FLOW LOADED CANINE CAROTID ARTERY: I. A Morphometric Study of Microfilament Bundles in Endothelial Cells

To estimate microfilament bundles in the endothelial cells of flow loaded arteries quantitatively, blood flow change was produced in the common carotid arteries of eleven female beagle dogs (1–2 years of age, 8–10 kg) employing the arteriovenous shunt method between the common carotid artery and external jugular vein. After 1 week, the amount of microfilament bundles was measured with a point counting method on transmission electron microscopical photographs. In the arteries loaded by highly elevated blood flow, the average thickness of microfilament bundles increased over 0.1 μm (non-operative control: 0.01–0.02 μ) and the average longitudinal cut area of a microfilament bundle grew over 0.7 μm² (non-operative control: 0.2 μm²). The number of bundles increased over 1.0 piece (non-operative control: 0.2 pieces) per unit length (μm) in transverse section and increased over 0.5 pieces (non-operative control: 0.2 pieces) in longitudinal section. The average thickness of the bundles significantly correlated to the blood flow increase ratio (the ratio of the blood flow rate of the final measurement to that before anastomosis). We consider that the microfilament bundles are the structure developed to combat the wall shear stress induced by the elevated blood flow.     

The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was ∼300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of ∼5 pS (∼7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 m m ), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (∼11 pS). This approach allowed us to obtain a maximum open probability ( P _o,max) value for the extrasynaptic receptors ( P _o,max= 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 μm², measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of ∼900 μm⁻² in the postsynaptic membrane.     

The structural effect of systemic NGF treatment on permanently axotomised dorsal root ganglion cells in adult rats

The effect of systemic NGF treatment on loss and shrinkage of dorsal root ganglion cells was studied in adult male rats after permanent axotomy. Nineteen 16 to 18-wk-old rats had their right 5th lumbar spinal nerve ligated and cut approximately 7 mm peripheral to the ganglion. Two days before the operation, treatment with subcutaneous injections of human recombinant NGF (1.0–0.5 mg/kg/day) was started in 9 test rats; 10 controls were given saline injections. After 1 mo the levels of substance P (SP) and calcitonin gene related peptide (CGRP) were significantly increased in intact sciatic nerve. The number and mean volume of perikarya were estimated using assumption-free stereological techniques including vertical sections, the Cavalieri principle, optical disectors, the planar rotator and systematic sampling techniques. Systemic NGF administration had no influence on survival of primary sensory neurons after axotomy. The number of perikarya was 14300 ( S.D. =1800) in axotomised ganglia in control rats versus 14700 ( S.D. =2100) in axotomised ganglia of NGF treated rats. The reduction of perikarya volume after axotomy was significantly less after NGF treatment (11600 μm³ in the control group versus 8000 μm³ in the NGF treated group). However, the apparent protection of NGF-treatment on perikaryal volume is explained by a hitherto unrecognised size effect on nonaxotomised dorsal root ganglion cells. The untreated rats had a mean volume of 24700 μm³ ( S.D. =2700 μm ³) whereas rats treated with NGF had a volume of 20400 μm³ ( S.D. =1700 μm³) on the nonaxotomised side. In conclusion, systemic NGF treatment in adult rats has no effect on dorsal root ganglion cell loss in permanent axotomy whereas perikaryal size of intact nonaxotomised cells is reduced.     

Morphometric studies of secretory granule formation in mouse pancreatic acinar cells. Dissecting the early structural changes following pilocarpine injection

Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2–4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178±0.028 μm³; at 4 h peak value, 0.535±0.109 μm³). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0–2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2–4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone.     

