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Mitochondrial diseases are a heterogeneous group of rare genetic disorders caused by mutations in nuclear or mitochondrial DNA (mtDNA). These diseases are frequently multisystemic, although mainly affect tissues that require large amounts of energy such as the brain. Mutations in mitochondrial transfer RNA (mt-tRNA) lead to defects in protein translation that may compromise some or all mtDNA-encoded proteins. Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) syndrome is mainly caused by the m.3243A>G mutation in the mt-tRNALeu(UUR) (MT-TL1) gene. Owing to the lack of proper animal models, several cellular models have been developed to study the disease, providing insight in the pathophysiological mechanisms of MELAS. In this study, we show a successful direct conversion of MELAS patient-derived fibroblasts into induced neurons (iNs) for the first time, as well as an electrophysiological characterization of iNs cocultured with astrocytes. In addition, we performed bioenergetics analysis to study the consequences of m.3243A>G mutation in this neuronal model of MELAS syndrome.

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle syn-thesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED su-per-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynap-tic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein MAP1B and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the pre-synaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high ex-pression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal. This work was supported by the Spanish Agencia Estatal de Investigación (grant number:PID2019-110272RB-100/AEI/10.13039/501100011033 (LT), SMA Europe (LT), Ministerio de Ciencia eInnovación/FEDER (grant: PID2021-122785OB-I00) (JC)), the Deutsche Forschungsgemeinschaft (Se697/7-1 (MS)), and the Maratóde TV3 Foundation (202005 (JC and LT))

The HERC protein family is one of three subfamilies of Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases. Six HERC genes have been described in humans, two of which encode Large HERC proteins -HERC1 and HERC2- with molecular weights above 520 kDa that are constitutively expressed in the brain. There is a large body of evidence that mutations in these Large HERC genes produce clinical syndromes in which key neurodevelopmental events are altered, resulting in intellectual disability and other neurological disorders like epileptic seizures, dementia and/or signs of autism. In line with these consequences in humans, two mice carrying mutations in the Large HERC genes have been studied quite intensely: the tambaleante mutant for Herc1 and the Herc2+/530 mutant for Herc2. In both these mutant mice there are clear signs that autophagy is dysregulated, eliciting cerebellar Purkinje cell death and impairing motor control. The tambaleante mouse was the first of these mice to appear and is the best studied, in which the Herc1 mutation elicits: (i) delayed neural transmission in the peripheral nervous system; (ii) impaired learning, memory and motor control; and (iii) altered presynaptic membrane dynamics. In this review, we discuss the information currently available on HERC proteins in the nervous system and their biological activity, the dysregulation of which could explain certain neurodevelopmental syndromes and/or neurodegenerative diseases., This work was funded by the following grants: EMP-V (DGICYT BFU2011-27207 and Spanish Junta de Andalucía CTS-2257); RR (Spanish Junta de Andalucía BIO-113 and SAF2015-64171-R); SB (Spanish Junta de Andalucía, Postdoctoral grant PAIDI-Doctor); JAA (Spanish Junta de Andalucía BIO-209 and DGICYT BFU2015-64536-R); JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00 / MCIN / AEI / 10.13039/501100011033).

Diversity within microglia, the resident brain immune cells, is reported. Whether microglial subsets constitute different subtypes with intrinsic properties and unique functions has not been fully elucidated. Here, we describe a microglial subtype characterized by the expression of the enzyme Arginase-1, i.e. Arg1+microglia, which is found predominantly in the cholinergic neuron-rich forebrain region during early postnatal development. Arg1+ microglia are frequently observed in close apposition to neurons and exhibit a distinctive molecular signature reflecting a reactive profile. Arg1 deficiency in microglia results in impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, and cognitive behavioural deficiencies in female mice. Our results expand on microglia diversity and provide insights into distinctive spatiotemporal functions exerted by microglial subtypes.

Critical periods of synaptic plasticity facilitate the reordering and refining of neural connections during development, allowing the definitive synaptic circuits responsible for correct adult physiology to be established. Presynaptic spike timing-dependent long-term depression (t-LTD) exists in the hippocampus, which depends on the activation of NMDARs and that probably fulfills a role in synaptic refinement. This t-LTD is present until the third postnatal week in mice, disappearing in the fourth week of postnatal development. We were interested in the mechanisms underlying this maturation related loss of t-LTD and we found that at CA3–CA1 synapses, presynaptic NMDA receptors (pre-NMDARs) are tonically active between P13 and P21, mediating an increase in glutamate release during this critical period of plasticity. Conversely, at the end of this critical period (P22–P30) and coinciding with the loss of t-LTD, these pre-NMDARs are no longer tonically active. Using immunogold electron microscopy, we demonstrated the existence of pre-NMDARs at Schaffer collateral synaptic boutons, where a decrease in the number of pre-NMDARs during development coincides with the loss of both tonic pre-NMDAR activation and t-LTD. Interestingly, this t-LTD can be completely recovered by antagonizing adenosine type 1 receptors (A1R), which also recovers the tonic activation of pre-NMDARs at P22–P30. By contrast, the induction of t-LTD was prevented at P13–P21 by an agonist of A1R, as was tonic pre-NMDAR activation. Furthermore, we found that the adenosine that mediated the loss of t-LTD during the fourth week of development is supplied by astrocytes. These results provide direct evidence for the mechanism that closes the window of plasticity associated with t-LTD, revealing novel events probably involved in synaptic remodeling during development.

