Reduced levels of Cacna1c attenuate mesolimbic dopamine system function.

Genetic variation in CACNA1C, which codes for the L-type calcium channel (LTCC) Cav 1.2, is associated with clinical diagnoses of bipolar disorder, depression and schizophrenia. Dysregulation of the mesolimbic-dopamine (ML-DA) system is linked to these syndromes and LTCCs are required for normal DAergic neurotransmission between the ventral tegmental area (VTA) and nucleus accumbens (NAc). It is unclear, however, how variations in CACNA1C genotype, and potential subsequent changes in expression levels in these regions, modify risk. Using constitutive and conditional knockout mice, and treatment with the LTCC antagonist nimodipine, we examined the role of Cacna1c in DA-mediated behaviors elicited by psychomotor stimulants. Using fast-scan cyclic voltammetry, DA release and reuptake in the NAc were measured. We find that subsecond DA release in Cacna1c haploinsufficient mice lacks normal sensitivity to inhibition of the DA transporter (DAT). Constitutive haploinsufficiency of Cacna1c led to attenuation of hyperlocomotion following acute administration of stimulants specific to DAT, and locomotor sensitization of these mice to the DAT antagonist GBR12909 did not reach the same level as wild-type mice. The maintenance of sensitization to GBR12909 was attenuated by administration of nimodipine. Sensitization to GBR12909 was attenuated in mice with reduced Cacna1c selectively in the VTA but not in the NAc. Our findings show that Cacna1c is crucial for normal behavioral responses to DA stimulants and that its activity in the VTA is required for behavioral sensitization. Cacna1c likely exerts these effects through modifications to presynaptic ML-DA system function.

An angle-supported foldable phakic intraocular lens for correction of myopia: A five-year follow-up. / Lente intraocular fáquica plegable acrílica de apoyo angular para la corrección de miopía: seguimiento de 5 años.

OBJECTIVE: To evaluate the efficacy and safety of an angle-supported foldable phakic intraocular lens (pIOL) for the correction of moderate to high myopia after 5 years follow-up. METHODS: Prospective and retrospective, observational, longitudinal, non-randomised consecutive series of cases conducted on a total of 100 eyes of 67 patients with moderate to high myopia implanted with an Acrysof Cachet pIOL (Alcon Laboratories Inc.) with the aim of minimising the refractive error. The ages ranged between 18 to 60years. Uncorrected distance visual acuity (UDVA), manifest refraction, corrected distance visual acuity (CDVA), endothelial cells density, pIOL position, intraocular pressure, and complications were recorded preoperatively and during the 5 year follow-up. RESULTS: Five years after implantation, the mean manifest spherical equivalent refraction reduced significantly from -11.62±3.35 dioptres (D) to -0.33±0.85D. UDVA was 20/20 or better in 5 of 25 cases (20%), and 20/40 or better in 22 cases (88%). CDVA was 20/20 or better in 17 cases (68%), and 20/32 or better in 23 cases (92%) of eyes. The residual refractive error was within ±0.50D of emmetropia in 12 cases (48%), and within ±1.00D in 19 cases (76%). Mean endothelial cell loss at 5 years was 11.8% central, and 13.7% peripheral. Mean endothelium-pIOL distance was 2.11±0.18mm, and mean pIOL-crystalline distance was 0.88±0.20mm. CONCLUSIONS: This angle supported pIOL provided a favourable refractive correction and predictability, as well as acceptable safety in patients with moderate to high myopia. Although endothelial cell density decreased over 5years, the results are within the range reported in previous studies with other pIOLs.

Sex-dependent mitophagy and neuronal death following rat neonatal hypoxia-ischemia.

