Ultrastructural Evidence for a Role of Astrocytes and Glycogen-Derived Lactate in Learning-Dependent Synaptic Stabilization.

Long-term memory formation (LTM) is a process accompanied by energy-demanding structural changes at synapses and increased spine density. Concomitant increases in both spine volume and postsynaptic density (PSD) surface area have been suggested but never quantified in vivo by clear-cut experimental evidence. Using novel object recognition in mice as a learning task followed by 3D electron microscopy analysis, we demonstrate that LTM induced all aforementioned synaptic changes, together with an increase in the size of astrocytic glycogen granules, which are a source of lactate for neurons. The selective inhibition of glycogen metabolism in astrocytes impaired learning, affecting all the related synaptic changes. Intrahippocampal administration of l-lactate rescued the behavioral phenotype, along with spine density within 24 hours. Spine dynamics in hippocampal organotypic slices undergoing theta burst-induced long-term potentiation was similarly affected by inhibition of glycogen metabolism and rescued by l-lactate. These results suggest that learning primes astrocytic energy stores and signaling to sustain synaptic plasticity via l-lactate.

Palmitoylation of cdc42 Promotes Spine Stabilization and Rescues Spine Density Deficit in a Mouse Model of 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11DS) is associated with learning and cognitive dysfunctions and a high risk of developing schizophrenia. It has become increasingly clear that dendritic spine plasticity is tightly linked to cognition. Thus, understanding how genes involved in cognitive disorders affect synaptic networks is a major challenge of modern biology. Several studies have pointed to a spine density deficit in 22q11DS transgenic mice models. Using the LgDel mouse model, we first quantified spine deficit at different stages using electron microscopy. Next we performed repetitive confocal imaging over several days on hippocampal organotypic cultures of LgDel mice. We show no imbalanced ratio between daily spine formation and spine elimination, but a decreased spine life expectancy. We corrected this impaired spine stabilization process by overexpressing ZDHHC8 palmitoyltransferase, whose gene belongs to the LgDel microdeletion. Overexpression of one of its substrates, the cdc42 brain-specific variant, under a constitutively active form (cdc42-palm-CA) led to the same result. Finally, we could rescue spine density in vivo, in adult LgDel mice, by injecting pups with a vector expressing cdc42-palm-CA. This study reveals a new role of ZDHHC8-cdc42-palm molecular pathway in postsynaptic structural plasticity and provides new evidence in favor of the dysconnectivity hypothesis for schizophrenia.

Spine dynamics and synapse remodeling during LTP and memory processes.

While changes in the efficacy of synaptic transmission are believed to represent the physiological bases of learning mechanisms, other recent studies have started to highlight the possibility that a structural reorganization of synaptic networks could also be involved. Morphological changes of the shape or size of dendritic spines or of the organization of postsynaptic densities have been described in several studies, as well as the growth and formation following stimulation of new protrusions. Confocal in vivo imaging experiments have further revealed that dendritic spines undergo a continuous turnover and replacement process that may vary as a function of development, but can be markedly enhanced by sensory activation or following brain damage. The implications of these new aspects of plasticity for learning and memory mechanisms are discussed.

WITHDRAWN: Role of NCAM in Spine Dynamics and Synaptogenesis.

Increasing evidence indicates that adhesion molecules are critically involved in the regulation of mechanisms of synaptic plasticity including synapse formation, but also synaptic remodeling associated to changes in synaptic strength. Among these, the Neural Cell Adhesion Molecule (NCAM) and its polysialylated form PSA-NCAM are important candidates. Here we review recent results that point to a possible role of these two molecules in regulating the structural properties of excitatory synapses and namely the composition and stability of the postsynaptic density, thereby accounting for their contribution to mechanisms of synaptogenesis and activity-dependent synaptic plasticity.

Analysis of instability patterns in acute scaphoid fractures by 4-dimensional computed tomographic imaging - A prospective cohort pilot study protocol.