A missense mutation in HERC1 provokes loss of cerebellar Purkinje cells, tremor, and unstable gait in tambaleante (tbl) mice. Recently, we have shown that before cerebellar degeneration takes place, the tbl mouse suffers from a reduction in the number of vesicles available for release at the neuromuscular junction (NMJ). The aim of the present work was to study to which extent the alteration in HERC1 may affect other cells in the nervous system and how this may influence the motor dysfunction observed in these mice. The functional analysis showed a consistent delay in the propagation of the action potential in mutant mice in comparison with control littermates. Morphological analyses of glial cells in motor axons revealed signs of compact myelin damage as tomacula and local hypermyelination foci. Moreover, we observed an alteration in non-myelinated terminal Schwann cells at the level of the NMJ. Additionally, we found a significant increment of phosphorylated Akt-2 in the sciatic nerve. Based on these findings, we propose a molecular model that could explain how mutated HERC1 in tbl mice affects the myelination process in the peripheral nervous system. Finally, since the myelin abnormalities found in tbl mice are histological hallmarks of neuropathic periphery diseases, tbl mutant mice could be considered as a new mouse model for this type of diseases.

Aim: to evaluate the safety and effectiveness of self-expandable metal stent placement for malignant gastric outlet obstruction (GOO). Methods: a retrospective, analytic cohort study at a single, tertiary-care center. Results: thirty-six patients that underwent stent placement for GOO of malignant origin were identified during the study period. Technical success was achieved in 36 (100 %) patients and clinical success was achieved in 31 patients (86.1 %). Before the procedure, 17 (54.8 %) patients had a gastric outlet obstruction score (GOOSS) of 0, which is a complete inability of oral intake. Twenty-three patients were alive 30 days after the procedure, two (8.6 %) patients had a GOOSS of 1, ten (43.3 %) had a GOOSS of 2 and eleven (47.9 %) had a GOOSS of 3. Abdominal pain was present in all 31 patients before the procedure and only seven (22.6 %) patients continued with abdominal pain 24 hours after the procedure. During follow-up, ten (30.3 %) patients developed complications related to the stents and none of them was fatal. Additional therapy due to partial occlusion of the stent was necessary in three patients. The stents functional duration had a median of 72 days (IQR 25-75 15-105 days) and was closely related to overall survival. Conclusion: palliative stenting for gastroduodenal obstruction is a safe, feasible and effective therapy to treat patients with malignant gastric outlet obstruction.

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efciency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics. Junta de Andalucía BIO-209 MINECO-DGICYT BFU2015-64536-R Junta de Andalucía BIO-122 MINECO-DGICYT BFU2015-64536-R MINECO-AEI/FEDER, UE, BFU2016-80295-R

Apoptotic caspases are thought to play critical roles in elimination of excessive and non-functional synapses and removal of extra cells during early developmental stages. Hence, an impairment of this process may thus constitute a basis for numerous neurological and psychiatric diseases. This view is especially relevant for dopamine due to its pleiotropic roles in motor control, motivation and reward processing. Here, we have analysed the effect of caspase-3 depletion on the development of catecholaminergic neurons and performed a wide array of neurochemical, ultrastructural and behavioural assays. To achieve this, we performed selective deletion of the Casp3 gene in tyrosine hydroxylase (TH)-expressing cells using Cre-loxP-mediated recombination. Histological evaluation of most relevant catecholaminergic nuclei revealed the ventral mesencephalon as the most affected region. Stereological analysis demonstrated an increase in the number of TH-positive neurons in both the substantia nigra and ventral tegmental area along with enlarged volume of the ventral midbrain. Analysis of main innervating tissues revealed a rather contrasting profile. In striatum, basal extracellular levels and potassium-evoked DA release were significantly reduced in mice lacking Casp3, a clear indication of dopaminergic hypofunction in dopaminergic innervating tissues. This view was sustained by analysis of TH-labelled dopaminergic terminals by confocal and electron microscopy. Remarkably, at a behavioural level, Casp3-deficient mice exhibited impaired social interaction, restrictive interests and repetitive stereotypies, which are considered the core symptoms of autism spectrum disorder (ASD). Our study revitalizes the potential involvement of dopaminergic transmission in ASD and provides an excellent model to get further insights in ASD pathogenesis., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (SAF2015-64171-R and RTI2018-098645-B-I00). J.A.A. and E.P.V. was supported by a MINECO (BFU2015-64536) grant. I.S.P., E.B. and L.B. were co-financed by the “Fondo Europeo de Desarrollo Regional” (FEDER)-UE “A way to build Europe” from the “Ministerio de Ciencia, Innovación y Universidades” (RTI2018-099778-B-I00) and the Ministerio de Salud-Instituto de Salud Carlos III (PI18/01691), as well as funding from the “Consejería de Salud de la Junta de Andalucía” (PI-0134-2018); the “Programa Operativo de Andalucía FEDER, Iniciativa Territorial Integrada ITI 2014-2020 Consejería Salud, Junta de Andalucía” (PI-0080-2017); the “Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía” (CTS-510); and the “Centro de Investigación Biomédica en Red de Salud Mental- CIBERSAM” (CB/07/09/0033).

