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Somatostatin-immunoreactive interneurons contribute to lateral inhibitory circuits in the dentate gyrus of control and epileptic rats.

Lateral inhibition, a feature of neuronal circuitry that enhances signaling specificity, has been demonstrated in the rat dentate gyrus. However, neither the underlying neuronal circuits, nor the ways in which these circuits are altered in temporal lobe epilepsy, are completely understood. This study examines the potential contribution of one class of inhibitory interneurons to lateral inhibitory circuits in the dentate gyrus of both control and epileptic rats. The retrograde tracer wheat germ ag-glutinin-apo-horse radish peroxidase-gold (WGA-apo-HRP-gold) was injected into the septal dentate gyrus. Neurons double-labeled for glutamic acid decarboxylase (GAD) and the retrograde tracer are concentrated in the hilus and may contribute to lateral inhibition. Neurons double-labeled for somatostatin and the retrograde tracer account for at least 28% of GAD-positive neurons with axon projections appropriate for generating lateral inhibition in control rats. Despite an overall loss of somatostatin-expressing cells in epileptic animals, the number of somatostatin-positive interneurons with axon projections appropriate for generating lateral inhibition is similar to that seen in controls. These findings suggest that somatostatinergic interneurons participate in lateral inhibitory circuits in the dentate gyrus of both control and epileptic rats, and that surviving somatostatinergic interneurons might sprout new axon collaterals in epileptic animals.

Animals↗

Improved assessment of lumbar vertebral body strength using supine lateral dual-energy x-ray absorptiometry.

Clinical and biomechanical investigations indicate that assessment of vertebral body bone mineral density (BMD) by anteroposterior dual-energy x-ray absorptiometry (DXA) is a useful index of vertebral body strength and fracture risk in osteoporosis. However, inclusion of non-force-bearing and small-force-bearing mineralized structures, such as the posterior elements and aortic calcifications, in the measurement of anterior BMD obscures the assessment of vertebral body mass by this technique. Indeed, such interference is particularly severe in the presence of posterior element degeneration or previous spinal surgery. Recent anatomic studies illustrate that the lateral view provides unobstructed visualization of the L3, L4, and possibly L2 vertebral bodies, suggesting that supine lateral BMD may more accurately assess vertebral body fracture risk. We evaluated this hypothesis in a blinded using human cadaver spines to compare the value of supine lateral and anteroposterior BMD in assessing vertebral body fracture force, average compressive stress, maximum stored strain energy, and strain at failure. Both measures of BMD significantly correlate with these biomechanical measures. However, statistical comparison of the methods using multiple and stepwise regression reveals that supine lateral BMD provides a better assessment of the vertebral body fracture properties than anteroposterior BMD. The enhanced predictive value of supine lateral BMD occurs because of the variable contribution of posterior element mineral to the anteroposterior BMD measurement. Evaluation to test the utility of supine lateral BMD for the assessment of fracture risk and a fracture threshold in patients with osteoporosis is therefore recommended.

Absorptiometry, Photon↗

Signal detection theory, lateral-line excitation patterns and prey capture behaviour of mottled sculpin.

The frequency with which blinded mottled sculpin, Cottus bairdi, oriented towards a dipole current source (50-Hz vibrating sphere) was measured as a function of source distance (2-18 cm) and azimuth (either 0 degrees in front or 90 degrees to the side of the fish). The orienting frequency declined from over 70% to under 50% as source distance increased from 4 to 12 cm for both frontal and lateral sources. When response biases (frequency of responding in the absence of the signal) were taken into account with the performance metric d', threshold distances (distances at which d' fell to 1) for frontal (12.5 cm) and lateral (11.6 cm) sources were 1.35-1.45 times the mean standard length of fish used in this study. At distances less than 8 cm, d' values were considerably higher (i.e. performance was better) for the lateral source, despite the fact that peak stimulus levels at the fish were twice as high for frontal as for lateral sources at any given distance. Performance differences may be related to differences in spatial excitation patterns, in particular the distribution of opposing pressure gradient directions along the lateral-line system, present for lateral sources, but absent for frontal sources. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗

Lateral clustering of cadherin-4 without homophilic interaction: possible involvement in the concentration process at cell-cell adhesion sites as well as in the cell adhesion activity.

