Neuroscience Institute of the Cavalieri Ottolenghi Foundation Regione Gonzole 10, 10046 Orbassano (Torino) - Italy Dept Anatomy, Pharmacology and Forensic Medicine Corso M. D'Azeglio 52 10126 Torino - Italy tel +39-011-6707700/6617
Abstract: Von Economo's neurons (VENs) are large, bipolar or corkscrew-shaped neurons located in layers III and V of the frontoinsular and the anterior cingulate cortices. VENs are reported to be altered in pathologies such as frontotemporal dementia and autism, in which the individual's self control is seriously compromised. To investigate the role of VENs in the active human brain, we have explored the functional connectivity of brain areas containing VENs by analyzing resting state functional connectivity (rsFC) in 20 healthy volunteers. Our results show that cortical areas containing VENs form a network of frontoparietal functional connectivity. With the use of fuzzy clustering techniques, we find that this network comprises four sub-networks: the first network cluster resembles a "saliency detection" attentional network, which includes superior frontal cortex (Brodmann's Area, BA 10), inferior parietal lobe, anterior insula, and dorsal anterior cingulate cortex; the second cluster, part of a "sensory-motor network", comprises the superior temporal, precentral and postcentral areas; the third cluster consists of frontal ventromedial and ventrodorsal areas constituted by parts of the "anterior default mode network"; and the fourth cluster encompasses dorsal anterior cingulate cortex, dorsomedial prefrontal, and superior frontal (BA 10) areas, resembling the anterior part of the "dorsal attentional network". Thus, the network that emerges from analyzing functional connectivity among areas that are known to contain VENs is primarily involved in functions of saliency detection and self-regulation. In addition, parts of this network constitute sub-networks that partially overlap with the default mode, the sensory-motor and the dorsal attentional networks.
Abstract: Kainic acid (KA) induced seizures provokes an extensive neuronal degeneration initiated by c-Jun N-terminal kinases (JNK) as central mediators of excitotoxicity. However, the actions of their individual isoforms in cellular organelles including mitochondria remain to be elucidated. Here, we have studied the activation of JNK1, JNK2 and JNK3 and their activators, mitogen-activated protein kinase kinase (MKK) 4/7, in brain mitochondria, cytosolic and nuclear fractions after KA seizures. In the mitochondrial fraction, KA significantly increased the presence of JNK1, JNK3 and MKK4 and stimulated their phosphorylation i.e. activation. The pro-apoptotic proteins, Bim and Bax were induced and, consequently, the ratio Bcl-2-Bax decreased. These changes were paralleled by the release of cytochrome c and cleavage of poly(ADP-ribose)-polymerase (PARP). The JNK peptide inhibitor, D-JNKI-1 (XG-102) reversed these pathological events in the mitochondria and almost completely abolished cytochrome c release and PARP cleavage. Importantly, JNK3, but not JNK1 or JNK2, was associated with Bim in mitochondria and D-JNKI-1 prevented the formation of this apoptotic complex. Apart from of the attenuation of c-Jun phosphorylation in the nucleus, D-JNKI-1 did not affect the level of JNK3 isoform in the nuclear and cytosolic fractions. These findings provide novel insights into the mode of action of individual JNK isoforms in cell organelles and points to the JNK3 pool in mitochondria as a target of the JNK inhibitor D-JNKI-1 to confer neuroprotection.
Abstract: Acute primary open angle glaucoma is an optic neuropathy characterized by the elevation of intraocular pressure, which causes retinal ischemia and neuronal death. Rat ischemia/reperfusion enhances endocytosis of both horseradish peroxidase (HRP) or fluorescent dextran into ganglion cell layer (GCL) neurons 24 h after the insult. We investigated the activation of autophagy in GCL-neurons following ischemia/reperfusion, using acid phosphatase (AP) histochemistry and immunofluorescence against LC3 and LAMP1. Retinal I/R lead to the appearance of AP-positive granules and LAMP1-positive vesicles 12 and 24 h after the insult, and LC3 labelling at 24 h, and induced a consistent retinal neuron death. At 48 h the retina was negative for autophagic markers. In addition, Western Blot analysis revealed an increase of LC3 levels after damage: the increase in the conjugated, LC3-II isoform is suggestive of autophagic activity. Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion. The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes. Treatment also prevented the increase in glial fibrillary acid protein immunoreactivity elicited by I/R. Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.
Abstract: The human insula is hidden in the depth of the cerebral hemisphere by the overlying frontal and temporal opercula, and consists of three cytoarchitectonically distinct regions: the anterior agranular area, posterior granular area, and the transitional dysgranular zone; each has distinct histochemical staining patterns and specific connectivity. Even though there are several studies reporting the functional connectivity of the insula with the cingulated cortex, its relationships with other brain areas remain elusive in humans. Therefore, we decided to use resting state functional connectivity to elucidate in details its connectivity, in terms of cortical and subcortical areas, and also of lateralization. We investigated correlations in BOLD fluctuations between specific regions of interest of the insula and other brain areas of right-handed healthy volunteers, on both sides of the brain. Our findings document two major complementary networks involving the ventral-anterior and dorsal-posterior insula: one network links the anterior insula to the middle and inferior temporal cortex and anterior cingulate cortex, and is primarily related to limbic regions which play a role in emotional aspects; the second links the middle-posterior insula to premotor, sensorimotor, supplementary motor and middle-posterior cingulate cortices, indicating a role for the insula in sensorimotor integration. The clear bipartition of the insula was confirmed by negative correlation analysis. Correlation maps are partially lateralized: the salience network, related to the ventral anterior insula, displays stronger connections with the anterior cingulate cortex on the right side, and with the frontal cortex on the left side; the posterior network has stronger connections with the superior temporal cortex and the occipital cortex on the right side. These results are in agreement with connectivity studies in primates, and support the use of resting state functional analysis to investigate connectivity in the living human brain.
Abstract: Spinal cord injury (SCI) often results in irreversible and permanent neurological deficits below the injury site and is considered a pathological state of functional damage to local neurons and axon fibers. There are several experimental treatments to minimize tissue damage, and recently cell transplantation has emerged as a promising approach in spinal cord repair. The authors undertook this study to evaluate grafting of neural tube precursors as a possible therapeutic strategy in a model of spinal cord compression in the mouse.