50 Páginas; 9 Figuras, Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2), which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease, such as intracellular iron and lipofuscin accumulation, increased oxidative stress, and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate., This work was supported by FIS PI16/00786 grant, Instituto de Salud Carlos III, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), Proyectos de Investigación de Excelencia de la Junta de Andalucía CTS-5725 and BIO-122, DGICYT BFU2015-64536-R, and by AEPMI (Asociación de Enfermos de Patología Mitocondrial) and ENACH (Asociación de Enfermos de Neurodegeneración con Acumulación Cerebral de Hierro).

The ubiquitin-proteasome system (UPS) plays a fundamental role in protein degradation in neurons, and there is strong evidence that it fulfills a key role in synaptic transmission. The aim of the present work was to study the implication of one component of the UPS, the HERC1 E3 Ubiquitin Ligase, in motor function and neuromuscular transmission. The tambaleante (tbl) mutant mouse carries a spontaneous mutation in HERC1 E3 Ubiquitin Ligase, provoking an ataxic phenotype that develops in the second month of life. Our results show that motor performance in mutant mice is altered at postnatal day 30, before the cerebellar neurodegeneration takes place. This defect is associated with by: (a) a reduction of the motor end-plate area, (b) less efficient neuromuscular activity in vivo, and (c) an impaired evoked neurotransmitter release. Together, these data suggest that the HERC1 E3 Ubiquitin Ligase is fundamental for normal muscle function and that it is essential for neurotransmitter release at the mouse neuromuscular junction.

Objective: To analyze the expression of transforming growth factor-s1 in heterotopic grafts of adult dental apical papillae. Methodology: Adult apical papillae of Wistar rats were grafted in the ear of the same donor rats. 1, 3, 7 and 14 days after grafting, rats were perfused and the tissue containing the graft was processed for histological conventional technique and for immunohistochemical detection of transforming growth factor-s1. Results: Heterotopically grafted apical papilla developed osteoid dentine. In an early post-grafting stage, odontoblast-like cells organized themselves in palisade and synthesized dentine. However, newly formed dentine possessed the structural appearance of reactive osteoid dentine, which was systematically destroyed by the activity of osteoclaste-like cells. Transforming Growth Factor-s1 was observed in mesenchymal cells, extracellular matrix of the graft and surrounding host tissue, while odontoblast-like cells were systematically devoid of immunoreactivity. Conclusion: The different expression of transforming growth factor-s1 between normal tissue and grafted tissue development suggests that in heterotopic graft conditions the inflammatory mediation of the transforming growth factor-s1 prevails against its morphogenetic role.

Tambaleante (tbl/tbl) is a mutant mouse that carries a spontaneous Gly483Glu substitution in the HERC1 (HECT domain and RCC1 domain) E3 ubiquitin ligase protein (HERC1). The tbl/tbl mutant suffers an ataxic syndrome given the almost complete loss of cerebellar Purkinje cells during adult life. More recent analyses have identified alterations at neuromuscular junctions in these mice, as well as in other neurons of the central nervous system, such as motor neurons in the spinal cord, or pyramidal neurons in the hippocampal CA3 region and the neocortex. Accordingly, the effect of the tbl/tbl mutation apparently extends to other regions of the nervous system far from the cerebellum. As HERC1 mutations in humans have been correlated with intellectual impairment, we studied the effect of the tbl/tbl mutation on learning. Using a behavioral test, ex vivo electrophysiological recordings, immunohistochemistry, and Golgi method, we analyzed the associative learning in the lateral amygdala of the tbl/tbl mouse. The tbl/tbl mice perform worse than wild-type animals in the passive avoidance test, and histologically, the tbl/tbl mice have more immature forms of dendritic spines. In addition, LTP cannot be detected in these animals and their STP is dampened, as is their glutamatergic input to the lateral amygdala. Together, these data suggest that HERC1 is probably involved in regulating synaptic function in the amygdala. Indeed, these results indicate that the tbl/tbl mutation is a good model to analyze the effect of alterations to the ubiquitin-proteasome pathway on the synaptic mechanisms involved in learning and its defects.

Investigations indicate that natural orifice translumenal endoscopic surgery (NOTES) procedures induce a less pronounced postoperative inflammatory response than open or laparoscopic surgery, inflicting less trauma. In NOTES procedures, no skin incision is performed. We compare the inflammatory response added by the type of incision by measuring C-reactive protein (CRP) and tumors necrosis factor-alfa (TNF-α).Twenty-seven pigs were randomized to open surgical, laparoscopic, or transgastric NOTES abdominal access. After completion of the accesses, no surgery was performed. All accesses were left open for 40 minutes followed by closure, animals were survived for 7 days. Blood samples were drawn at the start of the accesses, at 20 and 40 minutes during the procedure, and at postoperative day (POD) 1, 3, and 7. Analyses of CRP and TNF-α were performed.CRP increased in all animals until POD1. This increase was greater in the open group (P = .006). No significant differences in CRP-levels were found at POD 1, 3, or 7. TNF-α showed a peak during the procedure, at 20 and 40 minutes, with normalization at POD1 for 1/3 of the open and laparoscopic animals, but not for the NOTES animals. Due to variations within the groups, no statistical difference was shown between them. At postmortem, 1/3 of the pigs in the laparoscopic and open groups had wound infections, while no NOTES animals showed infections.This study provides no statistically significant differences in inflammatory response after the different abdominal accesses. However, the lack of a TNF-α-peak in the NOTES group might indicate a less pronounced response, supporting the initial theories.

