctions in chicken ears might be representative of other ears that also regenerate hair cells, we investigated inner ears from dogfish sharks, zebrafish, bullfrogs, Xenopus, turtles, and the lizard, Anolis. As in chickens, the supporting cells in adult zebrafish, Xenopus, and turtle ears retained thin circumferential F‐actin belts and expressed little E‐cadherin. Supporting cells in adult sharks and bullfrogs also retained thin belts, but were not tested for E‐cadherin. Supporting cells in adult Anolis exhibited wide, but porous webs of F‐actin and strong E‐cadherin expression. Anolis supporting cells also showed some cell cycle reentry when cultured. The results reveal that the association between thin F‐actin belts and low E‐cadherin is shared by supporting cells in anamniotes, turtles, and birds, which all can regenerate hair cells. Divergent junctional specializations in supporting cells appear to have arisen independently in Anolis and mammals. The presence of webs of F‐actin at the junctions in Anolis appears compatible with supporting cell proliferation, but the solid reinforcement of the F‐actin belts in mammals is associated with its absence. J. Comp. Neurol., 521:1430–1448, 2013. © 2012 Wiley Periodicals, Inc.     

The α2‐subunit of the nicotinic cholinergic receptor is specifically expressed in medial subpallium‐derived cells of mammalian amygdala

Nicotinic acetylcholine receptor (nAChR) subtypes are expressed in specific neuronal populations, which are involved in numerous neural functions such as sleep, fatigue, anxiety, and cognition, as well as the central processing of pain and food intake. Moreover, mutations in nAChRs subunits have been related to frontal lobe epilepsy, neurodegenerative diseases, and other neurological disorders, including schizophrenia and attention deficit and hyperactivity disorder (ADHD). Previous studies have shown that the α2‐subunit of the AChR (Chrna2) is expressed in the basal forebrain, in the septum, and in some amygdalar nuclei in the adult rodent brain. However, although the importance of this amygdalar expression in emotion‐related behavior and the physiopathology of neuropsychiatric disorders has been accepted, a detailed study of the Chrna2 expression pattern during development has been lacking. In this study we found that Chrna2 is specifically expressed in medial subpallium‐derived amygdalar nuclei from early developmental stages to adult. This finding could help us to better understand the role of Chrna2 in the differentiation and functional maturation of amygdalar neurons involved in cholinergic‐regulated emotional behavior. J. Comp. Neurol. 523:1608–1621, 2015. © 2015 Wiley Periodicals, Inc.     

Differential expression of protocadherin‐19, protocadherin‐17, and cadherin‐6 in adult zebrafish brain

Cell adhesion molecule cadherins play important roles in both development and maintenance of adult structures. Most studies on cadherin expression have been carried out in developing organisms, but information on cadherin distribution in adult vertebrate brains is limited. In this study we used in situ hybridization to examine mRNA expression of three cadherins, protocadherin‐19, protocadherin‐17, and cadherin‐6 in adult zebrafish brain. Each cadherin exhibits a distinct expression pattern in the fish brain, with protocadherin‐19 and protocadherin‐17 showing much wider and stronger expression than that of cadherin‐6. Both protocadherin‐19 and protocadherin‐17‐expressing cells occur throughout the brain, with strong expression in the ventromedial telencephalon, periventricular regions of the thalamus and anterior hypothalamus, stratum periventriculare of the optic tectum, dorsal tegmental nucleus, granular regions of the cerebellar body and valvula, and superficial layers of the facial and vagal lobes. Numerous sensory structures (e.g., auditory, gustatory, lateral line, olfactory, and visual nuclei) and motor nuclei (e.g., oculomotor, trochlear, trigeminal motor, abducens, and vagal motor nuclei) contain protocadherin‐19 and/or protocadherin‐17‐expressing cell. Expression of these two protocadherins is similar in the ventromedial telencephalon, thalamus, hypothalamus, facial, and vagal lobes, but substantially different in the dorsolateral telencephalon, intermediate layers of the optic tectum, and cerebellar valvula. In contrast to the two protocadherins, cadherin‐6 expression is much weaker and limited in the adult fish brain. J. Comp. Neurol. 523:1419–1442, 2015. © 2015 Wiley Periodicals, Inc.     

