The HERC1 E3 Ubiquitin Ligase is essential for normal development and for neurotransmission at the mouse neuromuscular junction.

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.

The Absence of Caspase-8 in the Dopaminergic System Leads to Mild Autism-like Behavior.

In the last decade, new non-apoptotic roles have been ascribed to apoptotic caspases. This family of proteins plays an important role in the sculpting of the brain in the early stages of development by eliminating excessive and nonfunctional synapses and extra cells. Consequently, impairments in this process can underlie many neurological and mental illnesses. This view is particularly relevant to dopamine because it plays a pleiotropic role in motor control, motivation, and reward processing. In this study, we analyze the effects of the elimination of caspase-8 (CASP8) on the development of catecholaminergic neurons using neurochemical, ultrastructural, and behavioral tests. To do this, we selectively delete the CASP8 gene in cells that express tyrosine hydroxylase with the help of recombination through the Cre-loxP system. Our results show that the number of dopaminergic neurons increases in the substantia nigra. In the striatum, the basal extracellular level of dopamine and potassium-evoked dopamine release decreased significantly in mice lacking CASP8, clearly showing the low dopamine functioning in tissues innervated by this neurotransmitter. This view is supported by electron microscopy analysis of striatal synapses. Interestingly, behavioral analysis demonstrates that mice lacking CASP8 show changes reminiscent of autism spectrum disorders (ASD). Our research reactivates the possible role of dopamine transmission in the pathogenesis of ASD and provides a mild model of autism.

Cellular and functional recovery of Parkinsonian rats after intrastriatal transplantation of carotid body cell aggregates.

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.

Analysis of CXCL12 3'UTR G>A polymorphism in colorectal cancer.

Colorectal cancer is one of the most prevalent cancers in developed countries. However, the genetic factors influencing its appearance remain far from being fully characterized. Recently, a G>A functional transition mapping the 3' untranslated region of the CXCL12 gene (rs1801157) has been found to be under-represented among rectal cancer patients when compared to colon cancer patients from a Swedish series. Here we present the results from an independent analysis of CXCL12 rs1801157 in a larger CRC series of Spanish origin in order to analyse the robustness of this association within a different European population. No significant difference was observed between controls and colon or rectal cancer patients. We were also unable to find a correlation between rs1801157 and different prognostic markers such as metastasis development or disease-free survival time. The epidemiologic data involving CXCL12 rs1801157 in colorectal cancer risk are discussed.

Diagnostic yield and safety of capsule endoscopy.

INTRODUCTION: the capsule endoscopy (CE), from his approval, has become a first line diagnostic procedure for the study of the small bowel disease. The aim of this study is to report our experience since the implantation of this technique in our hospital. MATERIAL AND METHODS: retrospective review of the CE undertaken in Department of Endoscopy. There was gathered in every case the age, sex, motive of consultation, previous diagnostic procedures, capsule endoscopy findings and complication of the technique. One took to end a descriptive and analytical analysis. RESULTS: there was achieved a total of 416 explorations in 388 patients. The obscure gastrointestinal bleeding was the most frequent indication (83.30%) followed by suspected Crohn s disease (7.5%). Angiodisplasia was the endoscopic lesion more frequently detected (42.2%), especially, in patients with digestive bleeding of obscure origin (OR 3.13 p < 0.001), followed by the flebectasia (10.6%) and the ulcer suspicious of Crohn s disease (9.9%). The global diagnostic yield as for the detection of injuries was 77.34% with a case of "not defecation of the capsule" and therefore need of laparotomy. CONCLUSIONS: the capsule endoscopy is a technique consolidated and as his potential is known, his indications are extended. The obscure gastrointestinal bleeding is the most frequent indication and the angiodisplasia the most identified injury. Once known his diagnostic yield, larger studies are needed that assess the influence of capsule endoscopy on clinical outcoumes.

Left Unilateral Inferior Pedunculotomy Prevents Neuronal Death During Postnatal Development of the Remaining Left Inferior Olivary Complex in the Rat.