The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tarnbaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity.

We developed a non-survival in vivo model for testing of gastric natural orifice translumenal endoscopic surgery (NOTES) closures based on the gastric yield pressure. The aim of this study was to test our model comparing different endoscopic closure techniques with surgical closure of a NOTES gastric incision.Laparotomy was performed in 30 pigs. One tube for air inflation and one manometry tube were inserted into the stomach via the pylorus, which was closed gas-tight, and the abdominal wall was closed. The stomach was inflated with air, and the gastroesophageal yield pressure was measured. A gastroscopy was performed, and a standard NOTES access was created followed by randomization to closure by surgical suturing, T-tags, Padlock-G over-the-scope (OTS)-clips, OVESCO OTS-clips, and traditional clips. All closures were tested twice with air insufflation. Gastric yield pressure or leak pressure of each closure was recorded.The mean baseline gastric yield pressure was 80.5 mm Hg. Post-closure yield pressure was 79.9 mm Hg. Leak test results after closure were as follows: surgery, 0/6 leaked; T-tags, 1/6 leaked before reaching yield pressure (56 mm Hg); Padlock-G, 2/5 leaked (71.5 mm Hg); OVESCO OTS-clips, 3/6 leaked (27.2 mm Hg); and traditional clips, 5/6 leaked (27.2 mm Hg). TAS T-tags and surgical closures leaked significantly less than the other groups (P=.01). Traditional clips and OVESCO OTS-clips leaked at significantly lower pressures than the other three groups (P=.007).This in vivo model using leak of the closure or the gastric yield pressure as endpoints for testing of the closure strength of a NOTES gastric access site seems to be reproducible. Our results support closure with T-tags and Padlock-G-clips over OVESCO OTS-clips and standard endoscopic clips.

Apart from the pioneering studies of Ramon y Cajal [1893] and Rochon-Duvigneaud [1943], few studies have been devoted to the detailed study of the cytological and biochemical structure of the chameleon retina. In the present study we analyzed the expression of calbindin (CB), calretinin (CR) and parvalbumin (PV) immunoreactivities in the chameleon retina, and compared their distribution with those found in the retinas of other vertebrate species. CB immunoreactivity is dense in photoreceptors, horizontal and some lower amacrine cells. The most intense immunoreactivity was observed for calretinin; CR-ir amacrine cells are distributed throughout the inner nuclear, inner plexiform, and ganglion cell layers of the retina. Horizontal cells also display immunoreactivity to CR. A few retinal interneurons are weakly PV-ir. Double-labeling shows that all PV-ir or CB-ir cells, except the photoreceptors, are also strongly CR-ir. The distributions of these calcium-binding proteins in the chameleon retina share similarities with those observed in mammalian and avian retinas. In addition, the widespread distribution and co-localization of CB and CR reinforces the idea that these proteins play a general role in buffering the intracellular calcium levels in retinal cells. Furthermore, CB- and CR-immunoreactivities have enabled us to identify for the first time axon-bearing horizontal cells in the peripheral retina of the chameleon, very similar to those described in mammals.

The diagnosis of colonic angiodysplasia is often challenging and relies on endoscopy or catheter angiography. We investigated whether computed tomographic angiography (CTA) contributes to the diagnosis of colonic angiodysplasia.Twenty-eight patients with suspected bleeding from colonic angiodysplasia were prospectively evaluated. Gastrointestinal bleeding was investigated by colonoscopy plus visceral angiography and by CTA. The level of agreement between CTA and the former procedures was determined.CTA images of diagnostic quality were obtained in 26 patients. Eighteen patients were diagnosed with colonic angiodysplasia by colonoscopy plus visceral angiography, and 14 by CTA (kappa = 0.68; P0.001). Sensitivity, specificity, and positive predictive values of CTA for detection of colonic angiodysplasia were 70%, 100%, and 100%, respectively. CTA signs including accumulation of vessels in the colonic wall, early filling vein, and supplying enlarged artery were present in 55%, 50%, and 22% of cases, respectively. None of these signs were present in the 8 patients with obscure gastrointestinal bleeding and negative diagnostic investigation of the digestive tract.CTA is a sensitive, specific, well-tolerated, and minimally invasive tool for the diagnosis of colonic angiodysplasia.

