Perforated patch-clamp analysis of the passive membrane properties of three classes of hippocampal neurons.

1. Perforated patch-clamp recordings were made from the three major classes of hippocampal neurons in conventional in vitro slices prepared from adult guinea pigs. This technique provided experimental estimates of passive membrane properties (input resistance, RN, and membrane time constant, tau m) determined in the absence of the leak conductance associated with microelectrode impalement or the washout of cytoplasmic constituents associated with conventional whole-cell recordings. 2. To facilitate comparison of our data with previous results and to determine the passive membrane properties under conditions as physiological as possible, recordings were made at the resting potential, in physiological saline, and without any added blockers of voltage-dependent conductances. 3. Membrane-potential responses to current steps were analyzed, and four criteria were used to identify voltage responses that were the least affected by activation of voltage-dependent conductances. tau m was estimated from the slowest component (tau 0) of multiexponential fits of responses deemed passive by these criteria. RN was estimated from the slope of the linear region in the hyperpolarizing direction of the voltage-current relation. 4. It was not possible to measure purely passive membrane properties that were completely independent of membrane potential in any of the three classes of hippocampal neurons. Changing the membrane potential by constant current injection resulted in changes in RN and tau 0; subthreshold depolarization produced an increase, and hyperpolarization a decrease, in both RN and tau 0 for all three classes of hippocampal neurons. 5. Each of the three classes of hippocampal neurons also displayed a depolarizing "sag" during larger hyperpolarizing voltage transients. To evaluate the effect of the conductances underlying this sag on passive membrane properties, 2-5 mM Cs+ was added to the physiological saline. Extracellular Cs+ effectively blocked the sag in all three classes of hippocampal neurons, but the effect of Cs+ on RN, tau 0, and the voltage dependence of these parameters was unique for each class of neurons. 6. CA1 pyramidal neurons had an RN of 104 +/- 10 (SE) M omega and tau 0 of 28 +/- 2 ms at a resting potential of -64 +/- 2 mV (n = 12). RN and tau 0 were larger at more depolarized potentials in these neurons, but the addition of Cs+ to the physiological saline reversed this voltage dependence. 7. CA3 pyramidal neurons had an RN of 135 +/- 8 M omega and tau 0 of 66 +/- 4 ms at a resting potential of -64 +/- 1 mV (n = 14).(ABSTRACT TRUNCATED AT 400 WORDS)     

The expanding phenotype of laminin alpha2 chain (merosin) abnormalities: case series and review

Initial reports of patients with laminin alpha2 chain (merosin) deficiency had a relatively homogeneous phenotype, with classical congenital muscular dystrophy (CMD) characterised by severe muscle weakness, inability to achieve independent ambulation, markedly raised creatine kinase, and characteristic white matter hypodensity on cerebral magnetic resonance imaging. We report a series of five patients with laminin alpha2 deficiency, only one of whom has this severe classical CMD phenotype, and review published reports to characterise the expanded phenotype of laminin alpha2 deficiency, as illustrated by this case series. While classical congenital muscular dystrophy with white matter abnormality is the commonest phenotype associated with laminin alpha2 deficiency, 12% of reported cases have later onset, slowly progressive weakness more accurately designated limb-girdle muscular dystrophy. In addition, the following clinical features are reported with increased frequency: mental retardation (~6%), seizures (~8%), subclinical cardiac involvement (3-35%), and neuronal migration defects (4%). At least 25% of patients achieve independent ambulation. Notably, three patients with laminin alpha2 deficiency were asymptomatic, 10 patients had normal MRI (four with LAMA2 mutations reported), and between 10-20% of cases had maximum recorded creatine kinase of less than 1000 U/l. LAMA2 mutations have been identified in 25% of cases. Sixty eight percent of these have the classical congenital muscular dystrophy, but this figure is likely to be affected by ascertainment bias. We conclude that all dystrophic muscle biopsies, regardless of clinical phenotype, should be studied with antibodies to laminin alpha2. In addition, the use of multiple antibodies to different regions of laminin alpha2 may increase the diagnostic yield and provide some correlation with severity of clinical phenotype.     

How many screens does a CT workstation need?

A considerable number of prototype and commerical workstations have been developed during the last 10 years for electronic display of computed tomographic (CT) images during clinical interpretation. These CT workstations have varied widely in the number and size of monitors available for the display of the medical images ranging from a single 1,024×1,204-pixel monitor, to eight 2,500×2,000-pixel monitors. Image display times also have varied considerably, ranging from as fast as. 11 seconds, to as slow as 26 seconds to fill a single monitor. No consensus has formed in the workstation community with regard to display area and response time requirements. To address this issue, we have constructed a time-motion model of CT interpretation. Model accuracy is experimentally verified with three workstations as well as with the film alternator. In general, CT interpretations with an electronic workstation become faster as display area increases and display time decreases. Results can be used by workstation designers and purchasers to roughly estimate differences in interpretation speeds among contending CT workstation designs.     