It is thought that the concentration of classic cadherins at cell-cell adhesion sites is essential for generating strong cell-cell adhesion activity, but the mechanism is not well understood. To clarify the structural basis of the concentration process and the cell adhesion activity, we constructed various mutants of cadherin-4 and examined the adhesion properties of the transfectants. A deletion mutant lacking the entire cytoplasmic domain had weak, but significant Ca(2+)-dependent cell adhesion activity. Interestingly, the deletion mutant showed intrinsic cluster formation in the absence of cell-cell adhesion, possible lateral cluster formation. The cytoplasmic domain-deleted cadherin-4 containing the mutation of Trp-2 to Ala, which is known to inhibit the strand dimer formation required for the cell-cell adhesion, retained the possible activity of lateral cluster formation, supporting this notion. These results suggest that the extracellular domain has intrinsic activity of lateral cluster formation. Indeed, deletion of a cadherin repeat in the extracellular domain significantly reduced or abolished the lateral cluster formation as well as the concentration of cadherin-4 at cell-cell contact sites and cell adhesion activity. When transfectants of the cytoplasmic domain-deleted cadherin-4 made cell-cell contact and formed intimate cell-cell adhesion, the lateral clusters of cadherin-4 initially gathered at cell-cell contact sites, and a smooth linear concentration was gradually formed along the cell-cell adhesion interface. The results suggest that the lateral cluster formation is involved in the concentration process of cadherin-4 at cell-cell adhesion sites, hence in the strong cell adhesion activity of cadherin-4 as well.

Amino Acid Substitution↗

Lateral bending of the lumbar spine during quadrupedalism in strepsirhines.

Much research has been devoted to spinal kinematics of nonmammalian vertebrates, while comparatively little is known about the locomotor role of spinal movements in mammals, especially primates. This study, conducted at the Duke University Primate Center, examines the function of lateral spinal bending during quadrupedal walking among a diverse sample of strepsirhines. The taxa studied include Loris tardigradus (1), Nycticebus coucang (1), N. pygmaeus (1), Cheirogaleus medius (2), Varecia variegata (2), Eulemur fulvus (2), and a total sample size of 261 strides. Lateral bending varies among the taxa with respect to both magnitude and effects of velocity, and does not appear to be correlated with body size. In addition, the timing of lateral bending during a stride appears to differ from that reported for other (nonmammalian) tetrapods. On average, maximum lateral flexion occurs just after ipsilateral foot touchdown, which may be functionally associated with touchdown of the contralateral forelimb during diagonal sequence gait. For some of the taxa, lateral flexion coincides more closely with foot touchdown as velocity increases, suggesting a functional role in increasing hindlimb stride length. Both of these timing patterns contrast with those reported for lizards. Finally, although lorids as a group have been described as having a "sinuous" gait, this study shows more pronounced lateral flexion in Nycticebus than in Loris.

Animals↗

Lack of evidence for connexin 43 gene mutations in human autosomal recessive lateralization defects.

Heterotaxy is the failure of the developing embryo to establish normal left-right asymmetry, which is often associated with multiple malformations. Previous studies have identified different mutations in the cytoplasmic tail of the connexin 43 (cx 43) gene in six patients from a series of six sporadic cases with defects of laterality and severe heart malformations. These cases showed that of the genes involved in lateralization defects with autosomal recessive transmission, cx 43 was the most important. This result was challenged by two different teams, which, on sequencing only the carboxyl terminal end of the cx 43 gene in 30 patients, found no mutations. To assess the responsibility of the cx 43 gene in human autosomal recessive lateralization defects, we tested its involvement in a selected group of 25 patients (19 familial cases) with a wide variety of lateralization defects and cardiovascular malformations. The whole coding sequence and direct flanking sequences were screened for mutations, both by single strand conformation analysis and direct fluorescent sequencing. We could only detect a single base pair insertion in the 3' untranslated region of one patient. To test the possibility of mutations in other parts of the cx 43 gene, the gene was located onto the physical map of chromosome 6, and flanking polymorphic markers were genotyped. Haplotype analysis excluded the cx 43 gene locus in nearly all of the familial cases of lateralization defects. Thus, our results do not support the suggestion that this gene is implicated in human autosomal recessive lateralization defects.