Abstract: Inflammation protects from dangerous stimuli, restoring normal tissue homeostasis. Inflammatory response in the nervous system ("neuroinflammation") has distinct features, which are shared in several diseases. The retina is an immune-privileged site, and the tight balance of immune reaction can be disrupted and lead to age-related macular disease (AMD) and to its peculiar sign, the druse. Excessive activation of inflammatory and immunological cascade with subsequent induction of damage, persistent activation of resident immune cells, accumulation of byproducts that exceeds the normal capacity of clearance giving origin to a chronic local inflammation, alterations in the activation of the complement system, infiltration of macrophages, T-lymphocytes and mast-cells from the bloodstream, participate in the mechanisms which originate the drusen. In addition, aging of the retina and AMD involve also para-inflammation, by which immune cells react to persistent stressful stimuli generating low-grade inflammation, aimed at restoring function and maintaining tissue homeostasis by varying the set point in relation to the new altered conditions. This mechanism is also seen in the normal aging retina, but, in the presence of noxious stimuli as in AMD, it can become chronic and have an adverse outcome. Finally, autophagy may provide new insights to understand AMD pathology, due to its contribution in the removal of defective proteins. Therefore, the AMD retina can represent a valuable model to study neuroinflammation, its mechanisms and therapy in a restricted and controllable environment. Targeting these pathways could represent a new way to treat and prevent both exudative and dry forms of AMD.
Abstract: A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/- C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.
Abstract: Low oxygen concentrations (hypoxia) occur in several physiological and pathological cellular situations such as embryogenesis and stem cell modulation (in terms of differentiation/proliferation), or ischemic stroke and cancer. On the other side of the coin, the generation of reactive oxygen species (ROS) is tightly controlled by the cell. ROS control redox sensitive signaling pathways and thus regulate cell physiology, such as programmed cell death, inflammation and/or stem cell modulation. Herein we analyze the role of hypoxia and ROS in the modulation of neuronal differentiation focusing on: (i) in vivo neurogenesis and (ii) in vitro neuronal differentiation from neural stem/precursor cells. In vivo, hypoxia promotes neurogenesis in embryos, newborns and adults, as well as in response to noxious stimuli such as ischemia. On the other hand, oxygen and ROS also play a role in in vitro neuronal differentiation. They further impact tumor growth by influencing cell proliferation and differentiation, such as in neuroblastoma development. Therefore, manipulating hypoxia and ROS production represents a useful therapeutic tool if one needs either to enhance or to modulate neurogenesis and neuronal differentiation, such as in cell replacement or in malignant cell proliferation.
Abstract: Myelin formation and maintenance are crucial for the proper function of the CNS and are orchestrated by a plethora of factors including growth factors, extracellular matrix components, metalloproteases and protease inhibitors. Hemopexin (Hx) is a plasma protein with high heme binding affinity, which is also locally produced in the CNS by ependymal cells, neurons and glial cells. We have recently reported that oligodendrocytes (OLs) are the type of cells in the brain that are most susceptible to lack of Hx, as the number of iron-overloaded OLs increases in Hx-null brain, leading to oxidative tissue damage. In the current study, we found that the expression of the Myelin Basic Protein along with the density of myelinated fibers in the basal ganglia and in the motor and somatosensory cortex of Hx-null mice were strongly reduced starting at 2 months and progressively decreased with age. Myelin abnormalities were confirmed by electron microscopy and, at the functional level, resulted in the inability of Hx-null mice to perform efficiently on the Rotarod. It is likely that the poor myelination in the brain of Hx-null mice was a consequence of defective maturation of OLs as we demonstrated that the number of mature OLs was significantly reduced in mutant mice whereas that of precursor cells was normal. Finally, in vitro experiments showed that Hx promotes OL differentiation. Thus, Hx may be considered a novel OL differentiation factor and the modulation of its expression in CNS may be an important factor in the pathogenesis of human neurodegenerative disorders.
Abstract: Recent evidence suggests that adult bone marrow stem cells reduce tissue damage and promote repair following CNS ischemic injury. Since granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells to the circulating compartment, here we tested whether administration of this drug modifies the outcome of a permanent occlusion of the middle cerebral artery in adult mice. To elucidate the behavior and fate of blood-borne cells in the ischemic brain, we produced chimeric animals, in which hematopoietic derivatives are genetically tagged. G-CSF administration enhances the proliferation of microglia in the uninjured CNS but has no effect on the amount of hematopoietic cells that infiltrate the ischemic tissue and on the size of the lesion. The blood-borne elements acquire different mesodermal identities but fail to adopt neural phenotypes, even though they occasionally fuse with Purkinje neurons. These results indicate that G-CSF treatment does not exert a significant beneficial effect on the ischemic injury.
Abstract: Systemic injections of kainic acid (KA) cause epileptic seizures with delayed neuronal damage in the limbic system, particularly in the hippocampus. KA excitotoxicity activates complex signal transduction events that trigger apoptotic cell death. The c-Jun N-terminal kinase (JNK) pathway plays an important role in cell death, and the peptide D-JNKI1, a competitive JNK inhibitor, is a potent neuroprotective agent. To analyse the role of JNK and the effects of D-JNKI1 administration on excitotoxic neuronal death, we induced epileptic seizures by intraperitoneal (i.p.) injection of KA in adult male Sprague-Dawley rats; a group of rats received i.p. D-JNKI1 2 h after KA. KA caused massive cell death in the hippocampus: in Nissl-stained sections, stereological counts showed a significant decrease in neuronal density in all CA fields, both at 1 and 5 days after seizures, which was partially prevented by D-JNKI1 treatment. These results were confirmed by counts of degenerating neurons in CA3 in FluoroJade B-stained sections. Seizure activity also induced marked gliosis as observed with glial fibrillary acidic protein (GFAP) immunohistochemistry. We also analysed c-Jun activation as a target of JNK and central transcriptional effector in the adult rat brain following KA injection. Phospho-c-Jun immunoreactivity was absent in the hippocampus of untreated animals, whereas strong nuclear neuronal labeling could be observed, starting from 3 h after KA administration, in microtubule-associated protein-2-positive neurons but not in GFAP-positive astrocytes. D-JNKI1 treatment also reduced the positivity for phospho-c-Jun in the hippocampus, thus confirming the specificity of the peptide in blocking JNK. Therefore, JNK is a promising target for blocking seizure-induced cell death.
Abstract: Even though several studies highlighted the role of maternal thyroid hormones (THs) during embryo-foetal development, direct evidence of their interaction with embryonic thyroid receptors (TRs) is still lacking. We generated a transgenic mouse model ubiquitously expressing a reporter gene tracing TH action during development. We engineered a construct (TRE2×) containing two TH-responsive elements controlling the expression of the LacZ reporter gene, which encodes β-galactosidase (β-gal). The specificity of the TRE2× activation by TH was evaluated in NIH3T3 cells by cotransfecting TRE2× along with TRs, retinoic or oestrogen receptors in the presence of their specific ligands. TRE2× transgene was microinjected into the zygotes, implanted in pseudopregnant BDF1 (a first-generation (F1) hybrid from a cross of C57BL/6 female and a DBA/2 male) mice and transgenic mouse models were developed. β-gal expression was assayed in tissue sections of transgenic mouse embryos at different stages of development. In vitro, TRE2× transactivation was observed only following physiological T3 stimulation, mediated exclusively by TRs. In vivo, β-gal staining, absent until embryonic day 9.5-10.5 (E9.5-E10.5), was observed as early as E11.5-E12.5 in different primordia (i.e. central nervous system, sense organs, intestine, etc.) of the TRE2× transgenic embryos, while the foetal thyroid function (FTF) was still inactive. Immunohistochemistry for TRs essentially colocalized with β-gal staining. No β-gal staining was detected in embryos of hypothyroid transgenic mice. Importantly, treatment with T3 in hypothyroid TRE2× transgenic mice rescued β-gal expression. Our results provide in vivo direct evidence that during embryonic life and before the onset of FTF, maternal THs are transcriptionally active through the action of embryonic TRs. This model may have clinical relevance and may be employed to design end-point assays for new molecules affecting THs action.