Distribution of tracheal and laryngeal mucous glands in some rodents and the rabbit

We used scanning electron microscopy to count the number of mucous gland openings in the tracheae and lower portion of the larynges of the rat, guinea pig, hamster, mouse and rabbit. Cells of the airway surface epithelium were removed by protease digestion better to visualise the gland openings. The distribution of glands was further studied by conventional histology and by PAS/Alcian blue staining of whole mounts. In all rodent species, gland openings in the larynx occurred with a frequency of 1–2 per mm². Mice had no gland openings in their tracheae, and hamsters, only a handful. Rat tracheae contained 126±42 gland openings (± S.D. ; n = 6) at a frequency of ∼ 0.6 per mm² at the top of the trachea and ∼ 0.15 per mm² at the bottom. Guinea pig tracheae contained 153±90 gland openings (± S.D. ; n = 5), with 54% being in the top 40% of the trachea. In both rat and guinea pig, tracheal glands were found in the ventral aspect between the cartilaginous rings, and were absent from the dorsal membranous portion. Gland openings in most species were simple circles of ∼ 50 μm diameter. However, glands in the rat trachea generally opened obliquely into shallow (∼ 20 μm deep) oval troughs (∼ 150×75 μm), which had their long axes oriented from head to tail. In the rabbit, there was no evidence of tracheal or laryngeal glands histologically. However, the tracheal and laryngeal surfaces contained numerous pits (∼ 30 μm diameter) distributed evenly over and between cartilages at a frequency of ∼ 4 per mm². These may correspond to the 'nests' of goblet cells described by others.     

Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide-binding domains (NBD1 and NBD2) that control its ATP-dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464 A) using the patch clamp technique. K464A mutants have an ATP dependence (EC₅₀≈ 60 μ m ) and opening rate at 2.75 m m ATP (∼ 2.1 s⁻¹) similar to wild type (EC₅₀≈ 97 μ m ; ∼ 2.0 s⁻¹). However, K464A's closing rate at 2.75 m m ATP (∼ 3.6 s⁻¹) is faster than that of wild type (∼ 2.1 s⁻¹), suggesting involvement of NBD1 in nucleotide-dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP's apparent on-rate and acceleration of its apparent off-rate (∼ 2- and ∼ 10-fold, respectively). Since the delay of closing by AMP-PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (∼ 0.055 s⁻¹) and closing (∼ 0.006 s⁻¹) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A-K1250A double mutants close ∼ 5-fold faster (∼ 0.029 s⁻¹) than K1250A but open with a similar rate (∼ 0.059 s⁻¹), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide-dependent closing, which provides important constraints for NBD-mediated gating models.     

Focal Segmental Glomerular Hyalinosis and/or Sclerosis in Rats with Congenital Unilateral Hydronephrosis

Focal segmental glomerular hyalinosis and/or sclerosis (FSHS) was observed in five Wistar-Imamichi rats with congenital unilateral hydronephrosis (CUH rats). Marked proteinuria (164.9+138.4mg/day) was observed in the CUH rats. Immunoperoxidase staining for IgM, C3 and IgG was positive in the glomerull, showing in a focal, segmental pattern that corresponded to the areas of FSHS seen by light microscopy. These glomerular findings were extremely similar to those of human focal glomerular sclerosis (FGS). FSHS was found to be common to both the hydronephrotic kidney and the contralateral kidney without hydronephrosis. Morphometry revealed that the glomerular area of the juxtamedullary glomeruli was greater than that of superficial glomeruli in control rats (11,037 μm² vs. 6,847 μm²). On the other hand, glomerular hypertrophy was observed in non-sclerotic glomeruli of CUH rats (superficial glomeruli; 12,477–16,123 μm², juxtamedullary glomeruli; 14,635–18,418 μm²). Also, a decreased in the number of glomeruli within the range 1.8-4.1 per unit area (1 mm²) was seen in CUH rats compared with control rats (mean 4.4). These results suggest that the increased rate of development of FSHS is based on hyperfiltration in the remaining functional nephrons. Acta Pathol Jpn 41: 653–660, 1991.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

High throughput protein expression screening in the nervous system – needs and limitations

The cellular complexity of the brain (some estimate that there are up to 10³ different cell types) is exceeded by the synaptic complexity, with each of the ∼10¹¹ neurons in the brain having around 10³–10⁴ synapses. Proteomic studies of the synapse have revealed that the postsynaptic density is the most complex multiprotein structure yet identified, with ∼10³ different proteins. Such studies, however, use brain tissue with many different regions and therefore different cell types, and there is clear potential for heterogeneity of protein content at different synapses within and between brain regions. Although large-scale mRNA-based assays are in progress to map this sort of complexity at the cellular level, and indeed all brain-expressed genes, analysis of protein distribution (at synapses and other structures) is still in the very early stages. We review existing large-scale protein expression studies and the specific technical obstacles that need to be overcome before applying the scaling used in nucleic acid based approaches.     