Abnormalities, Multiple↗

Lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility.

Consideration of the arrangement of actin filaments in the lamellipodia of crawling cells indicates that, in addition to a rearward flow of the actin cytoskeleton due to treadmilling, there is a lateral flow of filaments in both directions. The existence of such a lateral flow of actin filaments is supported by observation of the lateral movement of actin filament bundles in fibroblasts and by fluorescence photoactivation data. In addition to explaining the formation, dispersal and lateral movement of filament bundles, lateral filament flow could add a further velocity component to the centripetal flow of cytoplasm. As such, lateral filament flow may explain the discrepancies in the rates of the rearward flow of actin, cytoplasmic materials and dorsal particles.

Actins↗

Pattern of afferents to the lateral septum in the guinea pig.

The septal region represents an important telencephalic center integrating neuronal activity of cortical areas with autonomous processes. To support the functional analysis of this brain area in the guinea pig, the afferent connections to the lateral septal nucleus were investigated by the use of iontophoretically applied horseradish peroxidase (HRP). Retrogradely labeled perikarya were located in telencephalic, diencephalic, mesencephalic and metencephalic sites. The subnuclei of the lateral septum (pars dorsalis, intermedia, ventralis, posterior) receive afferents from the (i) medial septal nucleus, diagonal band of Broca (pars horizontalis and pars ventralis), and the principal nucleus of the stria terminalis, the hippocampus, and amygdala (nucleus medialis): (ii) the medial habenular nucleus, and the para- (peri-) ventricular, parataenial and reuniens nuclei of the thalamus; the anterior, lateral and posterior hypothalamic areas (in particular, the medial and lateral preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, premammillary, and supramammillary nuclei; (iii) the periaquaeductal grey, ventral tegmental area, nucleus interfascicularis, nucleus reticularis linearis, central linear nucleus, interpeduncular nucleus; (iv) dorsal and medial raphe complex, and locus coeruleus. Each subnucleus of the lateral septum displays an individual, differing pattern of afferents from the above-described regions. Based on a double-labeling method, the vasopressinergic and serotonergic afferents to the lateral septum were found to originate in the nucleus paraventricularis hypothalami and the raphe nuclei, respectively.

Animals↗

Lateral and medial sub-divisions within the olivocerebellar zones of the paravermal cortex in lobule Vb/c of the cat anterior lobe.

The olivocerebellar projection to the c1, c2 and c3 zones in the paravermal cortex of lobule Vb/c has been investigated in the cat using a combined electrophysiological/neuroanatomical tracing technique. The zonal boundaries in the paravermal cortex were located by recording, on the cerebellar surface, climbing fibre field potentials evoked in response to percutaneous stimulation of one or more paws. A small (10-30 nl) injection of WGA-HRP was then made either into the centre or into the medial or lateral geographical half of a chosen zone and the resultant distribution of retrogradely labelled cells within the contralateral inferior olive was plotted. The c1 and c3 zones were each found to consist of two mediolaterally oriented 'sub-zones' which could be distinguished by their olivocerebellar input. The medial part of the c1 zone received climbing fibre input from the rostromedial part of the dorsal accessory olive (DAO) while the lateral part of the c1 zone received climbing fibre input from middle/rostral regions of the medial accessory olive (MAO). Both medial and lateral 'sub-zones' within the c3 zone were found to receive climbing fibre input from the rostral pole of DAO but, whereas there was heavy overlap between the olivary territories projecting to the medial c1 and medial c3 subzones, olivary cells projecting to the lateral part of c3 were located more rostrally within DAO. The c2 zone was found not to be divisible into mediolaterally oriented subzones and to receive climbing fibre input from a region of MAO located rostral and somewhat lateral to the region projecting to the lateral part of the c1 zone. The sub-zonal organisation of the olivocerebellar projection to the c1, c2 and c3 zones is discussed in relation to the functional properties of the different zones.

Animals↗

Subcortical projections to the lateral geniculate body in the rat.