Abstract: THE SEARCH FOR THE FUNDAMENTAL BUILDING BLOCK OF THE CEREBRAL CORTEX HAS HIGHLIGHTED THREE STRUCTURES, PERPENDICULAR TO THE CORTICAL SURFACE: (i) columns of neurons with radially invariant response properties, e.g., receptive field position, sensory modality, stimulus orientation or direction, frequency tuning etc., (ii) minicolumns of radially aligned cell bodies and (iii) bundles, constituted by the apical dendrites of pyramidal neurons with cell bodies in different layers. The latter were described in detail, and sometimes quantitatively, in several species and areas. It was recently suggested that the dendritic bundles consist of apical dendrites belonging to neurons projecting their axons to specific targets. We review the concept above and suggest that another structural and computational unit of cerebral cortex is the cortical output unit, i.e., an assembly of bundles of apical dendrites and their parent cell bodies including each of the outputs to distant cortical or subcortical structures, of a given cortical locus (area or part of an area). This somato-dendritic assembly receives inputs some of which are common to the whole assembly and determine its radially invariant response properties, others are specific to one or more dendritic bundles, and determine the specific response signature of neurons in the different cortical layers and projecting to different targets.
Abstract: Type A Niemann-Pick is a severe neurological disease, caused by a mutation of the gene of acid sphingomyelinase (ASM) and reduced enzyme activity. Some studies reported neuropathological changes occurring in the central nervous system of ASM deficient transgenic (ASMKO) mice, while a detailed study on the peripheral nervous system (PNS) at different ages is currently lacking. The aim of our study was to examine the pathological changes occurring in the PNS and in the spinal cord in an AMSKO model of Niemann-Pick disease (NPD) Type A.
Abstract: Spinal cord injury (SCI) often results in permanent neurological deficits below the injury site. Serotonergic raphespinal projections promote functional recovery after SCI, but spontaneous regeneration of most severed axons is limited by the glial cyst and scar that form at the lesion site. Stem cell (SC) transplantation offers a promising approach for inducing regeneration through the damaged area. Here we compare the effects of transplantation of embryonic neural precursors (NPs) or adult mesenchymal SCs, both of which are potential candidates for SC therapy. The spinal cord was hemisected at the L2 neuromer in adult mice. Two weeks post-injury, we transplanted neural precursors or mesenchymal SCs into the cord, caudal to the hemisection. Injured mice without a graft served as controls. Mice were tested for functional recovery on a battery of motor tasks, then killed and analysed for survival of grafted cells, for effects of engraftment on the local cellular environment and for the sprouting of serotonergic axons. Both types of SCs survived and were integrated into the host tissue, but only the NPs expressed neuronal markers. All transplanted animals displayed an increased number of serotonin-positive fibres caudal to the hemisection, compared with untreated mice. And both cell types led to improved motor performance. These results point to a therapeutic potential for such cell grafting.
Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating incurable neurodegenerative disease that targets motor neurons, manifesting as a linear decline in muscular function and leading to death within 2 - 5 years of diagnosis. The vast majority of ALS cases are sporadic, the aetiopathology of which is incompletely understood. Recent data have implicated the microenvironment of the motor neuron as a primary target of the pathophysiology. Any experimental therapeutic approach to ALS is very difficult because of some peculiarities of the disease, such as the unknown origin, the spatial diffusion of motor neuron loss and the paucity of animal models. Despite such daunting challenges, in experimental models a number of potential benefits of stem cells in ALS therapy have been demonstrated: by providing non-compromised supporting cells such as astrocytes, microglia or growth factor-excreting cells, onset can be delayed and survival increased. Moreover, in animal models of acute or chronic motor neuron injury, neural stem cells implanted into the spinal cord have been shown to differentiate into motor neurons, with some evidence of axonal sprouting and formation of nerumuscular junctions with host muscle. Here we summarise and discuss current preclinical and clinical evidence regarding stem cells application in ALS, particularly focusing on methodological issues.
Abstract: Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer's disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer's disease and Parkinson's disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu-Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.
Abstract: Citron kinase (CIT-K), a ser/thr kinase, is required during neurogenesis for cytokinesis of neuronal precursors. Deletion of the cit-k gene in mice (cit-k(-/-) mice) leads to a severe malformative central nervous system syndrome characterized by microencephaly, ataxia, and epileptic seizures; affected mice die by the third week of postnatal life. We have used NADPH-diaphorase histochemistry, immunostaining for calbindin, calretinin, parvalbumin, and glutamic acid decarboxylase 67 (GAD67), and histological staining to undertake qualitative and quantitative analyses of the morphology and distribution of interneurons in the barrelfield cortex of cit-k(-/-) mice. By postnatal day 13, lack of CIT-K results in profoundly altered cortical cell morphology: the infragranular layers are populated by large, binucleate interneurons bearing many more dendrites than in control mice, an anatomical profile that has also been reported for the cortex of humans with cortical dysplasias and epilepsy. Tessellation analyses reveal that a clustered distribution of interneurons is maintained in cit-k(-/-) mice, but that their nearest neighbor distance is significantly increased, and thus their density is reduced; the overall number of interneurons is more dramatically decreased in the absence of CIT-K than would be predicted on the basis of the reduced brain size of affected mice. This reduction of inhibitory gamma-aminobutyric acid (GABA)ergic neurons likely underlies the occurrence of epileptic seizures in the cit-k(-/-) mice. Furthermore, the altered distribution of NADPH-diaphorase-positive interneurons could be responsible for an impaired coupling of cortical activity to blood flow, also affecting cortical growth and functioning.
