Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

Induction of growth cone formation by transient and localized increases of intracellular proteolytic activity.

The formation of a growth cone at the tip of a transected axon is a crucial step in the subsequent regeneration of the amputated axon. During this process, the transected axon is transformed from a static segment into a motile growth cone. Despite the importance of this process for regeneration of the severed axon, little is known about the mechanisms underlying this transformation. Recent studies have suggested that Ca2+-activated proteinases underlay the morphological remodeling of neurons after injury. However, this hypothesis was never tested directly. Here we tested the ability of transient and localized increases in intracellular proteolytic activity to induce growth cone formation and neuritogenesis. Minute amounts of the proteinase trypsin were microinjected into intact axonal segments or somata of cultured Aplysia neurons, transiently elevating the intracellular protease concentration to 13-130 nM in the vicinity of the injection site. Such microinjections were followed by the formation of ectopic growth cones and irreversible neuritogenesis. Growth cones were not formed after external application of trypsin, microinjection of the carrier solution, or inactivated trypsin. Growth cone formation was not preceded by increases in free intracellular Ca2+ or changes in passive membrane properties, and was blocked by inhibitors of actin and tubulin polymerization. Trypsin-induced neuritogenesis was associated with ultrastructural alterations similar to those observed by us after axotomy. We conclude that local and transient elevations of cytoplasmic proteolytic activity can induce growth cone formation and neuritogenesis, and suggest that localized proteolytic activity plays a role in growth cone formation after axotomy.

Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.

Axo-dendritic synaptogenesis was examined in live hippocampal cell cultures using the fluorescent dyes DiO to label dendrites and FM 4-64 to label functional presynaptic boutons. As the first functional synaptic boutons appeared in these cultures, numerous filopodia (up to 10 micron long) were observed to extend transiently (mean lifetime 9.5 min) from dendritic shafts. With progressively increasing numbers of boutons, there were coincident decreases in numbers of transient filopodia and increases in numbers of stable dendritic spines. Dendritic filopodia were observed to initiate physical contacts with nearby axons. This sometimes resulted in filopodial stabilization and formation of functional presynaptic boutons. These findings suggest that dendritic filopodia may actively initiate synaptogenic contacts with nearby (5-10 micron) axons and thereafter evolve into dendritic spines.

Potentiation of evoked vesicle turnover at individually resolved synaptic boutons.

We have studied synaptic plasticity in hippocampal cell cultures using a new imaging approach that allows unambiguous discrimination of presynaptic function at the level of single synaptic boutons. Employing a protocol designed to test for use-dependent plasticity resembling N-methyl-D-aspartate receptor-dependent long-term potentiation (NMDA-type LTP), we find that brief tetanic stimuli induce a potentiation of evoked synaptic vesicle turnover that lasts for at least 1 hr. Induction of this clearly presynaptic potentiation is blocked by putative postsynaptic glutamate receptor antagonists, suggesting that a retrograde induction signal might be involved. Potentiation appears to occur approximately equally at boutons of low and high initial release probabilities, and evidently does not involve an increase in the size of the total recycling synaptic vesicle pool.

Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment.

Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial axodendritic contact. Clusters of the presynaptic active zone protein Bassoon were present in all new boutons. Conversely, clusters of the postsynaptic molecule SAP90/PSD-95 and glutamate receptors were found on average only approximately 45 min after such boutons were first detected. AMPA- and NMDA-type glutamate receptors displayed similar clustering kinetics. These findings suggest that glutamatergic synapse assembly can occur within 1-2 hr after initial contact and that presynaptic differentiation may precede postsynaptic differentiation.

Assembling the presynaptic active zone: a characterization of an active one precursor vesicle.

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.

Principles of glutamatergic synapse formation: seeing the forest for the trees.

General principles regarding glutamatergic synapse formation in the central nervous system are beginning to emerge. These principles concern the specific roles that dendrites and axons play in the induction of synaptic differentiation, the modes of presynaptic and postsynaptic assembly, the time course of synapse formation and maturation, and the roles of synaptic activity in these processes.