INTRODUCTION: A scaphoid fracture is the most common carpal fracture. When healing of the fracture fails (nonunion), a specific pattern of osteoarthrosis occurs, resulting in pain, restricted wrist motion and disability. Scaphoid fracture classification systems recognize fragment displacement as an important cause of nonunion. The fracture is considered unstable if the fragments are displaced. However, whether and how displaced bone fragments move with respect to one another has not yet been investigated in vivo. With a four-dimensional (4D) computed tomographic (CT) imaging technique we aim to analyze the interfragmentary motion patterns of displaced and non-displaced scaphoid fragments. Furthermore, the correlation between fragment motion and the development of a scaphoid nonunion is investigated. We hypothesize that fragment displacement is not correlated to fragment instability; and concurrent nonunion is related to fragment instability and not to interfragmentary displacement. METHODS: In a prospective single-center cohort pilot study, patients with a one-sided acute scaphoid fracture and no history of trauma to the contralateral wrist are illegible for inclusion. Twelve patients with a non-displaced scaphoid fracture and 12 patients with a displaced scaphoid fracture are evaluated. Both wrists are scanned with 4D-CT imaging during active flexion-extension and radio-ulnar deviation motion. The contralateral wrist serves as kinematic reference. Relative displacement of the distal scaphoid fragment with respect to the proximal scaphoid fragment, is described by translations and rotations (the kinematic parameters), as a function of the position of the capitate. Non-displaced scaphoid fractures are treated conservatively, displaced scaphoid fractures receive intraoperative screw fixation. Follow-up with CT scans is conducted until consolidation at 1½, 3 and 6 months. This trial is registered in the Dutch Toetsingonline trial registration system, number: NL60680.018.17. ETHICS: This study is approved by the Medical Ethics Committee of the Academic Medical Center, Amsterdam.

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death.

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric ß-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.

Gastrointestinal blood loss after diflunisal and after aspirin: effect of ethanol.

Fecal blood loss was evaluated in normal subjects with 51Cr-labeled red cells. In a double-blind parallel study in 10 subjects, 250 mg diflunisal twice daily did not significantly increase blood loss in two consecutive treatment periods, while 750 mg acetylsalicylic acid (ASA) 4 times daily did so. In a double-blind crossover study in 2 subjects, diflunisal, 250 mg twice daily again did not significantly affect fecal blood loss during a 4-day treatment period, and there also was no significant effth diflunisal during two additional treatment days. ASA, 600 mg 4 times daily, induced an increase in blood loss and this effect was significantly enhanced by the addition of alcohol. The difference between treatments in the way they interact with alcohol was also statistically significant.

Vertebral crush fracture syndrome and reflex sympathetic dystrophy.

The association of reflex sympathetic dystrophy in one or more extremities with vertebral crush fracture syndrome is reported in six cases. In two of them the reflex sympathetic dystrophy preceded the vertebral crush fractures. The 99mTc-methylene diphosphonate scintigraphy results of the skeletons of 42 consecutive patients suffering from vertebral collapse and of 30 matched controls without osteoporosis have been evaluated by three independent observers for abnormal uptakes in the extremities. An abnormal radioactive bone tracer uptake in the extremities has been observed in 15 osteoporotics (36%) and 8 controls (29%). In most cases, osteoporotics and controls, the abnormal uptake was of the zonal type in a localized area, probably reflecting bone remodeling due to localized degenerative changes. An abnormal uptake of the segmental type involving multiple joints and even multiple extremities, as seen in reflex sympathetic dystrophy, was observed in 7 patients of the osteoporotic group (17%) and in none of the control group (P less than 0.05). The patients with an abnormal segmental uptake were younger and had a significantly higher mean 24 h calcium:creatinine ratio as well as a higher urinary hydroxyproline excretion. These observations and results suggest that in some cases of idiopathic osteoporosis there might be a relation between reflex sympathetic dystrophy and vertebral crush fracture syndrome, the vertebral crush fracture syndrome being the axial type of reflex sympathetic dystrophy.

Unilateral kidney irradiation and late retreatment with cis-dichlorodiammineplatinum (II): functional measurements with 99mtechnetium-dimercaptosuccinic acid.

A rat model was used to study renal function after unilateral kidney irradiations. The left kidney was irradiated with a single dose (6-14 Gy), 2 equal sized fractions (10-16 Gy total dose) or 4 equal sized fractions (12 to 21 Gy total dose). At regular time intervals after treatment, the left kidney function was assessed with the use of 99mTc-Dimercaptosuccinic acid for a period of about 1 year. It was found that renal function declined in a dose- and time-dependent manner. The threshold dose for detecting functional impairment was 8 Gy for 1 fraction, 12 Gy for 2 fraction and 15 Gy for 4 fraction irradiations, demonstrating the sensitivity of the isotope tracer test used in these experiments. Retreatment of the rats with a single i.p. dose of 5 mg/kg cis-Dichlorodiammineplatinum (II) (cis-DDP) given at about 1 year after X ray exposure revealed an important relative decrease in the function of the irradiated compared to the unirradiated kidney. Reductions in function of 11 to 70% (depending on radiation dose and schedule) compared with control values were observed at 11 weeks after drug injection. These results demonstrate that a previously irradiated kidney is more sensitive to a subsequent treatment with cis-DDP than the contralateral hypertrophied kidney in the same animal.