Abstract: STAT3 is a pleiotropic factor activated by many different signals including cytokines, growth factors and oncogenes. It is involved in a striking number of functions and can activate distinct repertoires of genes in different contexts. Like other STAT factors, STAT3 exists in two isoforms generated by alternative splicing, the full length STAT3alpha and the truncated STAT3beta, generally thought to act as a dominant negative factor. However, STAT3beta is not transcriptionally inactive and is able to both activate and repress genes depending on cellular environment. These unique properties of the STAT3beta isoform may contribute to the extraordinary functional complexity of STAT3 physiological and pathological actions, revealed by conditional mutagenesis studies and not yet fully understood. With this in mind, we try here to summarize what is known about the structure and function of the alpha and beta STAT3 isoforms, both in vitro and in vivo. In addition, we report unpublished data describing the phenotype of mice where the STAT3alpha isoform was specifically ablated.
Abstract: Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.
Abstract: Dendritic spines are highly dynamic protuberances that are thought to be crucial for learning and memory. Although it is well known that actin filaments and membrane dynamics regulate spine plasticity, how these two events are linked locally is less clear. Here, we provide evidence that Citron-N (CIT-N), a binding partner of the small GTPase RhoA, is associated with the actin filaments and Golgi compartments of dendritic spines. We also show that CIT-N is required for recruiting F-actin and Golgi membranes at spines of in vitro-grown neurons. Studies in knockout mice show that this protein is essential for the maturation of dendritic spines. We suggest that CIT-N might function as a scaffold protein in spine organization through its ability to bind to Golgi membranes and by affecting actin remodelling.
Abstract: The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.
Abstract: Giuseppe Levi (1872-1965), Professor of Anatomy at the University of Turin, had broad research interests and was a pioneer of in vitro studies on cultured cells. He provided a number of contributions on the nervous system, especially on the plasticity of sensory ganglion cells. An influential and magnetic teacher and mentor, he gathered around him a large group of brilliant students. He has the peculiar primate to count among his students three Nobel laureates in Physiology or Medicine: Salvador Luria, Renato Dulbecco, and Rita Levi-Montalcini. For all three of them, the internship in Levi's laboratory provided an exceptional initial stimulus. They remained in close contact with each other and with Levi even after the 1940s when they migrated to the United States for political and racial reasons, engaging in different fields of research. Rita Levi-Montalcini, who was awarded the Nobel Prize (1986) for the discovery of Nerve Growth Factor, was stimulated and assisted in her work by Giuseppe Levi during the difficult years of World War II. With Giuseppe Levi, she pursued early studies on the relationships between neural centers and their peripheral target of innervation, and she has witnessed in her writings the enthusiasm of her mentor.
Abstract: OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. MATERIALS AND METHODS: We tested hMSCs in three previously reported experimental conditions made of alpha-modified minimum essential medium (alpha-MEM)/1 mM beta-mercaptoethanol (betaME), 10 microM alpha-MEM/retinoic acid (RA) or alpha-MEM/2% dimethylsulfoxide (DMSO) + 200 microM beta-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). RESULTS: hMSCs were isolated from bone marrow and expanded for several passages. In betaME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K+ currents related to two ether-Ã -go-go K+ channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K+ channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. CONCLUSION: In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.
Abstract: Thanks to advances in the stem cell biology of the central nervous system, the previously unconceivable regeneration of the damaged spinal cord is approaching reality. A number of potential strategies aim to optimize functional recovery after spinal cord injury. They include minimizing the progression of secondary injury, manipulating the inhibitory environment of the spinal cord, replacing lost tissue with transplanted cells or peripheral nerve grafts, remyelinating denuded axons and maximizing the intrinsic regenerative potential of endogenous progenitor cells. We review the application of stem cell transplantation to the spinal cord, emphasizing the use of embryonic stem cells for remyelinating damaged axons. Recent advancements in neural injury and repair, and the progress towards development of neuroprotective and regenerative interventions are discussed.
Abstract: Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D'Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreER(T2) (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreER(T2) recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreER(T2) transgenic lines after tamoxifen mediated-CreER(T2) translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreER(T2) oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.
Abstract: AIM: To investigate the effects of troglitazone (TGZ), an anti-diabetic drug which activates peroxisome proliferator-activated receptor-gamma (PPAR-gamma), for liver tissue repair, and the development of ductular reaction, following common bile duct ligation (BDL) in rats. METHODS: Rats were supplemented with TGZ (0.2% w/w in the pelleted food) for 1 wk before BDL or sham operation. Animals were killed at 1, 2, or 4 wk after surgery. RESULTS: The development of liver fibrosis was reduced in rats receiving TGZ, as indicated by significant decreases of procollagen type I gene expression and liver hydroxy-proline levels. Accumulation of alpha-smooth-muscle actin (SMA)-expressing cells surrounding newly formed bile ducts following BDL, as well as total hepatic levels of SMA were partially inhibited by TGZ treatment, indicating the presence of a reduced number and/or activation of hepatic stellate cells (HSC) and myofibroblasts. Development of the ductular reaction was inhibited by TGZ, as indicated by histochemical evaluation and hepatic activity of gamma-glutamyl-transferase (GGT). CONCLUSION: Treatment with thiazolidinedione reduces ductular proliferation and fibrosis in a model of chronic cholestasis, and suggests that limiting cholangiocyte proliferation may contribute to the lower development of scarring in this system.
Abstract: Beta-nicotinamidedinucleotide phosphate diaphorase (NADPH-d) colocalizes with NOS in the central nervous system. Two types of NADPH-d-positive neurons are present in the primate cerebral cortex: type 1, intensely and Golgi-like labeled neurons, a subset of GABAergic interneurons; type 2, lightly labeled neurons (divided into two subclasses, a first one having a lightly stained cell body bearing only one short process, and a second one showing intense NADPH-d staining with short processes extending radially). We have analyzed the distribution of NADPH-d activity in human frontal, temporal, and occipital cortical areas, finding remarkable laminar and interareal differences in cell size and distribution of the different cell types. There was a clear bias for type 1 neurons in infragranular layers in all areas considered; both in supra- and infragranular layers, their density was highest in frontal, and lowest in temporal cortex. The density of type 2 neurons was lower supragranularly in temporal cortex and infragranularly in occipital cortex. The overall density of type 2 cells was remarkably higher in occipital cortex than in the temporal and frontal ones. Type 1 neurons were significantly larger than type 2, and were smaller in the supragranular than in the infragranular subzone in occipital and temporal cortex. Type 1 cells were significantly larger in frontal cortex than in occipital and temporal cortex, and type 2 cells were significantly smaller in occipital than in temporal and frontal cortex. These area-related differences might reflect differences between heterotypic and homotypic cortex in the regulation of cortical blood flow.