Reduced SNAP-25 increases PSD-95 mobility and impairs spine morphogenesis.

Impairment of synaptic function can lead to neuropsychiatric disorders collectively referred to as synaptopathies. The SNARE protein SNAP-25 is implicated in several brain pathologies and, indeed, brain areas of psychiatric patients often display reduced SNAP-25 expression. It has been recently found that acute downregulation of SNAP-25 in brain slices impairs long-term potentiation; however, the processes through which this occurs are still poorly defined. We show that in vivo acute downregulation of SNAP-25 in CA1 hippocampal region affects spine number. Consistently, hippocampal neurons from SNAP-25 heterozygous mice show reduced densities of dendritic spines and defective PSD-95 dynamics. Finally, we show that, in brain, SNAP-25 is part of a molecular complex including PSD-95 and p140Cap, with p140Cap being capable to bind to both SNAP-25 and PSD-95. These data demonstrate an unexpected role of SNAP-25 in controlling PSD-95 clustering and open the possibility that genetic reductions of the protein levels - as occurring in schizophrenia - may contribute to the pathology through an effect on postsynaptic function and plasticity.

Recruitment of synaptic molecules during synaptogenesis.

The glutamatergic synapse is the main type of excitatory synapse in the mammalian brain. The formation of each glutamatergic synapse is associated with the recruitment of numerous (probably hundreds) different molecules and their assimilation into functional assemblies. Intense research has revealed the identity of many of these molecules, provided information as to interactions they are involved in, and offered clues as to their roles in synaptic function. Recent work has also begun to shed light on fundamental mechanisms underlying molecule recruitment to developing glutamatergic synapses. Current data indicate that the formation of presynaptic active zones-sites of neurotransmitter release-may be realized by the insertion of precursor vesicles containing multiple active zone components, possibly in pre-assembled form. The assembly of the postsynaptic reception apparatus, on the other hand, seems to occur via the sequential recruitment of molecules to the postsynaptic membrane and their assimilation in situ. Several molecules and mechanisms have been identified that display a capacity for inducing pre- or postsynaptic differentiation. These exciting findings are starting to provide a rudimentary framework for understanding key processes underlying the formation of glutamatergic synaptic connections.

Familial chronic recurrent pancreatitis in identical twins. Case report and review of the literature.

Familial presentation of chronic recurrent pancreatitis in childhood is rare. The etiology of this illness is obscure, and its hereditary properties are not well defined. Simultaneous occurrence of chronic recurrent pancreatitis in identical twins with the same clinical presentation and similar typical pancreatographic abnormalities is exceptional. Twin sisters, aged 9 years, were admitted to the hospital because of recurrent attacks of pancreatitis. Ultrasound examination revealed an enlarged irregular pancreatic duct in both girls, and endoscopic retrograde cholangiopancreatography showed a distorted duct with multiple strictures and dilatations similar to a "chain of lakes" pattern. Both patients underwent longitudinal pancreatojejunostomy within a month. The therapeutic regimen and preoperative and surgical treatment of such patients are discussed, as is the optimal timing of intervention.

Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro.

The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.

Cervico-occipital teratoma in the newborn infant. Case report.

A case of cervico-occipital teratoma in a neonate is presented. This is the third reported case of teratoma in this unusual location and the first excised during the neonatal period. The radiographic and histological examination, treatment, and follow-up findings in this case are presented, along with a review of the literature.

Fractures in children who have Gaucher disease.

A series of twenty-three pathological fractures in nine children who had Gaucher disease was reviewed. Infiltration of the medullary space by Gaucher cells, erosion of bone, osteonecrosis in the area of the fracture, and disuse osteoporosis were the main etiological factors. Bone scans indicated that the osteonecrosis was due to osseous ischemia that occurred two to twelve months before the pathological fracture. Fracture-healing was prolonged, taking as long as two years for completion in some patients. Inadequate periods of immobilization and early weight-bearing led to malunion.