Normal brain perfusion pattern of technetium-99m-ethylcysteinate dimer in children.

UNLABELLED: The purpose of this study was to assess the normal perfusion pattern of the pediatric brain with 99mTc-ethylcysteinate dimer (99mTc-ECD). METHODS: Tomographic imaging was performed with a dedicated system with high sensitivity and resolution. Sixteen children, referred for brain imaging in the workup of seizure disorder, were included since they turned out negative after a 1-yr follow-up. A standardized brain presentation was obtained after reslicing and reorienting of the three-dimensional volumetric dataset. RESULTS: Quantitative analysis did not reveal significant left-right uptake differences per patient. Three age clusters were investigated that showed differences in regional uptake, mainly a relatively increased uptake in basal ganglia, visual and motor cortex. An uptake ratio or perfusion index was calculated after normalization. Normal limits were established for the children in the three groups. CONCLUSION: Technetium-99m-ECD is a safe agent for children and should be the radiopharmaceutical of choice for brain perfusion studies because of favorable radiation dosimetry and stability. The age dependence of perfusion necessitates a database comparison before concluding that the observed perfusion pattern is normal.

Intravenous erythromycin dramatically accelerates gastric emptying in gastroparesis diabeticorum and normals and abolishes the emptying discrimination between solids and liquids.

Erythromycin, a macrolide antibiotic, has recently been shown to have a motilin like effect on gastrointestinal muscle strips. In this study, we have evaluated the effect of erythromycin on patients with delayed gastric emptying and healthy subjects using the dual radionuclide technique. Twelve patients with gastroparesis diabeticorum and ten healthy age- and sex-matched controls were studied. Gastric emptying of solids and liquids was determined using 99mTc-SC scrambled egg and 111In-DTPA in water. Following a baseline study and on a separate day, each patient and control received a 15-min i.v. perfusion of erythromycin starting at meal ingestion. Eleven out of the 12 patients were restudied after a 3-wk oral administration. In patients and controls, i.v. erythromycin dramatically accelerated gastric emptying of both solids and liquids which were emptied at the same rate. After chronic oral administration, solid and liquid emptying remained significantly accelerated. Erythromycin appears to be a very powerful gastrokinetic drug. Derived compounds with the gastrokinetic effect and without the antibiotic activity could be useful in dyspeptic patients with delayed gastric emptying.

Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals.

L,L-ethylenedicysteine (L,L-EC) can be labeled efficiently with 99mTc at pH 12 to obtain a highly pure and very stable tracer agent (99mTc-L,L-EC). The biological behavior of 99mTc-L,L-EC was studied in mice and a baboon. In mice, 99mTc-L,L-EC demonstrated a more rapid urinary excretion and less retention in the kidneys, the liver, the intestines, and the blood than did 99mTc-MAG3 at 10 and 60 min p.i. Urinary excretion decreased in probenecid pretreated mice, which indicates active tubular transport. In the baboon, the renograms for 99mTc-MAG3 and 99mTc-L,L-EC were comparable. Plasma-protein binding of 99mTc-L,L-EC was lower than that of 99mTc-MAG3 while its distribution volume and 1-hr plasma clearance were clearly higher. The promising results of the animal experiments suggest that 99mTc-L,L-ethylenedicysteine may be a useful alternative to 99mTc-MAG3 for renal function studies in humans.

In vitro and in vivo evaluation of granulocyte labeling with [99mTc]d,1-HM-PAO.

The functional integrity of white blood cells labeled with [99mTc]d,1-HM-PAO containing variable amounts of the ligand or of the 99mTc activity was evaluated by enzymatic tests and by measuring random migration, chemotaxis, phagocytosis, killing, and adhesion. The ultrastructure of labeled cells was studied by electron microscopy. The tracer dose and the HM-PAO concentration did not significantly affect phagocytosis and killing. The results of the other tests remained normal. A maximum labeling efficiency of 80% was reached by incubating the granulocytes for 20 min with 10-20 mCi of [99mTc]d,1-HM-PAO containing 50 micrograms of the ligand in 1 ml of saline. There was only a slow washout of 20% of activity from the labeled cells in 24 hr. The ultrastructure was not influenced by the labeling technique. Proven infection sites of 17 orthopedic patients were clearly visualized. After a short transient lung uptake, there was a clear spleen and moderate liver uptake with early bladder and late prominent colon visualization. Because of the lower cost, favorable radiation dose and more suitable tracer characteristics, this technique is a promising alternative for 111In labeling of white blood cells.