Drug resistance in the fission yeast Schizosaccharomyces pombe: pleiotropic mutations affecting the oleic acid and sterol composition of cell membranes

Summary The whole cell lipid and sterol content of the drug resistant strains cyh1, cyh3 and cyh4 was compared with that of wild type by thin layer and gas liquid chromatography and by UV spectrophotometric analysis. The cyh3 and cyh4 strains had a decreased content of the unsaturated 18:1 fatty acid oleic acid, a decreased content of ergosterol and an increased content of 24,28 dehydroergosterol with respect to wild type. The cyh1 strain, however, only showed a decreased content of ergosterol and an increased content of 24,28 dehydro-ergosterol when compared to wild type.     

Selection for accelerated penetration in cell culture coselects for attenuated mutants of Venezuelan equine encephalitis virus

Previous studies with Sindbis virus (SB) suggested that a single point mutation in glycoprotein E2 (serine 114 to arginine 114) conferred three phenotypic alterations: attenuation in neonatal mice, accelerated penetration of cultured cells, and efficient neutralization by two E2-specific monoclonal antibodies (Davis, Fuller, Dougherty, Olmsted, and Johnston (1986) Proc. Natl. Acad. Sci. USA 83, 6771-6775). Moreover, selection for rapidly penetrating mutants of SB coselected for attenuation in vivo, indicating that a domain of SB E2 which influences penetration in culture overlaps an E2 domain which influences pathogenesis (Olmsted, Meyer, and Johnston (1986) Virology 148, 245-254). To test the possibility that overlapping penetration and pathogenesis domains exist in other alphaviruses, the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus (TRD-VEE) was serially passed in baby hamster kidney (BHK) cells under a stringent selective pressure for accelerated penetration. Isolates were biologically cloned from the first through the fourth passages and were characterized as to penetration time course in BHK cells and virulence in adult mice following intraperitoneal inoculation. Twenty-two of the 27 isolates segregated into two major categories: slowly penetrating and virulent (like the TRD-VEE parent) and rapidly penetrating and avirulent. Mice which received the avirulent mutants were positive for anti-VEE neutralizing antibody and were refractory to challenge with TRD-VEE. Of the seven mouse avirulent mutants, two also were attenuated in hamsters, indicating the presence of at least two genetic loci at which mutations may influence both pathogenesis and penetration.     

Contrast Limited Adaptive Histogram Equalization image processing to improve the detection of simulated spiculations in dense mammograms

The purpose of this project was to determine whether Contrast Limited Adaptive Histogram Equalization (CLAHE) improves detection of simulated spiculations in dense mammograms. Lines simulating the appearance of spiculations, a common marker of malignancy when visualized with masses, were embedded in dense mammograms digitized at 50 micron pixels, 12 bits deep. Film images with no CLAHE applied were compared to film images with nine different combinations of clip levels and region sizes applied. A simulated spiculation was embedded in a background of dense breast tissue, with the orientation of the spiculation varied. The key variables involved in each trial included the orientation of the spiculation, contrast level of the spiculation and the CLAHE settings applied to the image. Combining the 10 CLAHE conditions, 4 contrast levels and 4 orientations gave 160 combinations. The trials were constructed by pairing 160 combinations of key variables with 40 backgrounds. Twenty student observers were asked to detect the orientation of the spiculation in the image. There was a statistically significant improvement in detection performance for spiculations with CLAHE over unenhanced images when the region size was set at 32 with a clip level of 2, and when the region size was set at 32 with a clip level of 4. The selected CLAHE settings should be tested in the clinic with digital mammograms to determine whether detection of spiculations associated with masses detected at mammography can be improved.Key Words: mammography, image processing, contrast limited adaptive histogram equalization, observer studies, breast cancer, spiculations     

Development of action potentials and apamin-sensitive after-potentials in mouse vestibular nucleus neurones