Incidental diagnosis of asymptomatic neonatal cholelithiasis: case report and literature review.

A case of asymptomatic cholelithiasis detected incidentally at the age of 3 days in an otherwise healthy newborn infant is presented. The hospitalization was uneventful. At follow-up the biliary stones had completely resolved at the age of 3 months with no symptoms. Fourteen similar cases of incidentally diagnosed neonatal cholelithiasis were found in the literature. Clinical course was benign; 12 cases (86%) were asymptomatic and eight (57%) had spontaneous resolution by the age of 6 months. We conclude that incidentally diagnosed neonatal cholelithiasis usually has a benign course with about a 50% chance of spontaneous resolution during the first 6 months of life. In cases of persistent gallstones by age 6 months, no treatment is needed except for long-term follow-up.

Subtotal gastrectomy in infancy for perforating necrotizing gastritis.

Necrotizing gastritis is a rare pathology causing a high rate of morbidity and mortality in the infants. A 4-week-old baby, with right hypoplastic kidney and ectopic ureter, was admitted because of profound septic shock and "coffee ground" vomitus. Aggressive treatment was started and hemodynamic stabilization was achieved. On the fourth admission day, ascites was noted and on the eighth day in a plain abdominal x-ray, free air was shown. At urgent explorative laparotomy, double posterior gastric wall perforations with extensive gastric wall necrosis were found, which required subtotal gastrectomy. The etiology and pathophysiology of this rare process are discussed with an emphasis on the difficulty in diagnosis of posterior gastric perforation into the lesser sac.

Traumatic adrenal injury in children.

BACKGROUND: Multiple organ injury in children is an increasingly frequent phenomenon in the modern emergency room. Adrenal hemorrhage associated with this type of trauma has received little attention in the past. OBJECTIVES: Using computed tomography, we sought to determine the rate and nature of adrenal gland injury in children following blunt abdominal trauma due to motor vehicular accident. METHODS: A total of 121 children with blunt abdominal trauma were examined and total body CT was performed in cases of multi-organ trauma or severe neurological injury. RESULTS: Of all the children who presented with blunt abdominal trauma over a 51 month period, 6 (4.95%) had adrenal hemorrhage. In all cases only the right adrenal gland was affected. Coincidental injury to the chest and other abdominal organs was noted in 66.7% and 50% of patients, respectively. CONCLUSIONS: Traumatic adrenal injury in the pediatric population may be more common than previously suspected. Widespread application of the more sophisticated imaging modalities available today will improve the detection of damage to the smaller organs in major collision injuries and will help in directing attention to the mechanism of trauma.

Involvement of the foot and ankle in patients with Gaucher disease.

Of 48 children with type I Gaucher disease treated at our hospital, 11 had involvement of the foot and ankle that first appeared in adolescence. Follow-up ranged from 3 to 27 years (average, 10 years). We reviewed their clinical and radiographic histories. Patients presented with four types of pain: six (7 events of pain) complained of dull pain defined as nonspecific; seven (11 events) had severe pain caused by bone crisis; two (2 events) had moderate progressive pain caused by pathological fractures; and one had painful swollen ankles caused by degenerative arthritis. Greater awareness of these complications in patients with Gaucher disease will prevent misdiagnosis and lead to early treatment.

The details in the distributions: why and how to study phenotypic variability.

Phenotypic variability is present even when genetic and environmental differences between cells are reduced to the greatest possible extent. For example, genetically identical bacteria display differing levels of resistance to antibiotics, clonal yeast populations demonstrate morphological and growth-rate heterogeneity, and mouse blastomeres from the same embryo have stochastic differences in gene expression. However, the distributions of phenotypes present among isogenic organisms are often overlooked; instead, many studies focus on population aggregates such as the mean. The details of these distributions are relevant to major questions in diverse fields, including the evolution of antimicrobial-drug and chemotherapy resistance. We review emerging experimental and statistical techniques that allow rigorous analysis of phenotypic variability and thereby may lead to advances across the biological sciences.