Males are more susceptible than females to long-term cognitive deficits following neonatal hypoxic-ischemic encephalopathy (HIE). Mitochondrial dysfunction is implicated in the pathophysiology of cerebral hypoxia-ischemia (HI), but the influence of sex on mitochondrial quality control (MQC) after HI is unknown. Therefore, we tested the hypothesis that mitophagy is sexually dimorphic and neuroprotective 20-24h following the Rice-Vannucci model of rat neonatal HI at postnatal day 7 (PN7). Mitochondrial and lysosomal morphology and degree of co-localization were determined by immunofluorescence in the cerebral cortex. No difference in mitochondrial abundance was detected in the cortex after HI. However, net mitochondrial fission increased in both hemispheres of female brain, but was most extensive in the ipsilateral hemisphere of male brain following HI. Basal autophagy, assessed by immunoblot for the autophagosome marker LC3BI/II, was greater in males suggesting less intrinsic reserve capacity for autophagy following HI. Autophagosome formation, lysosome size, and TOM20/LAMP2 co-localization were increased in the contralateral hemisphere following HI in female, but not male brain. An accumulation of ubiquitinated mitochondrial protein was observed in male, but not female brain following HI. Moreover, neuronal cell death with NeuN/TUNEL co-staining occurred in both hemispheres of male brain, but only in the ipsilateral hemisphere of female brain after HI. In summary, mitophagy induction and neuronal cell death are sex dependent following HI. The deficit in elimination of damaged/dysfunctional mitochondria in the male brain following HI may contribute to male vulnerability to neuronal death and long-term neurobehavioral deficits following HIE.

TLR4 signaling in VTA dopaminergic neurons regulates impulsivity through tyrosine hydroxylase modulation.

Alcohol dependence is a complex disorder that initiates with episodes of excessive alcohol drinking known as binge drinking, and has a 50-60% risk contribution from inherited susceptibility genes. Cognitive impulsivity is a heritable trait that may set the stage for transition to alcohol dependence but its role in the ethanol-seeking behavior and the involved genes are still poorly understood. We have previously shown that alcohol-preferring P rats have innately elevated levels of a neuronal Toll-like receptor 4 (TLR4) signal in the ventral tegmental area (VTA) that controls the initiation of excessive alcohol drinking. Here we report that TLR4 is localized in dopaminergic (TH+) neurons and it upregulates the expression of tyrosine hydroxylase (TH) through a cAMP-dependent protein kinase (PKA)/cyclic AMP response element binding protein (CREB) signal. P rats have higher impulsivity than wild-type (WT) rats and VTA infusion of a non-replicating Herpes simplex virus (HSV) vector for TLR4-specific small interfering RNA (siRNA; pHSVsiTLR4) inhibits both impulsivity and TLR4/TH expression. A scrambled siRNA vector does not affect gene expression or impulsivity. The data suggest that TLR4 signaling in VTA dopaminergic neurons controls impulsivity related to the regulation of TH expression, likely contributing to the initiation of alcohol drinking and its transition to alcohol dependence.

Sensory experience selectively regulates transmitter synthesis enzymes in interglomerular circuits.

Sensory experience influences brain organization and function. A particularly striking example is in the olfactory bulb where reduction of odorant sensory signals profoundly down-regulates dopamine in glomerular neurons. There are two large populations of glomerular inhibitory interneurons: (1) GABAergic periglomerular (PG) cells, whose processes are limited to a single glomerulus, regulate intraglomerular processing and (2) DAergic-GABAergic short axon (SA) cells, whose processes contact multiple glomeruli, regulate interglomerular processing. The inhibitory neurotransmitter GABA is synthesized from L-glutamic acid by the enzyme glutamic acid decarboxylase (GAD) of which there are two major isoforms: GAD65 and GAD67. GAD65 is expressed in uniglomerular PG cells. GAD67 is expressed by SA cells, which also co-express the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH). Deafferentation or sensory deprivation decreases TH expression but it is not known if sensory input alters GAD isoforms. Here we report that either deafferentation or reduction of sensory input by nares occlusion significantly reduced GAD67 protein and the number of SA cells expressing GAD67. However, neither manipulation altered GAD65 protein or the number of GAD65 PG cells. These findings show that sensory experience strongly impacts transmitter regulation in the circuit that controls neural processing across glomeruli but not in the circuit that regulates intraglomerular processing.

Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R alpha-, TLR4-, and IFN-beta-dependent.

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed "Th1" and "Th2" cytokine storm. We hypothesized that differentiation of "alternatively activated" macrophages (AA-M phi) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4R alpha/STAT6-dependent AA-M phi differentiation, and significantly enhanced inflammation in the lungs of IL-4R alpha(-/-) mice. Adoptive transfer of wildtype macrophages to IL-4R alpha(-/-) mice restored RSV-inducible AA-M phi phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4(-/-) and interferon (IFN)-beta(-/-) macrophages and mice also failed to express AA-M phi markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptor gamma expression, a DNA-binding protein that induces AA-M phi genes, whereas IFN-beta regulates IL-4, IL-13, IL-4R alpha, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-M phi. These data suggest new treatment strategies for RSV that promote AA-M phi differentiation.

Effectiveness of the treatment with intravenous pamidronate in calcinosis in juvenile dermatomyositis.

OBJECTIVE: Calcinosis is a frequent finding in up to 40% of children with juvenile dermatomyositis (JDM). Different treatments (aluminum hydroxide, diltiazem, probenecid, alendronate, etc.) have been used in an attempt to clear calcinosis and to avoid the onset of new calcium deposition, but none has been clearly effective. Pamidronate is a nitrogen-containing bisphosphonate with a potent inhibiting bone resorption effect that has been used to treat osteoporosis in children. We report three children with JDM who developed calcinosis and who received intravenous pamidronate with good results. METHODS: All three patients met the Bohan and Peter diagnostic criteria for JDM. Intravenous pamidronate was given at 1 mg/kg/day on three consecutive days every three months according to the protocol established by Glorieux et al. for osteoporosis treatment in osteogenesis imperfecta. RESULTS: The calcinosis which developed in all three patients improved. No important adverse events were observed. CONCLUSION: In all three cases, calcinosis significantly decreased, and even totally cleared in patient 1. Total clearance of pre-existing calcinosis in JDM with pamidronate therapy has not been previously described with any of the aforementioned treatments. The advantage of treatment with pamidronate compared to treatment with alendronate is that intravenous administration does not produce esophagitis, the most frequent adverse event when orally administering bisphosphonates. Our results strongly suggest that therapy with intravenous pamidronate in conjunction with good disease control with DMARD therapy is an apparently safe and effective treatment for calcinosis management in JDM.

Two GABAergic intraglomerular circuits differentially regulate tonic and phasic presynaptic inhibition of olfactory nerve terminals.

Olfactory nerve axons terminate in olfactory bulb glomeruli forming excitatory synapses onto the dendrites of mitral/tufted (M/T) and juxtaglomerular cells, including external tufted (ET) and periglomerular (PG) cells. PG cells are heterogeneous in neurochemical expression and synaptic organization. We used a line of mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65-kDa gene (GAD65+) promoter to characterize a neurochemically identified subpopulation of PG cells by whole cell recording and subsequent morphological reconstruction. GAD65+ GABAergic PG cells form two functionally distinct populations: 33% are driven by monosynaptic olfactory nerve (ON) input (ON-driven PG cells), the remaining 67% receive their strongest drive from an ON-->ET-->PG circuit with no or weak monosynaptic ON input (ET-driven PG cells). In response to ON stimulation, ON-driven PG cells exhibit paired-pulse depression (PPD), which is partially reversed by GABA(B) receptor antagonists. The ON-->ET-->PG circuit exhibits phasic GABA(B)-R-independent PPD. ON input to both circuits is under tonic GABA(B)-R-dependent inhibition. We hypothesize that this tonic GABA(B)R-dependent presynaptic inhibition of olfactory nerve terminals is due to autonomous bursting of ET cells in the ON-->ET-->PG circuit, which drives tonic spontaneous GABA release from ET-driven PG cells. Both circuits likely produce tonic and phasic postsynaptic inhibition of other intraglomerular targets. Thus olfactory bulb glomeruli contain at least two functionally distinct GABAergic circuits that may play different roles in olfactory coding.