The distribution and ascending projections to the hypothalamic paraventricular nucleus of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive perikaria were studied in adult pigeons using a combination of retrograde transport of Fluorogold injected into the paraventricular nucleus, and double immunohistochemical procedures for PNMT, tyrosine hydroxylase and neuropeptide Y. PNMT-immunoreactive cell bodies were found in the subtrigeminal reticular nucleus of the ventrolateral medulla and in the nucleus of the solitary tract, mainly in the subnuclei: medialis superficialis, pars posterior, and medialis ventralis, pars posterior. PNMT-immunoreactive perikaria were also tyrosine hydroxylase immunoreactive, and are located within the rostral tyrosine hydroxylase immunoreactive cell groups of these areas. No perikaria double-labeled for neuropeptide Y and PNMT were found. Retrograde labeled cell bodies were observed in the subtrigeminal reticular nucleus and in the nucleus of the solitary tract. PNMT-immunoreactive retrogradely labeled cells were mainly observed in the subtrigeminal reticular nucleus. These data suggest the presence in the pigeon of medullary adrenergic cell groups partially comparable to mammalian C1 and C2 groups. Comparison of these results with data previously obtained in amphibians and reptiles suggests that the presence of a hypothalamically-projecting C1-like group might be a plesiomorphic medullary attribute in amniotes, whereas the variable presence of C2 and C3-like groups, as well as the content of NPY in the putative adrenergic perikaria, seem to be species-specific.

The possibility that peripheral exogenous pyrogens can activate brainstem nuclei by abdominal viscera aAerents was studied using the NADPH-diaphorase method in E. coli lipopolysaccharide treated rats with and without ibu- profen pre-treatment. 2. NADPH-d staining revealed: (i) a significant increase in the number of NADPH-d labeled neurons in the subpos- tremal area of the nucleus tractus solitarius lipopolysaccharide treated rats compared with control animals (p < 0.05), and (ii) an almost similar number of NADPH-d positive neurons in both control and ibuprofen pre- treated rats (p = 0.091). 3. These data indicate that peripheral administration of an exogenous pyrogen stimulates the synthesis of nitric oxide in the nucleus tractus solitarius, and are consistent with the hypothesis of a direct neural link between the periphery and the hypothalamus.

We have tested the suitability of chromaffin-like carotid body glomus cells for dopamine cell replacement in Parkinsonian rats. Intrastriatal grafting of cell aggregates resulted in almost optimal abolishment of motor asymmetries and deficits of sensorimotor orientation. Recovery of transplanted animals was apparent 10 days after surgery and progressed throughout the 3 months of the study. The behavioral effects were correlated with the long survival of glomus cells in the host brain. In host tissue, glomus cells were organized into glomerulus-like structures and retained the ability to secrete dopamine. Several weeks after transplantation, dopaminergic fibers emerged from the graft, reinnervating the striatal gray matter. The special durability of grafted glomus cells in the conditions of brain parenchyma could be related to their sensitivity to hypoxia, which is known to induce cell growth, excitability, and dopamine synthesis. This work should stimulate research on the clinical applicability of carotid body autotransplants in Parkinson's disease.

Naturally occurring neuronal death was found by in situ labelling of nuclear DNA fragmentation during the development of the chick inferior olive. Counting neuronal perikarya showed an evident loss of cells from embryonic day 18 to hatching. This reduction in neuronal numbers was followed by an increase of similar size from days 1–4 post-hatching. This biphasic evolution of the neuronal numbers is quite similar to that found in the inferior olive of rodents during the first two weeks of the postnatal life, a period also characterized by definitive synaptogenesis between climbing fibers and Pukinje cells in the cerebellum of the rodents. The similarity in the evolution of neuronal number in the inferior olive of both rodents and chicks, seems to indicate that definitive synaptogenesis between climbing fibers and Purkinje cells might occur from embryonic day 18 to postnatal day 3 in the chick cerebellum. Nevertheless, during the phase of cell loss the climbing fibers of chick have attained a more mature developmental stage than those of the rat. This difference suggests that naturally occurring neuronal death may be independent of the elimination of redundant axonic collaterals during the definitive climbing fibers-Purkinje cell synaptogenesis.

The use of horseradish peroxidase (HRP) and 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) as retrograde tracers, applied in vitro within the olivocerebellar tract of both embryos (9 to 21 days old) and postnatal (3–60 days old) chickens, has allowed the observation of a small population of neurons located ipsilaterally to the placement of the tracer. These neurons, whose morphology indicated that they belong to the inferior olive rather than to the reticular formation or the raphe nuclei, followed the same developmental steps as normally placed inferior olivary neurons. Furthermore, pedunculotomy experiments made on 3-day-old chickens demonstrated that ipsilateral neurons sent their axons through the cerebellar peduncle. In contrast to the completely crossed arrangement of the olivocerebellar projection, the present results show the existence, as in the rat, of a few ipsilateral inferior olivary neurons whose significance is unclear.

Homotopic and isochronic transplantation of the alar plate of the rhombomeres 7 and 8 was performed between chick and quail embryos at the stage of 10-14 somites. Analysis of the graft derivatives in 12-day-old chimeric embryos by means of the quail nucleolar marker showed that the ipsilateral inferior olive is formed from the transplanted neuroepithelium. In all embryos some cells originating from the graft were also found scattered throughout the contralateral inferior olive. The present results demonstrate that the inferior olive derives from the alar plate of the rhombomeres 7 and 8 and support the notion that a small contingent of inferior olivary neurones crosses the interolivary commissure during development.