The postnatal maturation of medial vestibular nucleus (MVN) neurones was examined in slices of the dorsal brainstem prepared from balb/c mice at specific stages during the first postnatal month. Using spike-shape averaging to analyse the intracellularly recorded action potentials and after-hyperpolarisations (AHPs) in each cell, all the MVN neurones recorded in the young adult (postnatal day 30; P30) mouse were shown to have either a single deep AHP (type A cells), or an early fast and a delayed slow AHP (type B cells). The relative proportions of the two subtypes were similar to those in the young adult rat. At P5, all the MVN cells recorded showed immature forms of either the type A or the type B action potential shape. Immature type A cells had broad spontaneous spikes, and the characteristic single AHP was small in amplitude. Immature type B cells had somewhat narrower spontaneous spikes that were followed by a delayed, apamin-sensitive AHP. The delayed AHP was separated from the repolarisation phase of the spike by a period of isopotentiality. Over the period P10–P15, the mean resting potentials of the MVN cells became more negative, their action potential fall-times became shorter, the single AHP in type A cells became deeper, and the early fast AHP appeared in type B cells. Until P15 cells of varying degrees of electrophysiological maturity were found in the MVN but by P30 all MVN cells recorded were typical adult type A or type B cells. Exposure to the selective blocker of SK-type Ca-activated K channels, apamin (0.3 μM), induced depolarising plateaux and burst firing in immature type B cells at rest. The duration of the apamin-induced bursts and the spike frequency during the bursts were reduced but not abolished after blockade of Ca channels in Ca-free artificial cerebrospinal fluid containing Cd²⁺. By contrast, in mature type B cells at rest apamin selectively abolished the delayed slow AHP but did not induce bursting activity. Apamin had no effect on the action potential shape of immature type A cells. These data show that the apamin-sensitive I _AHP is one of the first ionic conductances to appear in type B cells, and that it plays an important role in regulating the intrinsic rhythmicity and excitability of these cells. Received: 19 November 1996 / Accepted: 30 June 1997     

Occurrence and distribution of putative neurotransmitters in the frog-lung parasiteHaplometra cylindracea (Trematoda: Digenea)

The localistion and distribution of the cholinergic, serotoninergic and peptidergic components of the nervous system of the frog-lung flukeHaplometra cylindracea have been determined by the application of standard enzyme cytochemical and immunocytochemical techniques to cryostat sections and whole-mount preparations. Cholinesterase activity (ChE), as indicative of acetylcholine, has been demonstrated cytochemically in the CNS and PNS; however, the anterior ganglia were notably unreactive. The occurrence of serotonin was examined by an indirect immunofluorescence technique, and immunoreactivity (IR) was demonstrable in small, paired anterior ganglia and in fine nerve fibres associated with the somatic muscle, cirrus and gonopore. The peptidergic protion of the nervous system was investigated using antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide, and was visualised by both indirect immunofluorescence and confocal scanning laser microscopy. Positive immunostaining occurred with antisera raised against pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP), peptide histidine isoleucine (PHI) and FMRFamide. Immunoreactivity to PP, PYY and FMRFamide was widespread throughout the nervous system and was evident in large, paired anterior ganglia, the dorsal commissure, main nerve tracts and the extensive array of small fibres that constitute the PNS. In contrast, the distribution of nerves immunoreactive to SP and PHI was less apparent, with PHI-IR occurring exclusively within the fibrous neuropile of the ganglia and in fibres of the ventral nerve cord. Results are discussed with respect to the distribution of the various neurochemical elements and their roles as putative neurotransmitters and/or regulatory molecules.     

Effect of glycine and glycine receptor antagonists on NMDA-induced brain injury

In postnatal day 7 rats, a unilateral intrastriatal injection of 12.5 nmol of N-methyl-D-aspartate (NMDA) reproducibly injures the ipsilateral striatum, adjacent hippocampus and overlying cortex. The severity of injury can be quantified by comparing cerebral hemisphere weights in animals sacrificed 5 days after the injection. Co-injection of NMDA and the glycine receptor antagonists kynurenic acid (KYN) or 7-chlorokynurenic acid (7-CKA) reduced the severity of NMDA-induced damage in a dose-dependent fashion. One hundred nmol of KYN with 12.5 nmol of NMDA reduced average % damage from 19.3 +/- 0.9% (n = 9) to 2.3 +/- 0.5% (n = 6), P less than 0.001, ANOVA. Co-injection of 40 nmol of 7-CKA with 12.5 nmol of NMDA (n = 6) reduced average % damage from 17.1 +/- 1.6% (n = 15) to 3.0 +/- 0.6%, P less than 0.001, ANOVA. Concurrent injection of 1000 nmol glycine with 5 nmol NMDA did not increase the extent of NMDA-induced damage. Our results demonstrate that glycine receptor antagonists attenuate NMDA-induced brain injury in vivo.