Characterization of technetium-99m-L,L-ECD for brain perfusion imaging, Part 2: Biodistribution and brain imaging in humans.

The safety, biodistribution and kinetics of a new perfusion imaging agent [99mTc-L,L]-ethyl cysteinate dimer (ECD) was evaluated in normal volunteers. Technetium-99m-L,L-ECD is a neutral, lipophilic complex, which is radiochemically pure and stable. Twelve healthy adults were injected with 25-30 mCi of 99mTc-L,L-ECD and imaged periodically for up to 24 hr. Planar imaging showed rapid brain uptake with a peak concentration of 4.9% injected dose and very slow brain washout (approximately 6% per hour during the first 6 hr). Repeat or dynamic tomographic imaging of the brain using either a rotating gamma camera or a multidetector system was performed up to 6 hr postinjection. The distribution of 99mTc-L,L-ECD in the brain did not change and was similar to the pattern seen with other perfusion agents. Background facial areas and lungs cleared rapidly. Peak blood activity was below 10% injected dose at all times and 99mTc-L,L-ECD cleared rapidly through the kidneys. Vital signs, blood and urine chemistries were normal in all volunteers and no adverse reactions were noted. These results suggest that 99mTc-L,L-ECD should be useful for routine assessment of cerebral perfusion in humans.

Comparison of modified technetium-99m albumin and technetium-99m red blood cells for equilibrium ventriculography.

UNLABELLED: A newly developed modified form of 99mTc-labeled human serum albumin reconstituted from a kit (99mTc-dimercaptopropionyl-human serum albumin; 99mTc-DMP-HSA) was prospectively compared to 99mTc-labeled red blood cells (RBC) in patients referred for equilibrium radionuclide ventriculography at rest to evaluate its potential use as a blood-pool imaging agent. METHODS: A Paired comparison between 99mTc-DMP-HSA and either in vitro or in vivo 99mTc-labeled RBC was performed within 2 days in 20 patients'. For each study, two sets of images were acquired, starting at 15 min and 180 min postinjection, respectively. Each set consisted of a gated blood-pool cardiac study and a planar static image centered on the patient's thorax. All data were processed by two independent observers. Early and late postinjection parameters were calculated: ejection fraction (EF) value, activity within the main organs surrounding the left ventricle (LV), ratio of activity between the LV and these surrounding organs for each study separately, and temporal (late/early) evolution of the intraorgan activities and of the LV/organ ratios after decay correction. RESULTS: The images and the visual wall-motion analysis were of good quality with both agents in most patients, without significant image degradation at 180 min postinjection. Calculated EF values were highly comparable with the two tracers. Interobserver variability was 0.17% (RBC) and 1.08% (DMP-HSA) for the early EF value (EF1), and 0.62% (RBC) and 0.27% (DMP-HSA) for the late EF (EF2). Mean difference between EF2 and EF1 was 0.74% (Observer 1) and 0.28% (Observer 2) for 99mTc-RBC, and -2.88% (Observer 1) and -2.07% (Observer 2) for 99mTc-DMP-HSA. When comparing 99mTc-DMP-HSA to 99mTc-RBC the mean difference was 1.27% (Observer 1) and 0.36% (Observer 2) for EF1, and -2.35% (Observer 1) and -1.99% (Observer 2) for EF2. Also, the biodistribution and temporal evolution of the organ repartition of both compounds were stable and similar, with values of late/early activity ratios very close to one for all the studied organs [mean intraorgan ratio: 0.946 for 99mTc-RBC (range: 0.881-1.086) and 0.979 for 99mTc-DMP-HSA (range: 0.914-1.141); mean late/early LV/organ ratio: 0.964 for 99mTc-RBC (range: 0.919-1.016) and 0.967 for 99mTc-DMP-HSA (range: 0.912-1.035)]. CONCLUSION: Paired comparison of kit-prepared 99mTc-DMP-HSA to 99mTc-labeled RBC demonstrated that both agents were very closely related regarding as well the calculated EF value as the in vivo stability up to more than 3 hr postinjection. Technetium-99m-DMP-HSA may constitute a practical and useful replacement for 99mTc-labeled RBC.

Fluoride kinetics of the axial skeleton measured in vivo with fluorine-18-fluoride PET.