Macrophage inflammatory protein-1 (MIP-1) evokes an intense fever, independent of a prostaglandin mechanism, and is now thought to play an important role in the defence response to bacterial pyrogens. The purpose of this study was 2-fold: (i) to determine whether the potent doublet of this cytokine, MIP-1beta, is actually produced in the brain in response to a pyrogenic dose of a lipopolysaccharide of Escherichia coli and (ii) to determine the anatomical site of synthesis of this cytokine in the brain. Following the intense fever produced by intraperitoneal administration of lipopolysaccharide in the unrestrained rat, MIP-1beta immunoreactivity was identified post mortem in two regions of the brain implicated in fever: the organum vasculosum laminae terminalis (OVLT) and the anterior hypothalamic, preoptic area (AH/POA). Microinjection of goat anti-mouse MIP-1beta antibody (anti-MIP-1beta) directly int the AH/POA markedly suppressed fever in rats in response to lipopolysaccharide. Further anti-MIP-1beta administered 180 min after the injection of lipopolysaccharide acted as an antipyretic and reversed the fever induced by the endotoxin. anti-MIP-1beta or control immunoglobulin G antibody microinjected into the hypothalamus immediately before the intraperitoneal injection of the control saline did not alter the temperature of the rats. Taken together, the present results demonstrate that MIP-1beta is produced in the brain in response to a bacterial endotoxin. These observations, in the light of earlier data on fever induced by MIP-1beta, further support the hypothesis that endogenously synthesized MIP-1beta acts as an intermediary factor in the evocation of fever by acting on the thermosensitive cells of the brain.

Demyelinating disorders such as leukodystrophies and multiple sclerosis are neurodegenerative diseases characterized by the progressive loss of myelin that may lead toward a chronic demyelination of the brain’s white matter, impairing normal axonal conduction velocity and ultimately causing neurodegeneration. Current treatments modifying the pathological mechanisms are capable of ameliorating the disease; however, frequently, these therapies are not sufficient to repress the progressive demyelination into a chronic condition and permanent loss of function. To this end, we analyzed the effect that bone marrow-derived mesenchymal stromal cell (BM-MSC) grafts exert in a chronically demyelinated mouse brain. As a result, oligodendrocyte progenitors were recruited surrounding the graft due to the expression of various trophic signals by the grafted MSCs. Although there was no significant reaction in the non-grafted side, in the grafted regions oligodendrocyte progenitors were detected. These progenitors were derived from the nearby tissue as well as from the neurogenic niches, including the subependymal zone and dentate gyrus. Once near the graft site, the cells matured to myelinating oligodendrocytes. Finally, electrophysiological studies demonstrated that axonal conduction velocity was significantly increased in the grafted side of the fimbria. In conclusion, we demonstrate here that in chronic demyelinated white matter, BM-MSC transplantation activates oligodendrocyte progenitors and induces remyelination in the tissue surrounding the stem cell graft., This work was supported by MINISTERIO CIENCIA E INNOVACION (MICINN BFU2010-27326), DIGESIC-MEC BFU2008-00588, Ingenio 2010 MEC-CONSOLIDER CSD2007-00023, from the GVA Prometeo grant 2009/028 and ISCIII Cibersam and Tercel (RD06/0010/0023), as well as ELA Foundation, Fundación Diogenes-Elche city government and Rotary Club Elche-Illice and Fundació Gent per Gent grant 24 NEURO.

The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.

The presence of ipsilateral inferior olivary neurons during postnatal development of the olivocerebellar projection in the rat was investigated by two in vitro axonal tracing methods and by the axotomy of one olivocerebellar tract. The experiments were carried out before (P1), during (P5–P10) and after (P20) the period of multiple innervation of Purkinje cells by climbing fibers. According to present results: (1) ipsilateral inferior olivary neurons are distributed, on all analyzed days, throughout the entire inferior olive; (2) cell counts after axotomy experiments demonstrated that they represent a small population of inferior olivary neurons, whose number oscillated between 271 ± 30 in young animlas (pedunculotomized at P1 and killed at P7) and 26 ± 12 in older ones (pedunculotomized at P20 and killed at P40). This experiment confirmed that most of these neurons are eliminated during the regressive events that take place during normal development of the olivocerebellar projection; and (3) few ipsilateral inferior olivary neurons, however, survive at P40, but their significance is still unclear. © 1994 Wiley-Liss, Inc.

The membrane properties of the neurons located in the ventral part of the periaqueductal grey (PAG) of the guinea-pig were studied using an in vitro slice preparation. Cells had low values of resting membrane potential (-53.3 +/- 1.3 mV, mean +/- standard error), high input resistance (195. +/- 16.2 M ohm) and moderate values of membrane time constant (12.6 +/- 0.7 ms). The last two parameters changed as recordings were made along the sagittal axis, higher values corresponding to the more rostral cells. Three main neuronal types-fusiform, triangular and stellate-were found in the ventral PAG using intracellular injection of Lucifer yellow. A study of the cell number and cell density was carried out in coronal and sagittal sections of the ventral PAG. This analysis showed a clear gradient of size in this region arising from the gradual disappearance of large (17 to 40 microm) neurons in the caudorostral direction. The neuronal density also increased in this direction. Therefore, some electrotonic and morphological parameters differ along the sagittal axis. These findings suggest a larger neuronal heterogeneity of the caudal part of the PAG, and might contribute to a functional segregation of this region.