Abstract: NADPH-diaphorase (NADPH-d) histochemistry labels a subpopulation of nitric oxide-synthesizing amacrine cells in the inner nuclear layer of the rat retina. We have studied their morphology and distribution in postnatal and adult rats in whole-mounted retinae. NAPDH-d-positive neurons are detected as early as postnatal day (P)5, especially in the peripheral retina; intense labeling of somata and long lengths of dendrites is obtained between P10 and P18, after which only the somata exhibit NADPH-d activity. The density and number of these cells increase progressively from P7 to P14, with a significantly higher density in the central retina as compared to the periphery. The sociology of these cells was analyzed quantitatively studying the Voronoi domains: a polygon area can be drawn that delineates the territory of the map that is closer to the cell than to any other cell of the map. In addition, we calculated the conformity ratio of Cook, i.e., the mean nearest neighbor distance/standard deviation of all the nearest neighbor distances, in order to reveal whether or not these cells are regularly distributed through the retina. We find that the distribution of the NADPH-d-positive cells tends to be regular throughout the retina: the local coefficient of variation (obtained by comparing the size of each Voronoi polygon area to those of its neighbors) tends to regularity at P14 and remains unaltered through maturity. Therefore, as other cell types, NADPH-d-positive amacrine cells are almost regularly distributed from the time of eye opening and nitric oxide may play a role in the development of retinal circuitry and in the regulation of retinal blood flow.
Abstract: The apical dendrites of the pyramidal neurons of the cerebral cortex form radial bundles in all species and areas. Using microtubule-associated protein (MAP)2 immunostaining and Voronoi tessellation analysis in the rat visual cortex, we obtained objective criteria to define dendritic bundles in tangential sections: in supragranular layers of the rat visual cortex we found bundles of 6-6.4 dendrites, at a density of 1929 bundles/mm(2) and a centre-to-centre distance of 27 micro m. Using lipophilic tracers to label different pyramidal cell populations, based on the same criteria as in MAP2-immunostained material, we found that in the rat visual cortex the bundles consist of neurons with specific targets. Neurons projecting to the ipsi- or contralateral cortex form bundles together and with neurons projecting to the striatum, but not with those projecting to the superior colliculus, dorsal division of the lateral geniculate nucleus or through the cerebral peduncle. The latter neurons form bundles with neurons projecting to the striatum. Thus, the cerebral cortex is organized in minicolumns of output neurons visible at the earliest ages studied (P3), which might have a higher probability of being interconnected than those outside.
Abstract: Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.
Abstract: Small GTPases of the rho family regulate the extensive rearrangements of the cytoskeleton that characterize neuronal differentiation. Citron kinase is a target molecule for activated rhoA, previously implicated in control of cytokinesis. We have found that, in addition, it could play an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, we showed that citron kinase is involved in the morphologic differentiation of N1E-115 neuroblastoma cells induced by serum starvation. More importantly, quantitative analysis of citron kinase knockout cerebral cortex displayed that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons.
Abstract: We investigated the molecular mechanisms of cell death in the dorsal lateral geniculate nucleus of the rat, following suction lesion of the visual cortex at birth or in the third postnatal week, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique and immunohistochemistry for caspase-3, -7, -8, and cleaved poly(ADP-ribose) polymerase.Following lesion at birth, TUNEL-positive neurons were found in the dorsal lateral geniculate nucleus between 24 h and 3 days after lesion, with a peak on the second day. Shorter survival times (12-18 h) resulted in labeling of very few neurons in dorsal lateral geniculate nucleus and of several neurons in the perilesional cortex. Activated caspase-3 was expressed from the first to the third days after lesion, whereas cleaved poly(ADP-ribose) polymerase and activated caspase-8 were expressed on the second and third day. Activated caspase-7 was expressed mainly in pretectal nuclei. Caspase-3 activation coincided with the appearance of TUNEL-positive profiles, but decreased earlier than TUNEL. In the ipsi- and contralateral cerebral cortex, all parameters were unchanged. In animals lesioned in the third week, rare apoptotic thalamic neurons were detected as TUNEL- and activated caspase-3-positive profiles 2 days after cortical ablation, and were still present 1 week after lesion.Thus, early target ablation has dramatic effects on neonatal thalamic neurons, which die following activation of caspases 3 and 8. In contrast, cortical neurons are relatively unaffected by target deprivation. Compared with early lesions, late lesions induce a limited thalamic cell death, that persists over time.
Abstract: Genetic and epigenetic factors may alter the normal development of cerebral cortex, producing laminar and cellular abnormalities and heterotopiae, major causes of juvenile, drug-resistant epilepsy. Experimentally-induced migration disorders provide interesting insights in the mechanisms of the determination of neuronal phenotype and connectivity, of congenital cortical dysgenesis and the pathophysiology of associated neurological disorders, such as epilepsy. We investigated the effects of E14 administration of methylazoxymethanol acetate (MAM), which induces microencephaly by ablating dividing cells. Brains from newborn and adult rats were reacted for NADPH-d and CO histochemistry. Moreover, callosally-projecting neurons were retrogradely labeled with DiI at P9 or with BDA in adults. MAM-treated rats displayed a remarkable reduction in cortical thickness, mainly due to reduction in layer IV and in supragranular layers. Heterotopic nodules appeared in the supragranular layers and in the hippocampus. CO-positive barrels in somatosensory cortex were almost absent. The distribution of NADPH-d-positive neurons was regular, but they were rare in heterotopic nodules. Callosally-projecting neurons displayed abnormal orientation of the apical dendrite and increase in the basal dendritic length. Alterations in the dendritic arborization of pyramidal neurons may be one of the substrates for the increased sensitivity to drugs which induce epileptic seizures in these animals.
Abstract: The apical dendrites of pyramidal neurons in the cerebral cortex form vertical bundles whose distribution and density vary across species and areas. To understand their relationships with cortical columns, we labeled retrogradely neurons from the white matter underlying the visual cortex with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) at P3 and P10 and with biotinylated dextran amine at P30. We also mapped the distribution of apical dendrites in tangential sections, immunostained for microtubule-associated proteins (MAP2). Their composition and distribution were studied with Neurolucida and NeuroExplorer software. The apical dendrites of pyramidal neurons formed different bundle types: at P3 we found bundles formed (a) by neurons located in cortical plate; (b) by layer V neurons; and (c) by upper layer V neurons and cortical plate neurons. At P10, the amount of supragranular neurons participating in the bundles increased. The inter-dendritic and inter-bundle distances increased with age. These findings confirm that dendritic bundles are present in the rat visual cortex early in development and are formed by neurons belonging to different cortical layers. The existence of different types of bundles relative to the layer of location of their parent neurons suggests that they are heterogeneous from each other in nature and in the pattern of connectivity.