UNLABELLED: The aim of this study was to quantify regional bone blood flow and influx rate with PET and [18F]fluoride in patients with metabolic bone disorders. METHODS: Dynamic imaging of the spine or pelvis was performed after administration of 300-370 MBq of 18F-. Plasma clearance of 18F- was determined in blood sampled from the radial artery. A three-compartment model was used to estimate the regional flow and fluoride influx rate. RESULTS: In this preliminary study, fluoride flux (in micromol/min/liter) could be measured regionally. The flux was consistent with the pathophysiology of the studied metabolic disorders and allowed the various disease states to be distinguished. Bone blood flow and influx rate were low in osteoporosis (in the "normal-appearing" bone) and high in Paget's disease. CONCLUSION: With PET and [18F]fluoride, local bone blood flow and fluoride influx rate can be quantified in patients in vivo. Metabolically active zones have an increased influx rate and an accordingly increased flow. In principle, this technique permits classification of bone disorders and has potential for the monitoring of therapy response in metabolic bone disease.

Dynamic antral scintigraphy to characterize gastric antral motility in functional dyspepsia.

UNLABELLED: We evaluated intragastric food distribution and antral motor activity in patients with functional dyspepsia. METHODS: A standard gastric emptying test and dynamic imaging of the antrum were used to characterize gastric antral motility disturbances and to correlate them with total and compartmental gastric emptying in 25 dyspeptic patients. RESULTS: We found a 40% prevalence of gastroparesis in functional dyspepsia. Solid gastric emptying delay is indicated by a prolonged lag phase and an increase in frequency and amplitude of gastric contractions, resulting in nonexpulsive antral contractions and/or antropyloric dyscoordination. Food retention in the distal stomach and antral distention appears to account for patients' dyspeptic symptoms. CONCLUSION: This study demonstrates that scintigraphy not only detects abnormalities of food distribution in the stomach but also provides information on antral motor activity noninvasively. Dynamic antral scintigraphy and compartmental gastric emptying are useful tools to define the pathophysiology of dyspeptic patients with or without gastroparesis.

Ocular penetration of (125I)IVDU, a radiolabeled analogue of bromovinyldeoxyuridine.

Following topical application of (125)IVDU, the radiolabeled analogue of bromovinyldeoxyuridine ([E]-5-[2-bromovinyl]-2'-deoxyuridine), as 0.5% or 0.3% eyedrops, to rabbits, (125I)IVDU appeared in the anterior chamber fluid at drug levels well above the minimum concentration (0.01 microgram/mL) required for inhibition of herpes simplex virus type 1 replication. These findings are consistent with the efficacy of 0.5% bromovinyldeoxyuridine eyedrops in the topical treatment of herpes simplex uveitis.

Scintigraphic evaluation of shape of lung and chest in upright and head-down posture.

We determined the configuration of lungs and chest in six healthy young subjects using anteroposterior and lateral technetium-99m-labeled scintigraphic images obtained in upright and in 90 degree head-down posture at 0, 25, 50, 75, and 100% vital capacity (VC). The lung shape was evaluated from curves relating vertical height vs. cumulative volume of 20 apicodiaphragmatic lung zones of equal height. S-shaped curves were obtained, which, after size normalization, were largely independent of volume or posture (P greater than 0.1). However, the apical zones tended to become relatively wider and the diaphragmatic zones relatively smaller with increasing volume, especially between 0 and 25% VC in upright posture and 0-50% VC in head-down posture. Changing posture from upright to head-down also tended to slightly widen the apical zones and to narrow the diaphragmatic zones, which is in line with a greater intrathoracic penetration of the diaphragm/abdomen. The shape of the chest was evaluated from the ratio of the transverse-thoracic and anteroposterior distances over height. These ratios did not clearly change with posture (P greater than or equal to 0.05) but increased by approximately 30% with decreasing volume (P less than 0.01). The fact that these shape changes of the chest were not accompanied by similar changes in lung shape can be explained mainly by widening of the mediastinum when volume decreases. In conclusion, the shape of the lung and chest are similar in head-down and upright humans, in contrast to the reversal of the apicodiaphragmatic differences in alveolar expansion and in transpulmonary pressure.

Clinical evaluation of 99mTc-DMSA renogram.

Two hundred-two 99mTc-DMSA renograms for urologic problems were evaluated. Some technical aspects of the examination and the value of the scintigraphic depth estimation are discussed. Pre- and postoperative uptake values in patients with renal surgery and sequential postoperative examinations are considered. The value of DMSA renograms in predicting recovery in obstructive uropathy and in deciding to opt for conservative therapy or nephrectomy is discussed.