The subcortical projections to the lateral geniculate body (LGB) in the rat were studied by means of discrete HRP iontophoretic deposits in the dorsal or the ventral LGB; the labelling was compared to that resulting from HRP deposits in neighboring nuclei. After injecting HRP in the dorsal LGB, labelled cells appeared bilaterally in the ventral LGB, pretectum, superior colliculus, lateral groups of the dorsal raphe nucleus and locus coeruleus. Ipsilaterally, labelled cells were found in the lateral posterior thalamus, nucleus of the posterior commissure and deep mesencephalic reticular nucleus. After injecting HRP into the ventral LGB, labelled cells were observed bilaterally in the pretectum, superior colliculus and dorsal raphe nucleus (lateral groups). Contralateral labelling appeared in the ventral LGB and parabigeminal nucleus. Ipsilateral labelling was found in the zona incerta, lateral posterior thalamus, lateral and medial mesencephalic reticular formation, vestibular and dorsal tegmental nuclei. These findings provide evidence of subcortical projections to the LGB arising in visually-related areas as well as extravisual areas, which might be related to the LGB boutons that survive complete cortical and retinal ablations.

Animals↗

Lateral asymmetry of constitutive heterochromatin in human chromosomes.

Variations in lateral asymmetry of constitutive heterochromatin were studied in 30 normal individuals with reference to the chromosomal regions 1q12, 9q12, 15p11, 16q12 and Yq12. The technique consisted of growing human lymphocytes for one cell cycle in BrdU, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 x SSC, and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin in these chromosomal regions. Chromosomes 15, 16, and Y showed simple lateral asymmetry, whereas chromosome 1 showed both simple and compound asymmetry. In 15 cases, compound lateral asymmetry was evident in both homologues of chromosome 1, 12 cases showed compound lateral asymmetry in one homologue and simple lateral asymmetry in both the homologues. The centromere region of chromosome 9 stained symmetrically with this technique. The lateral asymmetry is presumed to reflect the strand bias in the distribution of thymine in satellite DNA fractions.

Bromodeoxyuridine↗

Postnatal sequential development of dopaminergic and enkephalinergic perineuronal formations in the lateral septal nucleus of the rat correlated with local neuronal maturation.

Tyrosine-hydroxylase (TH-IR) and methionine-enkephalin like immunoreactivity (MetE-IR) were analyzed in the lateral septal nucleus (LSN) of the rat from birth (PO) to adulthood. TH-IR labeled specifically the dopaminergic (DA) pericellular arrangements of the LSN, as checked by negative dopamine-beta-hydroxylase and phenylethanolamine-N-methyl transferase-IR. TH-IR and Met-IR processes were present at birth in the medial LSN and extended lateralwards and caudalwards from P0 to P6 to constitute two main DA terminal fields (medial and lateral) surrounding a MetE one. Within these fields, the development of perineuronal baskets followed a similar medial to lateral sequence: DA axons first surrounded a few neuronal cell bodies at P3 in the medial part of the intermediate LSN; at P6, Met-IR axons encircled more laterally located perikarya, and only at P9, some neurons located along the ventricle in the lateral DA field became surrounded. The initial aspect of TH-IR baskets consisting of few axons surrounding the cell body rapidly evolved in a positive network encapsulating the perikaryon and long segments of the proximal dendrites, whereas MetE-IR varicosities remained restricted around the perikaryon and the initial dendritic segments. Ultrastructural study at P14 revealed numerous TH-IR and MetE-IR axosomatic and axodendritic profiles. TH-IR axosomatic varicosities exhibited asymmetrical synapses, whereas MetE-IR ones displayed rare symmetrical contacts. The medio-lateral gradient of development of the perineuronal baskets was parallel to the postnatal neuronal development of the LSN as evaluated by cytological criteria: neuronal density, cell size and Nissl staining. Therefore, the formation of DA and MetE perineuronal arrangements in the LSN does not seem to be subordinate to the nature of the neurotransmitter they contain but related to the level of differentiation of their target neurons. A similar sequential set-up in the development of afferences paralleling the neuronal differentiation is discussed.

Animals↗

The efferent connections of the lateral septal nucleus in the guinea pig: projections to the diencephalon and brainstem.