Abstract: In the spinal cord, nitric oxide pathways are involved in hyperalgesia, and nitric oxide synthase, the enzyme responsible for its synthesis, is upregulated following several noxious and lesion stimuli. Since the histochemical reaction for NADPH-diaphorase colocalizes with NOS, we decided to study the effects of infusion of bacterial lipopolysaccharides close to the sciatic nerve on the expression of NADPH-d in the dorsal root ganglia and spinal cord of the rat. The percentage of NADPH-d-positive neurons in the L4 dorsal root ganglia increased 7-10 times on the treated side of LPS-treated rats (12.5-17.5%, compared to 0.5-2.5% of control side), whereas sham operation had no effects. The cross-sectional area of NADPH-d-positive neuronal profiles in all the dorsal root ganglia considered was consistently smaller than that of those which were negative to the histochemical reaction. In animals treated with LPS the NADPH-d-positive neurons were significantly (p = 0.02) smaller on the treated side (520 +/- 100 microns) than on the control one (679 +/- 135 microns), whereas those which were negative were of similar sizes on the two sides (1170 +/- 256 microns on the treated side vs 1214 +/- 371 microns on the control side). On the contrary, in control animals, there were no differences between untreated and sham operated sides, but differences between the sizes of NADPH-d-positive and negative neurons persisted. Therefore, LPS treatment on the sciatic nerve upregulates NADPH-d expression in the corresponding dorsal root ganglion, thus indicating an increased rate of NO production. Moreover, NADPH-d is upregulated mainly in small sized neurons, thus suggesting that it may be related with pain transmission.
Abstract: The neurotoxicant trimethyltin (TMT) induces massive neuronal loss in vivo in the hippocampus of rodents, accompanied by behavioral alterations. The present study investigates the pattern of cell death after in vivo administration of TMT to adult mice. In the granular cell layer of the Dentate Gyrus, TUNEL staining detected DNA fragmentation, and apoptotic bodies were also evident. In addition, a ladder pattern of internucleosomal DNA fragmentation was shown in agarose gel electrophoresis. We show that activated caspase-3, which is known to play a pivotal role in apoptotic processes, is clearly expressed by degenerating neurons. Inducible cyclooxygenase is also expressed at cytoplasmic level by degenerating granular neurons, suggesting that this enzyme may participate in TMT-induced neurodegeneration.
Abstract: Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.
Abstract: Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are alpha/beta heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.
Abstract: Synthesis of nitric oxide (NO) occurs downstream from activation of NMDA receptors and NO acts as a retrograde messenger, influencing the refinement and stabilization of coactive afferent terminals. Cells and neuropil in the rat superior colliculus (SC) and lateral geniculate body (LGB) show intense, developmentally regulated activity for NO synthase (NOS). To study the role of NO in the development of retinogeniculate and retinotectal axon arbors, we examined primary visual projections of rats that had received daily i.p. injections of L-NoArg (an NOS inhibitor) for 4-6 weeks starting from postnatal day 0. Retinal fibers labeled by intraocular injection of the B subunit of cholera toxin were revealed immunohistochemically and the density of fibers in the superficial SC and in the dorsal LGB was measured by computerized image analysis. Single retinocollicular terminal arbors were reconstructed at the computer (Neurolucida). Treated rats showed significant alterations in ipsilateral retinotectal projections, in the mediolateral and anteroposterior axes: there was an increase in the density of fibers entering the SC, in branch length, and in numbers of boutons on retinotectal arbors in the treated group. Ipsilaterally projecting retinal axons also showed an increase in density and distribution in the dorsal nucleus of the LGB. If animals were allowed to survive for several months after stopping treatment, similar changes were also noted, but these were much less striking. Our results suggest that, in the mammalian visual system, NO released from target neurons in the SC and LGB serves as a retrograde signal which feeds back on retinal afferents, influencing their growth.
Abstract: BACKGROUND: We analysed changes in nitric oxide synthase (NOS) and cytochrome oxidase (CO) activities in the tumoural and peritumoural cerebral cortex in order to investigate: a) the role of NO in tumourigenesis, in TBF regulation, and in vasogenetic PBE; b) the metabolic changes caused by the neoplasm in the surrounding tissues. METHOD: Intra-operative samples of cerebral cortex were studied by means of immunohistochemistry for nNOS and iNOS, and by histochemistry for NADPH-diaphorase (NADPH-d) and CO. FINDINGS: In contrast with normal cortex, reactive glial cells and the endothelium of small blood vessels displayed strong NADPH-d and iNOS activities in oedematous peritumoural tissue. In the tumoural cortex, NADPH-d and nNOS-positive neurones were reduced in number and their dendrites were thin and interrupted, and infiltrates of NADPH-d and iNOS-positive tumoural cells were frequent. CO activity was decreased in the deep layers of peritumoural cortex, and it was almost absent in the tumoural cortex. INTERPRETATION: In peritumoural and tumoural cortex changes in NOS and CO activities suggest that the coupling between neuronal activity and blood flow is impaired in the damaged cerebral cortex, and that the increase in NOS activity may play a role in tumour vascularization and progression.
Abstract: In the central nervous system, NOS activity is involved in several physiological events, such as refinement of afferent connections in development, or linking cerebral blood flow to neural activity in adulthood, and also in many pathological events, such as cell death in brain ischemia and regulation of vasospasm in hemorrhage. Therefore, we studied NOS activity in the CNS. We describe a fast and accurate method in which we use HPLC analysis to identify and quantify citrulline eluted by ion-exchange chromatography, thus implementing the current method to evaluate NOS activity. This technique could be readily applied for NOS activity determination not only in brain, but also in all other tissues.
Abstract: Synthesis of nitric oxide (NO) occurs downstream from activation of N-methyl-D-aspartate (NMDA) receptors; NO reportedly acts as a retrograde messenger, influencing the refinement and stabilization of coactive afferent terminals. Cells and neuropil in the rat superior colliculus (SC) and lateral geniculate body (LGB) show intense, developmentally regulated activity for NO synthase (NOS). To study the role of NO in the development of retinogeniculate and retinotectal axon arbors, we examined primary visual projections of rats that had received intraperitoneal injections of Nomega-nitro-L-arginine (L-NoArg, an NOS inhibitor) on postnatal day 0, and daily thereafter for 4-6 weeks. Treated rats showed significant alterations in ipsilateral retinotectal projections, in the mediolateral and anteroposterior axes; there was an increase in the density of fibres entering the SC, in branch length, and in the numbers of boutons on retinotectal arbors in the treated group. Ipsilaterally projecting retinal axons also showed an increase in density and distribution in the dorsal nucleus of the LGB. If animals were allowed to survive for several months after stopping treatment, similar changes were also noted, but these were much less striking. Our results support the hypothesis that, in the mammalian visual system, NO released from target neurons in the SC and LGB serves as a retrograde signal which feeds back on retinal afferents, influencing their growth. The effects of NOS inhibition are partially reversed after treatment is stopped, indicating that lack of NO synthesis delays the maturation of retinofugal connections, and also that NO plays a constitutive role in their development.