Naturally occurring neuronal death plays a substantial developmental role in the building of the neural circuitries. The neuronal death caused by different cerebellar mutations is mostly of an apoptotic nature. Apart from the identity of the intrinsic mechanisms of the mutations, adult cerebellar mutants are a powerful tool to causally study the development of the cerebellar connectivity. Thus, studies on adult cerebellar neuronal cell death occurring in mouse mutants elucidate: (i) the dependence of the postsynaptic neurons on their partners, (ii) the 'en cascade' postsynaptic transneuronal degeneration after target-deprivation, and (iii) the close relationship between the molecular modular organization of the cerebellar cortex and dying Purkinje cells. Neuronal cell death has been extensively studied in developing olivocerebellar system. However, less data are available on the occurrence of naturally occurring neuronal death during the in vivo normal development of the Purkinje cells and the mossy fiber system neurons. The developmental role of neuronal death during the establishment and refinement of the olivocerebellar projection is currently discussed. Moreover, the occurrence of neuronal death during the development of the basilar pontine nuclei and its role in the acquisition of the adult pontocerebellar projection is still poorly understood. In the present review, we correlate the dates of Purkinje cells death with the inferior olivary and basilar pontine neuronal apoptosis, discussing their developmental relationships during the elaboration of the fine-grained maps of the cerebellar afferent connections.

Several indices evaluate disease activity in ulcerative colitis, but most have drawbacks to their application (invasiveness, complexity, or lack of specificity), and discrepancies between them are frequent. Assuming that desquamation of epithelial and inflammatory cells increases in damaged colonic mucosa, we hypothesized that fecal excretion of human DNA is an index of mucosal inflammation and damage. The aim of our study was to determine whether excretion of human DNA is useful in the evaluation of disease activity in ulcerative colitis.Thirty-one controls and 36 ulcerative colitis patients were included. Ulcerative colitis patients and controls underwent colonoscopic examination after preparation by gut lavage with polyethylene-glycol electrolyte solution. In patients, disease activity was established using the clinical index of Rachmilewitz and an endoscopic score. Feces and gut lavage fluid were obtained and DNA levels were measured by quantitative polymerase chain reaction of the human beta-globin gene.Fecal DNA excretion correlated with the clinical index (r = 0.59, P0.05) and the endoscopic score (r = 0.76, P0.01). Gut lavage fluid DNA levels also correlated with clinical and endoscopic activity scores (r = 0.41 and 0.51, respectively, P0.05). Fecal DNA excretion was significantly higher in patients with endoscopically or clinically active disease than in controls or patients in remission. Fecal DNA excretion discriminates between endoscopically active disease and remission (sensitivity 0.67, specificity 1.00, P0.01).Excretion of human DNA in feces, as an expression of cellular desquamation, is a novel noninvasive technique to objectively assess disease activity in ulcerative colitis.

Sympathoadrenal (SA) cell lineage encompasses neural crest derivatives such as sympathetic neurons, small intensely fluorescent (SIF) cells of sympathetic ganglia and adrenal medulla, and chromaffin cells of adrenal medulla and extra-adrenal paraganglia. SA autografts have been used for transplantation in Parkinson's disease (PD) for three reasons: (i) as autologous donor tissue avoids graft rejection and the need for immunosuppressant therapy, (ii) SA cells express dopaminotrophic factors such as GNDF and TGFbetas, and (iii) although most of SA cells release noradrenaline, some of them are able to produce and release dopamine. Adrenal chromaffin cells were the first SA transplanted cells in both animal models of PD and PD patients. However, these autografts have met limited success because long-term cell survival is very poor, and this approach is no longer pursued clinically. Sympathetic neurons from the superior cervical ganglion have been also grafted in PD animal models and PD patients. Poor survival into brain parenchyma of grafted tissue is a serious disadvantage for its clinical application. However, cultured sympathetic cell grafts present a better survival rate, and they reduce the need for levodopa medication in PD patients by facilitating the conversion of exogenous levodopa. SA extra-adrenal chromaffin cells are located on paraganglia (i.e., the Zuckerkandl's organ), and have been used for grafting in a rodent model of PD. Preliminary results indicate that long-term survival of these cells is better than for other SA cells, exerting a more prolonged restorative neurotrophic action on denervated host striatum. The ability of SA extra-adrenal cells to respond to hypoxia, differently to SA sympathetic neurons or adrenal medulla cells, could explain their good survival rate after brain transplantation.