Neuronal death in the inferior olivary complex (IOC) was studied in control and unilaterally pedunculotomized newborn rats, from postnatal day 1 (P1) to P30, in order to test whether the approximately two-fold increase in available specific targets (i.e. Purkinje cells) that is theoretically provided by sectioning one inferior cerebellar peduncle to the developing climbing fibres of the remaining IOC could prevent the loss of inferior olivary neurons taking place during the first 2 weeks of postnatal life in the rat. Numerical estimation of the number of inferior olivary neurons in control and experimental rats showed that (i) in pedunculotomized rats, the number of inferior olivary neurons of the remaining inferior olivary complex was always greater than that encountered in control rats, (ii) the consistent decrease in the number of inferior olivary neurons observed in control animals between P2 and P8 was absent in cell counts of the pedunculotomized rats, and (iii) the increase in olivary cell number following the phase of cell decrease was also absent in pedunculotomized rats. It is concluded that the increase of available Purkinje cells during early postnatal development of the olivocerebellar projection prevents neuronal death in the remaining inferior olivary complex following pedunculotomy.

The Study of Passive Membrane Properties and Morphology Reveals Neuronal Differences Along the Sagittal Axis of the Ventral Periaqueductal Grey Matter.

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.

Morphological evidence for the presence of ipsilateral inferior olivary neurons during postnatal development of the olivocerebellar projection in the rat.

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 animals (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.

Transient ipsilateral innervation of the cerebellum by developing olivocerebellar neurons. A retrograde double-labelling study with fast blue and diamidino yellow.

In neonatal rats the injection of Fast Blue and Diamidino Yellow retrograde fluorescent tracers, each into separate cerebellar 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 hemicerebellar 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 cerebellar 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 ipsilateral 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.

Involvement of cerebellar cortex and nuclei in the genesis and control of unconditioned and conditioned eyelid motor responses.

The eyelid motor system of the cat was used here 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 were recorded in alert cats in simultaneity with unitary and field electrical activity of cerebellar cortex and nuclear zones related to the eyelid motor system. Results indicate that nuclear unitary activity does not precede unconditioned or conditioned lid responses, but that cerebellar nuclei are directly involved in the performance of the late components of reflex lid movements and in the acquisition of conditioned lid responses.

Pattern of degeneration of the rat inferior olivary complex after the early postnatal axotomy of the olivocerebellar projection.

Neuronal death of inferior olivary neurons after early axotomy of the olivocerebellar tract was studied in newborn (P1) hemicerebellectomized rats during the first six days after lesion. The degeneration of the inferior olive showed a topographic pattern from one (P2) to six days after axotomy (P7), after which this complex had almost completely disappeared. The first degenerative changes were observed in the principal olive (P2), while the medial accessory olive was the later-degenerated area (P5). The analysis of these degenerative changes provides a reference for future experimental studies. Furthermore, the topographic study of the degenerative process demonstrated that: i) the most vulnerable neurons were dorsolaterally located, whereas the most resistant ones occupied the medial aspect of the inferior olivary complex, ii) the comparison between the topographical arrangement of the inferior olivary neurons according to their birth dates, and the rate of degenerative changes observed after hemicerebellectomy, open the possibility that the neuronal generation date and the response to the axotomy of the inferior olivary neurons could be related.

The avian inferior olive derives from the alar neuroepithelium of the rhombomeres 7 and 8: an analysis by using chick-quail chimeric embryos.

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.

Comparison of plethysmographic methods for obtaining thoracic gas volume in anesthetized dogs.

Thoracic gas volume (TGV) cannot be measured in the body plethysmograph by the standard spontaneous breathing technique (SB) when there is a significant respiratory center depression. In this case, either external compression of the chest wall (EC) or phrenic nerve stimulation (PhN) can be used to induce the pressure-volume changes necessary to calculate TGV. In the present study we compared TGV measured in eight pentobarbital-anesthetized dogs by SB, EC, PhN, and the standard He-dilution method. EC and simulated PhN were also used to measure the volume of a lung model, and EC was used to measure the volume of isolated lung lobes. A method for fast and accurate plethysmographic calibration is also described. In the intact dogs, SB, PhN, and He-dilution techniques gave similar results, but EC overestimated TGV. In the isolated lobes and lung model EC accurately measured volume. We speculate that EC induces substantial intersegmental and/or interlobal gas movement in intact lungs and that the pressure drop due to airway resistance causes proximal airway pressure to underestimate alveolar pressure changes, which induces overestimation in calculated TGV. We conclude that PhN is the method of choice to measure plethysmographic TGV when the respiratory center is depressed and that EC overestimates TGV in intact dogs.