The anterograde Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing technique was used to determine the distribution of efferent fibers originating in the lateral septal nucleus of the guinea pig. For complementary detection of the chemical identity of the target neurons, double-labeling immunocytochemistry was performed with antibodies to PHA-L and to vasopressin, oxytocin, vasoactive intestinal polypeptide, serotonin or dopamine beta-hydroxylase, respectively. The hypothalamus received the majority of the PHA-L-stained septofugal fibers. Here, a specific topography was observed. (1) The medial and lateral preoptic area, (2) the anterior, lateral, dorsal, posterior hypothalamic and retrochiasmatic area, (3) the supraoptic, paraventricular, suprachiasmatic, dorsomedial, caudal ventromedial and arcuate nuclei, and (4) the tuberomammillary, medial and lateral supramammillary, dorsal and ventral premammillary nuclei always contained PHA-L-labeled fibers. The rostral portion of the ventromedial nucleus and the medial and lateral mammillary nucleus only occasionally showed weak terminal labeling. In other diencephalic areas, termination of PHA-L-labeled fibers was observed in the epithalamus and the nuclei of the midline region of the thalamus. In the mesencephalon, terminal varicosities occurred in the ventral tegmental area, interfascicular and interpeduncular nucleus, and periaqueductal gray. In addition, the dorsal and medial raphe nuclei of the metencephalon, together with the locus coeruleus and the dorsal tegmental nucleus, received lateral septal efferents.

Animals↗

The lateral line mechanoreceptive mesencephalic, diencephalic, and telencephalic regions in the thornback ray, Platyrhinoidis triseriata (Elasmobranchii).

Central lateral line pathways were mapped in the thronback ray, Platyrhinoidis triseriata, by analyzing depth profiles of averaged evoked potentials (AEPs), multiunit activity (MUA), and single unit recordings. Neural activity evoked by contra- or ipsilateral posterior lateral line nerve (pLLN) shock is restricted to the tectum mesencephali, the dorsomedial nucleus (DMN) and anterior nucleus (AN) of the mesencephalic nuclear complex, the posterior central thalamic nucleus (PCT), the lateral tuberal nucleus of the hypothalamus, and the deep medial pallium of the telencephalon (Figs. 2, 3, 4, 6, 7). Neural responses (AEPs and MUA) recorded in different lateral line areas differ with respect to shape, dynamic response properties, and/or latencies (Figs. 9, 10 and Table 1). Ipsilaterally recorded mesencephalic and diencephalic AEPs are less pronounced and of longer latency than their contralateral counterpart (Fig. 9 and Table 1). In contrast, AEP recorded in the telencephalon show a weak ipsilateral preference. If stimulated with a low amplitude water wave most DMN, AN, and tectal lateral line units respond in the frequency range 6.5 Hz to 200 Hz. Best frequencies (in terms of least displacement) are 75-150 Hz with a peak-to-peak water displacement of 0.04 micron sufficient to evoke a response in the most sensitive units (Fig. 11A, B, C). DMN and AN lateral line units have small excitatory receptive fields (RFs). Anterior, middle, and posterior body surfaces map onto the rostral, middle, and posterior brain surfaces of the contralateral DMN (Fig. 12). Some units recorded in the PCT are bimodal; they respond to a hydrodynamic flow field--generated with a ruler approaching the fish--only if the light is on and the eye facing the ruler is left uncovered (Fig. 13).

Animals↗

Physiology of lateral line mechanoreceptive regions in the elasmobranch brain.