Developmental regulation of metabotropic glutamate receptor 1 splice variants in olfactory bulb mitral cells.

Alternative splicing of the metabotropic glutamate receptor 1 (mGluR1) receptor gene generates two major receptor isoforms, mGluR1a and mGluR1b, differing in intracellular function and distribution. However, little is known on the expression profiles of these variants during development. We examined the mRNA expression profile of mGluR1a/b in microdissected layers and acutely isolated mitral cells in the developing mouse olfactory bulb. This analysis showed that the two mGluR1 variants are differentially regulated within each bulb layer. During the first postnatal week, the mGluR1a isoform replaces GluR1b in the microdissected mitral cell layer (MCL) and in isolated identified mitral cells, coinciding with a developmental epoch of mitral cell dendritic reorganization. Although mGluR1a mRNA is expressed at high levels in both the adult external plexiform layer (EPL) and MCL, Western blotting analysis reveals a marked reduction of the mGluR1a protein in the MCL, where mitral cell bodies are located, and strong labeling in the EPL, which contains mitral cell dendrites. This suggests that there is increased dendritic trafficking efficiency of the receptor in adult. The temporal and spatial shift in mGluR1b/a expression suggests distinct roles of the mGluR1 isoforms, with mGluR1b potentially involved in the early mitral cell maturation and mGluR1a in dendritic and synapse function.

Quantitative analysis of neuronal diversity in the mouse olfactory bulb.

Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D-28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67-kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types.

"Benign" shaken baby syndrome. Case report.

The authors report an infant with clinical and neuroimaging findings of shaken baby syndrome. The pitfalls encountered in the assessment on the cause of the bilateral frontal and interhemispheric subdural hematomas in this child are also briefly discussed. We have called this condition "benign" shaken baby syndrome and emphasize that not always acute subdural hematomas are of non-accidental nature.

"Benign" shaken baby syndrome. Case report

The authors report an infant with clinical and neuroimaging findings of shaken baby syndrome. The pitfalls encountered in the assessment on the cause of the bilateral frontal and interhemispheric subdural hematomas in this child are also briefly discussed. We have called this condition "benign" shaken baby syndrome and emphasize that not always acute subdural hematomas are of non-accidental nature

Los autores describen un lactante con hallazgos clínicos y de neuroimagen sugestivos del síndrome del niño maltratado. Se discuten brevemente las dificultades para la identificación de la causa del hematomasubdural agudo frontal bilateral e interhemisférico que el niño presentaba. Hemos denominado a esta entidad síndrome "benigno" del niño maltratado para destacar que el hallazgo de un hematoma subdural agudo en un niño pequeño no significa necesariamente maltrato infantil

GABAergic phenotypic differentiation of a subpopulation of subventricular derived migrating progenitors.

Olfactory bulb interneurons are continuously generated throughout development and in adulthood. These neurons are born in the subventricular zone (SVZ) and migrate along the rostral migratory stream into the olfactory bulb where they differentiate into local interneurons. To investigate the differentiation of GABAergic interneurons of the olfactory bulb we used a transgenic mouse which expresses green fluorescent protein (GFP) under the control of the glutamic acid decarboxylase 65 kDa (GAD65) promoter. During development and in adulthood GFP was expressed by cells in the SVZ and along the entire length of its rostral extension including the distal portion within the olfactory bulb. The occurrence of GAD65 mRNA in these zones was confirmed by PCR analysis on microdissected regions along the pathway. Polysialic acid neural cell adhesion molecule, a marker of migrating neuroblasts in adults, was coexpressed by the majority of the GFP-positive SVZ-derived progenitor cells. Cell tracer injections into the SVZ indicated that approximately 26% of migrating progenitor cells expressed GFP. These data show the early differentiation of migrating SVZ-derived progenitors into a GAD65-GFP-positive phenotype. These cells could represent a restricted lineage giving rise to GAD65-positive GABAergic olfactory bulb interneurons.

Centre-surround inhibition among olfactory bulb glomeruli.