Ipsilaterally located olivocerebellar projection neurons of the chick.

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.

Naturally occurring neuronal death during the development of the inferior olive in the chick.

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.

A fast and easy fluorescent counterstaining method for neuroanatomical studies by using Acridine Orange.

Acridine Orange is commonly used as a fluorescent counterstain in fluorescent tract tracing techniques. Here we describe a method in which the substitution of the standard washing solutions (i.e., 0.9% saline) for a diluted solution of Acridine Orange (0.001%) during the perfusion of the animal before fixation provides a fluorescent counterstaining compatible with Fast Blue fluorescent retrograde labeling. In contrast to other fluorescent counterstaining methods, this procedure minimizes the diminution in the fluorescence of the tracer during the handling of sections.

Antibodies to macrophage inflammatory protein-1beta in preoptic area of rats fail to suppress PGE2 hyperthermia.

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.

Early dendritic development of Purkinje cells in the rat cerebellum. A light and electron microscopic study using axonal tracing in 'in vitro' slices.

The early stages in the formation of Purkinje cell dendritic arbors have been analyzed using the horseradish peroxidase (HRP) 'in vitro' axonal tracing method, from embryonic day 19 (E19) to postnatal day 6 (P6). These stages comprise the transition from the bipolar Purkinje cell, at the end of its migration, to the phase of stellate cell with disoriented dendrites. Postmigratory Purkinje cells in the cortical plate exhibit poorly elaborated bipolar shapes, here named 'simple-fusiform' cells. They constitute the vast majority of labeled cells up to P0, and thereafter they decrease in number until P4. As a result of continuous outgrowth of new primary dendrites emerging from the apical pole but also from the basal and lateral aspects of the cell bodies, the Purkinje cells enter the 'complex-fusiform' phase, which peaks by P1 and slowly disappears by P6. The disappearance of 'complex-fusiform' cells is the result of an intense regressive process with resorption or retraction of the long dendrites that reaches a maximum by P3. We have called this stage: the Purkinje cell with 'regressive-atrophic' dendrites. This regression marks the initiation of the phase of the stellate cell, characterized by the explosive outgrowth of shorter perisomatic protrusions emerging in all directions. By P6, almost all the labeled Purkinje cells have attained this phase. The ultrastructural study of the labeled Purkinje cells has revealed that the transient dendrites of the fusiform cells have all the cytologic features of mature dendrites, particularly cytoskeletal elements (microtubules) and free polyribosomes. More importantly, axon terminals of unknown origin establish a few, constantly present, mature-like synaptic contacts on the dendritic shafts and spinous protrusions from P0, the earliest studied age. Their frequency increases on the Purkinje cells which enter the phase of stellate cell. Our results emphasize that the transformation of bipolar postmigratory Purkinje cells into the stellate cell stage results from a complex cascade of alternating creative and destructive processes, taking place in parallel with the formation and regression of mature synaptic contacts, between the remodelling dendritic arbors and unidentified afferent inputs. Purkinje cells, in all the different transitional stages, are present side by side in the same folial regions, at least until P4, and receive a similar contingent of synaptic input. This indicates that the dendritic remodelling is not driven by the synaptic inputs, but obeys either neural interactions that lead Purkinje cells to assume their monocellular layer configuration, or an internal clock depending on the Purkinje cell birthdate, or an interplay between these two kinds of mechanisms.

Displaced horizontal cells in the chick retina.

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.

Developmental study of axon formation in the horizontal neurons of the retina of the chick embryo.

Among the types of horizontal cells of the avian retina, one has been described that has an axon terminating in a typical structure. The present study analyses the histogenesis of this axon whose initial outgrowth occurs on day 14 of incubation (HH-40). The axon terminal is first detectable, towards day 15 of incubation (HH-41), in the form of a varicose thickening possessing short filopodia. The formation of the axon and the growth of the axon terminal is coincident with a retraction of the perikaryal process. The axon usually originates from one of the principal dendrites and in these stages shows short and fine filopodia throughout its length. From day 16 onwards (synaptic) spines may be distinguished, both in the dendritic field and on the axon terminal. The growth of the axon, in the phase when the axon terminal still has not formed, may exhibit deflections and deviations in its course, the possible cause and mechanism of which are discussed.