Abstract: Over the last 20 years, the choice of neural tracers has increased manyfold, and includes newly introduced anterograde tracers that allow quantitation of single-axon morphologies, and retrograde tracers that can be combined with intracellular fills for the study of dendritic arbors of neurons which have a specific projection pattern. The combination of several different tracers now permits the comparison of multiple connections in the same animal, both quantitatively and qualitatively. Moreover, the finding of new virus strains, which infect neural cells without killing them, provides a tool for studying multisynaptic connections that participate in a circuit. In this paper, the labeling characteristics, mechanism of transport and advantages/disadvantages of use are discussed for the following recently introduced neural tracers: carbocyanine dyes, fluorescent latex microspheres, fluorescent dextrans, biocytin, dextran amines, Phaseolus vulgaris leucoagglutinin, cholera toxin and viruses. We also suggest the choice of specific tracers, depending on the experimental animal, age and type of connection to be studied, and discuss quantitative methodologies.
Abstract: The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.
Abstract: Histochemical detection of NADPH-d activity in rat barrel-field cortex reveals four types of distributions. (i) A transient, diffuse neuropil staining is visible in the cortical plate and in deeper layers until postnatal day (P) 4. Thereafter, until P15, it is segregated in whisker-specific patches in layer IV, then the pattern gradually disappears, becoming virtually indistinct by P21. This transient patterning of diffuse NADPH-d activity in layer IV disappears after cortical injections of kainic acid and is affected by neonatal damage to the contralateral snout. An intense labeling (ii) of scattered cells and (iii) of a plexus of fibers is present. With maturation, the cells become localized mostly in layers II/III, in the lower part of layer V, and in layer VI. They are sparse in layer I, in upper layer V, and in layer IV where their somata are located primarily in the interbarrel septa. (iv) Light staining of cortical neurons is detected mostly in layers II-IV but occasionally also in layers V-VI. Cytochrome c oxidase (CO)-positive patches associated with barrels are first detected in layer IV around P4-P5; their staining density increases with development, then stays high. In the adult, CO activity is moderate in supragranular layers, highest in the barrels in layer IV, low in upper layer V, medium dense in the deeper half of layer V, and low in lamina VI. Thus, NADPH-d and CO activities are not necessarily colocalized in the rodent barrel-field cortex. The varied (transient and long-lasting) distributions of NADPH-d activity indicate that the enzyme and its associated production of NO serve multiple roles in developing and adult barrel-field cortex.
Abstract: Dendritic spines are a key structure in neuronal plasticity. Enhanced activity is commonly associated with an increase in spine size and density. Purkinje cell dendrites are characterized by a proximal and a distal compartment on which climbing fibers and parallel fibers, respectively, impinge. The proximal region has a very low spine density, whereas the distal region has a high density. Previous experiments showed that after climbing fiber deletion, Purkinje cells become hyperactive, and a large number of spines develop on the proximal dendrites. Here we show that the same hyperspiny transformation occurs in the proximal dendrites of adult Purkinje cells by depressing electrical activity with tetrodotoxin. Thus, spines in different dendritic compartments are created or maintained independently from the level of Purkinje cell-firing rate and when the afferent activity is blocked. This conclusion supports the view that spinogenesis is the expression of an intrinsic program and the two regions of the dendritic tree respond differently to activity block because of differences in the inputs that they receive. On tetrodotoxin treatment, climbing fibers become atrophic and may sprout thin collateral ramifications directed mainly toward the granular layer. All changes are reversible on tetrodotoxin removal. Therefore, Purkinje cells provide a model where spines in different compartments of the same neuron are differently regulated by the activity of their local afferents. In addition, electrical activity is also essential to maintain the full climbing fiber innervation.
Abstract: The rat adrenal gland contains nitric oxide-producing ganglion cells, contributing to its innervation. In a previous study postnatal number and morphology of these adrenal neurons were analyzed by NADPH-diaphorase histochemistry in the two sexes. A transient sex-related difference in the number of NADPH-diaphorase positive neurons per adrenal gland was found at postnatal day 10, when the number of stained neurons in males was nearly twice that found in females. In the present work we studied the effects of perinatal hormonal manipulation on the number of adrenal NADPH-diaphorase-positive neurons during the second postnatal week. The number of labeled adrenal neurons at postnatal day 10 was higher in females receiving perinatal androgen treatment than in control untreated females, and was similar to that of control untreated males. In contrast, in males that underwent perinatal deprivation of testosterone the number of labeled adrenal neurons was lower than in control males, and similar to that of control females. These differences were found in both the adrenal cortex and medulla. In males and in testosterone-treated females there was a higher proportion of stained multipolar neurons than in females and in androgen-deprived males. No intergroup differences were found in the size of stained neurons. Thus, we demonstrated that the postnatal difference in the number of NADPH-diaphorase-positive adrenal neurons in the two sexes is related to the epigenetic action of gonadal hormones during perinatal maturation.
Abstract: The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.
Abstract: BACKGROUND: Primary open-angle glaucoma is commonly treated with long-term hypotensive medical therapy. When this approach becomes inadequate, therapy proceeds with surgery. The present study investigates morphological changes in the conjunctival and subconjunctival tissues induced by short- and long-term topical medical therapy of primary open-angle glaucoma. METHODS: Comparisons were made between biopsy specimens from glaucomatous patients, who received specific eyedrop therapy (timolol and pilocarpine) for various periods of time, and control patients with no conjunctival pathology or topical treatment. Histological, immunohistochemical and ultrastructural parameters were investigated. RESULTS: The morphometric analysis of histological sections and immunohistochemistry (anti-fibronectin antibody) in medium- and long-term therapy patients showed: (a) significant increases in the thickness and number of epithelial cell layers; (b) significant increases in the fibroblast density in both subepithelial and deep connective tissue; and (c) a more compact connective tissue, richer in collagen fibers arranged in whirls, with some inflammatory elements. These findings were confirmed by the ultrastructural analysis. In the same patients, the other immunohistochemical parameters investigated (anti-HLA-DR, anti-CD1a, anti-CD4, anti-CD8, anti-IL2 and C3b antibodies) revealed a tendency to chronic inflammation. Following specific surgery, this tendency manifested itself in a diffuse immune response, especially in those patients who underwent medium- and long-term medical therapy. CONCLUSION: According to these results, antiglaucomatous surgery should be rehabilitated and considered as an alternative to long-term medical therapy in the first-instance treatment of primary open-angle glaucoma.