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice

Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2^–/–A3^–/– mice by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU⁺ cells in MC derived from A2^–/–A3^–/– mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho^–/–) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2^–/–A3^–/– , Rho^–/– and Rho^–/–A2^–/–A3^–/– mice and the numbers of EdU⁺ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho^–/– mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2^–/–A3^–/– mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU⁺ cells in the retinas of adult ephrin-A2^–/–A3^–/– mice mainly in the inner nuclear layer; and these EdU⁺ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho^–/–A2^–/–A3^–/– mice), a significantly greater amount of EdU⁺ cells were located in the ciliary body, retina and RPE than that of Rho^–/– mice. Comparing between 6 and 12 weeks old Rho^–/–A2^–/–A3^–/– mice, we recorded more EdU⁺ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

Chinese Library Classification No. R459.9; R364; R774     

Cell‐ and region‐specific expression of depression‐related protein p11 (S100a10) in the brain

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion, and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11‐KO) in mice results in a depressive‐like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that, although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and nonneuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry, and whole‐tissue volume imaging. Overall, expression spans multiple brain regions, structures, and cell types, suggesting a complex role of p11 in depression. J. Comp. Neurol. 525:955–975, 2017. © 2016 Wiley Periodicals, Inc.     

Dopamine D3 receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type‐B and ‐C cells in adult subventricular zone

The neurotransmitter dopamine acts on the subventricular zone (SVZ) to regulate both prenatal and postnatal neurogenesis, in particular through D₃ receptor (D₃R) subtype. In this study, we explored the cellular mechanism(s) underlying D₃R‐mediated cell proliferation and tested if systemic delivery of a D₃R agonist would induce SVZ multipotent neural stem/precursor cell (NSC/NPC) proliferation in vivo. We found that treatment with the D₃R agonist, 7‐OH‐DPAT, enhances cell proliferation in a dose‐dependent manner in cultured SVZ neurospheres from wild‐type, but not D₃R knock‐out mice. Furthermore, D₃R activation also stimulates S‐phase and enhances mRNA and protein levels of cyclin D1 in wild‐type neurospheres, a process which requires cellular Akt and ERK1/2 signaling. Moreover, chronic treatment with low dose 7‐OH‐DAPT in vivo increases BrdU⁺ cell numbers in the adult SVZ, but this effect was not seen in D₃R KO mice. Additionally, we probed the cell type specificity of D₃R agonist‐mediated cell proliferation. We found that in adult SVZ, GFAP⁺ astrocytes, type‐B GFAP⁺/nestin⁺ and type‐C EGF receptor (EGFR⁺)/nestin⁺ cells express D₃R mRNA, but type‐A Doublecortin (Dcx)⁺ neuroblasts do not. Using flow cytometry and immunofluorescence, we demonstrated that D₃R activation increases GFAP⁺ type‐B and EGFR⁺ type‐C cell numbers, and the newly divided Dcx⁺ type‐A cells. However, BrdU⁺/Dcx⁺ cell numbers were decreased in D₃R KO mice compared to wildtype, suggesting that D₃R maintains constitutive NSC/NPCs population in the adult SVZ. Overall, we demonstrate that D₃R activation induces NSC/NPC proliferation through Akt and ERK1/2 signaling and increases the numbers of type‐B and ‐C NSC/NPCs in the adult SVZ. © 2013 Wiley Periodicals, Inc.     

Absence of laminin α1 chain in the skeletal muscle of dystrophic dy/dy mice

In Duchenne muscular dystrophy (DMD) and laminin α2 defective congenital muscular dystrophies (CMD) there are reports of an induction of laminin α1 chain in regenerating muscle fibers. These studies are based on immunohistochemistry data with one monoclonal antibody alone. Based on these data we sought to establish if the laminin α1 chain is induced in the muscle of dy/dy mice. We found no evidence of induction of laminin α1 chain protein or mRNA in dystrophic dy/dy skeletal muscle fibers as determined by immunohistochemistry, Western blotting, Northern blotting, or PCR analysis. Our data point to the need for additional immunological reagents specific for human laminin-α1 to resolve whether the conflicting data on laminin-α1 distribution in human and mouse tissues is due to species differences or, alternatively, due to differences in reagent specificity. Our data might be important when designing therapy strategies for CMD. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1515–1524, 1997     

Localization of nectin‐2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage‐gated A‐type K+ channel Kv4.2 in the medial habenula

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin‐like cell adhesion molecules (CAMs) nectin‐2α and nectin‐2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin‐2α, but not nectin‐2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage‐gated A‐type K⁺ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin‐2α at this boundary was not colocalized with the nectin‐2α‐binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin‐2α forms a cell adhesion apparatus different from the Kv4.2‐associated MSs. Genetic ablation of nectin‐2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin‐2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.     

Mid‐life environmental enrichment increases synaptic density in CA1 in a mouse model of Aβ‐associated pathology and positively influences synaptic and cognitive health in healthy ageing

Early‐life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later‐life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later‐life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APP_SWE/PS1_dE9) from 6 months of age. After 6 months of housing in standard laboratory cages, APP_SWE/PS1_dE9 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APP_SWE/PS1_dE9 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APP_SWE/PS1_dE9 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APP_SWE/PS1_dE9 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later‐life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.     