Inflammatory bowel disease impairs health-related quality of life. Therefore, it is very important to develop adequate instruments to measure the disease impact, but these instruments need to be practical as well as accurate. Our aim was to determine whether a short questionnaire obtained from the reduction of the 36-item version of the inflammatory bowel disease questionnaire (IBDQ-36) accurately reflects the impact of clinical and endoscopic activity on health-related quality of life.To this purpose the original IBDQ-36 and a reduced version, composed of only 9 items (IBDQ-9), were administered to 68 patients with inflammatory bowel disease. Disease activity was established by standard clinical activity indices (Rachmilewitz for ulcerative colitis (UC) and Harvey-Bradshaw for Crohn's disease (CD)) and by information gathered at colonoscopy.In UC patients the Spearman's correlation coefficients between IBDQ-9 and clinical and colonoscopic indices were statistically significant (-0.67 and -0.70, respectively, p0.01) and similar to those obtained with IBDQ-36 (-0.61 and -0.67). In CD patients IBDQ-9 also correlated well with the clinical index (-0.59, p0.05) but less with the colonoscopic index (-0.30, p= 0.1). In CD patients, the correlation of the IBDQ-36 with clinical and colonoscopic indices gave similar results to the IBDQ-9 (-0.58 and -0.21, respectively). The IBDQ-9 power to discriminate between clinical relapse and remission was statistically significant (p0.01) both for UC (55 (48-57) vs 69 (63-75) and CD (58 (51-63) vs 69 (64-83)) patients. Similar results were obtained for conoloscopic indices of endoscopic relapse and remission (56 (52-65) vs 70 (66-77) in UC and 58 (52-63) vs 68 (62-73) in CD).Quality of life impairment produced by relapses of inflammatory bowel disease can be reliably assessed with a short questionnaire, with considerable savings in time and expense.

This study determined whether macrophage inflammatory protein-1beta (MIP-1beta) plays a role in the hyperthermia caused by prostaglandin E2 (PGE2) given intracerebroventricularly (i.c.v.) in the rat. In these experiments, anti-murine MIP-1beta antibody (anti-MIP-1beta) was micro-injected in the anterior hypothalamic, preoptic area (AH/POA) just before i.c.v. PGE2. The results showed that anti-MIP-1beta failed to alter the PGE2 hyperthermia. However, immunocytochemical studies revealed MIP-1beta immunoreactivity detectable in both the organum vasculosum laminae terminalis (OVLT) and AH/POA in the febrile rat. These data thus demonstrate that MIP-1beta is sequestered in diencephalic structures underlying thermoregulation even though it is not involved in PGE2 hyperthermia. This dissociation supports the viewpoint that at least two distinct systems exist in the brain which underlie a febrile response: MIP-1beta underlies one component whereas PGE2 comprises the other.

Publisher Summary This chapter summarizes some of the recent findings regarding the use of the eyelid motor system as a model for the study of reflex and learned motor responses and on the role played by cerebellar structures in the genesis and control of both types of movement. The eyelid motor system of the cat is used for the study of the kinetic properties of reflex and conditioned lid movements and of the role played by the cerebellum in the acquisition and/or performance of both types of motor responses. Spontaneous blinks, eyelid reflex responses, eye-guided lid movements, and conditioned lid responses are recorded in alert cats in simultaneity with unitary and field electrical activity of cerebellar cortex and nuclear zones related to the eyelid motor system. The metric properties of eyeblinks seem to depend largely on the sensory modality used and the precise neural circuits involved in each particular movement. This diversity in eyelid motor responses can only be fully appreciated when lid position is recorded with the search-coil in a magnetic field technique.

In neonatal rats the injection of Fast Blue and Diamidino Yellow retrograde fluorescent tracers, each into separate cellebellar hemispheres, reveals the presence of double-labelled neurons positioned bilaterally in the inferior olivary complex during the early postnatal period (postnatal day 0 to postnatal day 5). This suggests that those neurons whose axons are able to take up both tracers project to both hemicerebella during this period of postnatal development. Double-labelled neurons were observed in one-and five-day-old injected postnatal rats, but were absent in older animals (10 and 30 days old). The presence of these neurons coincides with a transient period of poly-innervation of Purkinje cells by climbing fibres. They may thus be participating in transitory interactions preceding the formation of definitive climbing fibre synaptic arrangements in the cerebeller cortex. The technique employed is unable to clearly define the pathway of this transient olivocerebellar projection into the ipsilateral cerebellum; however, in direct evidence—like the topographic distribution of double-labelled neurons relative to tracer injection sites, and the small number of single-labelled neurons within the ipisilateral olivary complex, together with previous data on the axonogenesis of olivary neurons [ Bourrat and Sotelo (1988) Devl Brain Res . 39, 19–37]—suggests that these fibres reach the cerebellum through the contralateral inferior cerebellar peduncle and give rise to collaterals, some of which subsequently decussate again within the cerebellum. These fibres probably represent transient collaterals of the normally contralateral olivocerebellar fibres that cross the cerebellar midline and reach mirror-image loci within the ipsilateral hemicerebellum.

The retina of the chick contains retinal cells of a morphology very similar to that of the horizontal cells, but the perikarya, axons, and axon terminals lie in the inner plexiform layer. The discovery of this neuronal ectopia appears to support the idea that some horizontal and amacrine cells derive from a common, freely migrating cell.

In this work we analyse the effect produced by reserpine on the development of thickness and cell number in the external granular layer in the cerebellum of chick embryo. A striking 48-hour histo genetic delay is observed in the treated embryos relative to controls, as show by greater thickness and cell density of this layer in the former, as well as by retarded appearance of a typical radial morphological organization of the external granular layer.