Centre-surround inhibition--the suppression of activity of neighbouring cells by a central group of neurons--is a fundamental mechanism that increases contrast in patterned sensory processing. The initial stage of neural processing in olfaction occurs in olfactory bulb glomeruli, but evidence for functional interactions between glomeruli is fragmentary. Here we show that the so-called 'short axon' cells, contrary to their name, send interglomerular axons over long distances to form excitatory synapses with inhibitory periglomerular neurons up to 20-30 glomeruli away. Interglomerular excitation of these periglomerular cells potently inhibits mitral cells and forms an on-centre, off-surround circuit. This interglomerular centre-surround inhibitory network, along with the well-established mitral-granule-mitral inhibitory circuit, forms a serial, two-stage inhibitory circuit that could enhance spatiotemporal responses to odours.

Unique neuronal tracers show migration and differentiation of SVZ progenitors in organotypic slices.

Continual neurogenesis in the subventricular zone (SVZ) of postnatal and adult mammalian forebrain has been well documented, but the mechanisms underlying cell migration and differentiation in this region are poorly understood. We have developed novel in vivo and in vitro methods to investigate these processes. Using stereotaxic injections of a variety of tracers/tracker [Cholera Toxin beta subunit (CTb-), Fluorogold (FG), and Cell Tracker Green (CTG)], we could efficiently label SVZ cells. Over several days, labeled cells migrate along the rostral migratory stream (RMS) to their final differentiation site in the olfactory bulb (OB). The compatibility of these tracers/trackers with immunohistochemistry allows for cell labeling with multiple dyes (e.g., CTb and CTG) and/or specific cell antigens. To investigate the dynamics of migration we labeled SVZ progenitor cells with small injections of CTG and monitored the movements of individual cells in fresh parasagittal brain slices over several hours using time-lapse confocal microscopy. Our observations suggest that tangential cell migration along the RMS occurs more rapidly than radial cell migration into the OB granule cell layer. To investigate migration over longer time periods, we developed an in vitro organotypic slice in which labeled SVZ progenitors migrate along the RMS and differentiate within the OB. The phenotypic characteristics of these cells in vitro were equivalent to those observed in vivo. Taken together, these methods provide useful tools investigating cell migration and differentiation in a preparation that maintains the anatomical organization of the RMS.

Radial glia development in the mouse olfactory bulb.

Radial glia are critical for cell migration and lamination of the cortex. In most developing cortical structures, radial glia, as their name suggests, extend processes from the ventricle to the pia in regular parallel arrangements. However, immunohistochemical labeling from several laboratories suggests that radial glia have a more branched morphology in the olfactory bulb. To investigate the morphology of radial glia in the mouse olfactory bulb we (1) labeled radial glia and olfactory receptor neuron axons at 24-hour intervals by immunohistochemistry; and (2) developed a novel method of generating and applying "nanocrystals" of 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) to the ventricle surface such that the processes of single olfactory bulb radial glia are labeled in the embryonic olfactory bulb. We examined the structure and interactions of radial glia with ingrowing olfactory receptor neuron (ORN) axons in late embryonic olfactory bulb development. These results showed that olfactory bulb radial glia do not form straight parallel structures as do radial glia in the neocortex but rather have a convoluted trajectory from the ventricle to the bulb surface. Moreover, olfactory bulb radial glia consistently extend tangential branches at the level of the internal plexiform layer. Beginning at embryonic day 17.5, two types of radial glia can be distinguished: type I radial glia have a process that extends from the ventricle into the glomerular layer. These apical processes form highly restricted tufts, or "glial glomeruli" at the same time that ORN axons are forming "axonal glomeruli." In type II radial glia the apical process does not enter the glomerular layer but instead ramifies within the external plexiform layer. The tight spatiotemporal relationship between the glomerulization of radial glia processes and ORN axons during development suggest that radial glia processes could play a role in the formation and/or stabilization of mammalian glomeruli.

Expression of semaphorins in developing and regenerating olfactory epithelium.

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration.

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10(-11) M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.