The physiology of mechanoreceptive lateral line areas was investigated in the thornback guitarfish, Platyrhinoidis triseriata, from medulla to telecephalon, using averaged evoked potentials (AEPs) and unit responses as windows to brain functions. Responses were analysed with respect to frequency sensitivity, intensity functions, influence of stimulus repetition rate, response latency, receptive field (RF) organization and multimodal interaction. 1. Following a quasi-natural vibrating sphere stimulus, neural responses were recorded in the medullary medial octavolateralis nucleus (MON), the dorsal (DMN) and anterior (AN) nucleus of the mesencephalic nuclear complex, the diencephalic lateral tuberal nucleus (LTN), and a telencephalic area which may correspond to the medial pallium (Figs. 2, 3, 13, 14, 15, 16). 2. Within the test range of 6.5-200 Hz all lateral line areas investigated responded to minute water vibrations. Best frequencies (in terms of displacement) were between 75 and 200 Hz with threshold values for AEPs as low as 0.005 microns peak-to-peak (p-p) water displacement calculated at the skin surface (Fig. 6). 3. AEP-responses to a vibrating sphere stimulus recorded in the MON are tonic or phasic-tonic, i.e., responses are strongest at stimulus onset but last for the whole stimulus duration in form of a frequency following response (Fig. 3). DMN and AN responses are phasic or phasic-tonic. Units recorded in the MON are phase coupled to the stimulus, those recorded in the DMN, AN or LTN are usually not (Figs. 5, 8, 9). Diencephalic LTN and telencephalic lateral line responses (AEPs) often are purely phasic. However, in the diencephalic LTN tonic and/or off-responses can be recorded (Fig. 11). 4. For the frequencies 25, 50, and 100 Hz, the dynamic intensity range of lateral line areas varies from 12.8 to at least 91.6 dB (AEP) respectively 8.9 and 92 dB (few unit and single unit recordings) (Fig. 7). 5. Mesencephalic, diencephalic, and telecephalic RFs, based on the evaluation of AEPs or multiunit activity (MUA), are usually contralateral (AN and LTN) or ipsi- and contralateral (telencephalon) and often complex (Figs. 10, 12, 16). 6. In many cases no obvious interactions between different modalities (vibrating sphere, electric field stimulus, and/or a light flash) were seen. However, some recording sites in the mesencephalic AN and the diencephalic LTN showed bimodal interactions in that an electric field stimulus decreased or increased the amplitude of a lateral line response and vice versa (Fig. 13 B).

Action Potentials↗

Microsurgical anatomy and operative technique for extreme lateral lumbar disc herniations.

The anatomy of the lateral aspect of the lumbar spine and our lateral microsurgical technique for extreme lateral lumbar disc herniations (ELLDH) is described. This study was based on the microdissection of 4 cadavers, on the morphometric evaluation of these as well as 6 dried cadaver spines and 8 lumbar CT scans, and on the use of this technique on over 200 cases. Level dependent changes in the posterior arch cause a shift of the disc space distally relative to the facet joint, an increasing amount of bone to overlie the intervertebral foramen, and a decreasing amount of working space within the exposure in the caudal direction. Therefore, more bone removal from the lateral aspect of the pars interarticularis and supero-lateral aspect of the facet joint is required in the lower lumbar spine. When the exposed ligamentum flavum is resected, the dorsal root ganglion is seen and access to the herniation and disc space is achieved. Level dependent changes in the pedicles and transverse processes lead to an alteration in the course and relationships of the nerves, thereby influencing the pathophysiology of and surgical technique for the ELLDH. The operative target is the lateral aspect of the pars interarticularis and not the intertransverse space as has been previously described. Our techniques allows for the early identification of the nerve with minimal risks of injury to it, to the adjacent vessels and to the structural integrity of the facet joint and pars interarticularis.

Humans↗

Radiographic assessment of venous catheter position in children: value of the lateral view.

Frontal chest radiographs can be misleading in the evaluation of central venous catheter placement. Lateral chest radiographs were obtained, in addition to the standard frontal radiographs, in 25 young children. In five (20%) of the children, the catheters were found to be malpositioned, and the frontal radiograph showed evidence of the abnormality in all five. The lateral radiographs also showed evidence of the abnormality in all five children; however, in three of the five, the lateral radiographs provided additional, more specific, diagnostic information. The lateral chest radiographs also demonstrated satisfactory catheter positioning in three other children in whom the frontal radiographs suggested abnormal positioning. Accurate assessment of catheter position was possible in all patients using both frontal and lateral chest radiographs. Injection of contrast material was not necessary to locate malpositioned catheters. Lateral radiographs are recommended whenever an abnormal catheter position is suspected clinically or from findings on the routine frontal radiograph.

Catheterization↗

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium.

The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Normarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with 125I. The basal-lateral components yielded a hetero-disperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochrome c reductase activities, were separated from the radio-iodine labeled by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 x g x 1 hr after removal of the mitochrondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.

Animals↗