Abstract: Dihydronicotinamide adenine-dinucleotide phosphate diaphorase (NADPH-d) positive neurons in the superficial layers of superior colliculus (SC) were studied in the adult rat after eye enucleation at postnatal day 5 (P5). Bilaterally, NADPH-d histochemistry revealed either weakly or intensely labeled neurons. In the SC contralateral to the enucleation, the volume of superficial layers decreased significantly, whereas the total number of NADPH-d positive neurons was only slightly reduced, thus resulting in an increased cell density. Bilaterally, the number of NADPH-d positive neurons was around 20% of Nissl-stained neurons. While the number of neurons which were weakly positive for NADPH-d was unchanged contralateral to the enucleation (thus resulting in a significant increase in their percentage on the overall NADPH-d population), the number of intensely labeled neurons decreased by 30%. Intensely labeled neurons were classified with respect to cell size and dendritic distribution. Some (126) were reconstructed and analyzed on the computer, in order to quantitate morphological differences in dendritic distribution in the denervated and control SC. The percent of neurons which could be assigned to some classes (marginal, stellate, narrow field vertical and wide field vertical) was reduced contralateral to the enucleation. In addition, vertically-oriented neurons (narrow field vertical, wide field vertical and pyriform) showed a significant decrease in soma size, dendritic length and number of branch points. And finally, the overall orientation of dendrites on narrow and wide field vertical neurons was more dispersed, when compared to the control colliculus. Thus, P5 eye enucleation affects the adult morphology of NADPH-d positive neurons in the superficial layers of the rat SC, resulting in increased cell density, changed relative number of cells in each morphological type, and altered soma size, dendritic length and orientation in specific neurons.
Abstract: In kittens, callosally projecting neurons were labeled by retrograde transport of FITC- (fluorescein isothiocyanate)- and TRITC- (tetramethylrhodamine isothiocyanate)-conjugated latex microspheres injected in two different visual areas (17, 17/18, 19, or postero-medial lateral suprasylvian; PMLS) at postnatal day 3. At postnatal day 57 more than 1200 labeled neurons in visual cortical areas were intracellularly injected with 3% lucifer yellow (LY) in perfusion-fixed slices of the contralateral hemisphere. The distribution of labeled neurons was charted, and LY-filled neurons were classified on the basis of their area and layer of location, and dendritic pattern. The dendritic arbors of 120 neurons were computer reconstructed. For the basal dendrites of supragranular pyramidal neurons a statistical analysis of number of nodes, internodal and terminal segment lengths, and total dendritic length was run relative to the area of location and axonal projection. Connections were stronger between homotopic than between heterotopic areas. Overall tangential and laminar distributions depended on the area injected. Qualitative morphological differences were found among callosally projecting neurons, related to the area of location, not to that of projection. In all projections from areas 17 and 18, pyramidal and spinous stellate neurons were found in supragranular layers. In contrast, spinous stellate neurons lacked in projections from area 19, 21a, PMLS and postero-lateral lateral suprasylvian (PLLS). In all areas, the infragranular neurons showed heterogeneous typology, but in PMLS no fusiform cells were found. Quantitative analysis of basal dendrites did not reveal significant differences in total dendritic length, terminal, or intermediate segment length among neurons in area 17 or 18, and this was related to whether they projected to contralateral areas 17-18 or PMLS. All injections produced exuberant labeling in area 17. No differences could be found between neurons in area 17 (with transient axons through the corpus callosum) and neurons near the 17/18 border (which maintain projections to the corpus callosum). In conclusion, morphology of callosally projecting neurons seems to relate more to intrinsic specificities in the cellular composition of each area than to the area of contralateral axonal projection or the fate of callosal axons.
Abstract: Callosally projecting neurons in areas 17 and 18 of the adult cat can be classified into two types on the basis of their dendritic morphology: pyramidal and stellate cells. The latter are nearly exclusively of the spinous type and are predominantly located in upper layer IV. Retrograde transport of the carbocyanine dye DiI, applied to the corpus callosum, showed that, up to P6, all callosally projecting neurons resemble pyramids in the possession of an apical dendrite reaching layer I. At P10, however, callosally projecting neurons with stellate morphology were found. A study was designed to distinguish whether these neurons are late in extending their axons to the corpus callosum or, alternatively, have transient apical dendrites. To this end, callosally projecting neurons were retrogradely labeled by fluorescent beads injected in areas 17 and 18 at P1-P3 and then either relabeled with DiI applied to the corpus callosum at P10 or intracellularly injected with Lucifer Yellow at P57. Double-labeled stellate and pyramidal cells were found in similar proportions to those found for the total, single-labeled population of callosally projecting neurons. It is therefore concluded that callosally projecting spiny stellate cells initially possess an apical dendrite and a pyramidal morphology. At P6, i.e. close to the time when stellate cells appear, layer IV neurons with an atrophic apical dendrite were found, suggestive of an apical dendrite in the process of being eliminated.
Abstract: The costo-uterine muscle provides a skeletal attachment to the longitudinal myometrial layer of the uterine horn. In this study we investigated the possibility that the muscle is responsive to sex steroid hormones. In rats of 4 weeks of age, injected with oestradiol for 5 days, the cross-sectional area of nucleated muscle cell profiles was significantly increased. A significant increase in the sectional area of muscle cells was also demonstrated in the costo-uterine muscle of 16-week-old rats, on the 20th day of gestation, compared with non-pregnant rats in dioestrus and of the same age. In oestrogen-treated and in pregnant rats, there was also an increase in muscle cell length. As to the innervation of the costo-uterine muscle, in glyoxylic acid-treated whole-mount and cryostat preparations, we found not only perivascular nerve fibres, but also a few nerve fibres innervating the muscle proper. The pattern of innervation was unchanged after oestrogen treatment and during pregnancy. In the electron microscope, axonal varicosities were observed in the proximity of both vascular and non-vascular muscle cells.
Abstract: The location, number and size of the motoneurons innervating the ischiocavernosus muscle, identified by means of horseradish-peroxidase (HRP) retrograde transport, were studied (1) in adult untreated male rats, (2) in adult male rats castrated before puberty, and (3) in adult male rats castrated before puberty and injected with testosterone from the day of castration. After injection of HRP into the ischiocavernosus muscle, labeled motoneurons were found in the dorsolateral and dorsomedial columns of the lamina IX, at the level of L6 and S1 segments of the spinal cord. Morphometric analysis demonstrated that prepubertal castration induces a statistically significant reduction in the somatic and nuclear areas (40% and 35%, respectively, if compared to those of the control rats) of both the dorsolateral and dorsomedial motoneurons, but does not affect their number. The effects of castration are prevented by exogenous testosterone.
Abstract: The mean area of the neuromuscular endplates and the acetylcholinesterase (AChE) activity at the myotendinous junction of the ischiocavernosus muscle were studied in normal, castrated and testosterone-treated castrated Wistar rats by the Koelle method. The mean endplate area was found to be smaller in castrated rats, compared to normal ones (p less than 0.001), while testosterone treatment restored its normal size (0.8 greater than p greater than 0.7). The terminal AChE activity in castrated rats was as strong and spread as in juvenile ones, while it was almost absent in normal and in testosterone-treated castrated rats. The same parameters were examined in the tibialis anterior muscle of the same rats, chosen as a specimen of 'nonhormone-dependent' muscle, without finding any difference among the single groups.