Expression of cell fate determinants and plastic changes after neurotoxic lesion of adult mice spinal cord by cholera toxin‐B saporin

Recent studies have attempted to repair the damaged spinal cord (SC) by stimulating neurogenesis or neuroplasticity. Sonic hedgehog (Shh), Notch‐1 and Numb are involved in the stem cell functioning. Additionally, Notch‐1 has a role as modulator of synaptic plasticity. However, little is known about the role of these proteins in the adult SC after removal of motoneurons. In this study, we have injected cholera toxin‐B saporin into the gastrocnemius muscle to induce a depletion of motoneurons within the lumbar SC of adult mice, and analysed the expression of choline acetyltransferase (ChAT), Synapsin‐I, Shh, Notch‐1 and Numb proteins. The functional outcome of the lesion was monitored by grid walk and rotarod tasks. The neurotoxin lesion determined a motoneuron depletion and a transient decrease of ChAT, Synapsin‐I, Shh and Numb levels in the lumbar SC. ChAT was associated with Synapsin‐I expression and motor performance at 1 week but not 1 month after lesion, suggesting that the recovery of locomotion could depend on synaptic plasticity, at least in an early phase. Shh and Notch‐1 were associated with Synapsin‐I levels, suggesting a role in modulating synaptic plasticity. Numb expression also appeared reduced after lesion and linked to motor performance. Moreover, unlike other lesion models, we observed glial reaction but no evidence of cell proliferation within the depleted SC. Given the mentioned roles of Shh, Notch‐1 and Numb, we believe that an in vivo manipulation of their signalling after lesion could represent a suitable way to improve functional recovery by modulating synaptic plasticity and/or neurogenesis.     

Extrinsic regulation of injury/growth-related gene expression in the inferior olive of the adult rat

Successful axon regeneration relies on the capability of the lesioned neurons to up-regulate a specific set of injury/growth-associated genes. In the adult central nervous system, the strength of the cell body response is generally related to the distance of the injury site from the perikaryon, being stronger for proximal lesions. Nevertheless, inferior olive (IO) cells react to injury and regenerate their axons even after distal transections. To investigate the mechanisms that regulate the IO growth properties, we examined the expression of injury/growth markers (nitric oxide synthase, growth-associated protein 43 and c-Jun) after target deletion or axotomy performed at different sites along the olivocerebellar pathway. Both axon injury and target loss disclose two subsets of IO neurons distributed within precise subnuclei: one subset up-regulates all markers in all conditions, whereas the other shows a mild c-Jun expression but remains unresponsive even after a very proximal axotomy. These observations indicate that distinct subpopulations of IO cells respond to different regulatory strategies. Unresponsive neurons appear insensitive to environmental positive or negative cues, suggesting that they are intrinsically unable to set up a cellular reaction to injury. In contrast, cell body changes in reactive neurons are elicited after the removal of retrogradely transported target-derived inhibitory signals. Target loss also induces degeneration of IO cells, whose survival remains partially dependent on Purkinje targets in adulthood. Thus, the intrinsic regenerative potential of a functionally homogeneous population is regulated by multiple mechanisms, specific for distinct neuronal subsets.     

Nectin‐1 spots as a novel adhesion apparatus that tethers mitral cell lateral dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb

Mitral cells project lateral dendrites that contact the lateral and primary dendrites of other mitral cells and granule cell dendrites in the external plexiform layer (EPL) of the olfactory bulb. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. In immunofluorescence microscopy, the immunofluorescence signal for the cell adhesion molecule nectin‐1 was concentrated on mitral cell lateral dendrites in the EPL of the developing mouse olfactory bulb. In electron microscopy, the immunogold particles for nectin‐1 were symmetrically localized on the plasma membranes at the contacts between mitral cell lateral dendrites, which showed bilateral darkening without dense cytoskeletal undercoats characteristic of puncta adherentia junctions. We named the contacts where the immunogold particles for nectin‐1 were symmetrically accumulated "nectin‐1 spots." The nectin‐1 spots were 0.21 μm in length on average and the distance between the plasma membranes was 20.8 nm on average. In 3D reconstruction of serial sections, clusters of the nectin‐1 spots formed a disc‐like structure. In the mitral cell lateral dendrites of nectin‐1‐knockout mice, the immunogold particles for nectin‐1 were undetectable and the plasma membrane darkening was electron‐microscopically normalized, but the plasma membranes were partly separated from each other. The nectin‐1 spots were further identified between mitral cell lateral and primary dendrites and between mitral cell lateral dendrites and granule cell dendritic spine necks. These results indicate that the nectin‐1 spots constitute a novel adhesion apparatus that tethers mitral cell dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb. J. Comp. Neurol. 523:1824–1839, 2015. © 2015 Wiley Periodicals, Inc.     

Characterization of the mitofusin 2 R94W mutation in a knock‐in mouse model

Charcot‐Marie‐Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre‐clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock‐in mouse model expressing Mfn2^R94W, which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2^R94W heterozygous mice show histopathology and age‐dependent open‐field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.