Size of neocortical neurons in control subjects and in Alzheimer's disease

The aim of the present study was to estimate mean neuronal volume and absolute size distributions of the neocortical neurons in brains from controls and AD patients using stereological methods based on unbiased principles to determine whether changes in absolute cell size are part of the neuropathological pattern of Alzheimer's disease. The neocortex of 8 patients with Alzheimer's disease (AD), mean age 81·1 (68–94) y was compared with 9 nondemented controls, mean age 80·9 (65–101) y. The brains came from Johns Hopkins University Hospital (JHUH) in Baltimore, USA, the Netherlands Brain Bank (NBB), and from a large brain repository in Denmark. The rotator method was used to obtain an estimate of cell volumes providing absolute size distributions of the volume of both cell perikaryon and cell nuclei. The geometric mean volume of cell nuclei in neocortical neurons was 328 μm³ (interindividual CV = 0·15) in the Alzheimer group compared with 277 μm³ (interindividual CV = 0·17) in controls which was a statistically significant increase ( P = 0·049). The perikaryal volume was 1117 μm³ in the Alzheimer group compared with 999 μm³ in controls which was a nonsignificant difference ( P = 0·20). There was a highly significant correlation between the nuclear and perikaryal volumes in all individuals. The average slope of the regression lines was significantly higher in the Alzheimer patients than in the controls, illustrating that nuclear hypertrophy was more pronounced in the largest neurons.     

Mast cell-cholinergic nerve interaction in mouse airways

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 μg ml⁻¹) caused histamine release (∼70% total tissue content), and smooth muscle contraction that was rapid in onset and short-lived ( t _1/2 < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 μ m -ketanserin (5-HT₂ receptor antagonist) and strongly inhibited by atropine (1 μ m ). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient ( sash −/−) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient ( sash −/−) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT₂ receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.     

Action potential initiation and propagation in hippocampal mossy fibre axons

Dentate gyrus granule cells transmit action potentials (APs) along their unmyelinated mossy fibre axons to the CA3 region. Although the initiation and propagation of APs are fundamental steps during neural computation, little is known about the site of AP initiation and the speed of propagation in mossy fibre axons. To address these questions, we performed simultaneous somatic and axonal whole-cell recordings from granule cells in acute hippocampal slices of adult mice at ∼23°C. Injection of short current pulses or synaptic stimulation evoked axonal and somatic APs with similar amplitudes. By contrast, the time course was significantly different, as axonal APs had a higher maximal rate of rise (464 ± 30 V s⁻¹ in the axon versus 297 ± 12 V s⁻¹ in the soma, mean ± s.e.m. ). Furthermore, analysis of latencies between the axonal and somatic signals showed that APs were initiated in the proximal axon at ∼20–30 μm distance from the soma, and propagated orthodromically with a velocity of 0.24 m s⁻¹. Qualitatively similar results were obtained at a recording temperature of ∼34°C. Modelling of AP propagation in detailed cable models of granule cells suggested that a ∼4 times higher Na⁺ channel density (∼1000 pS μm⁻²) in the axon might account for both the higher rate of rise of axonal APs and the robust AP initiation in the proximal mossy fibre axon. This may be of critical importance to separate dendritic integration of thousands of synaptic inputs from the generation and transmission of a common AP output.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Sensitivity of coherent oscillations in rat hippocampus to AC electric fields

The sensitivity of brain tissue to weak extracellular electric fields is important in assessing potential public health risks of extremely low frequency (ELF) fields, and potential roles of endogenous fields in brain function. Here we determine the effect of applied electric fields on membrane potentials and coherent network oscillations. Applied DC electric fields change transmembrane potentials in CA3 pyramidal cell somata by 0.18 mV per V m⁻¹ applied. AC sinusoidal electric fields have smaller effects on transmembrane potentials: sensitivity drops as an exponential decay function of frequency. At 50 and 60 Hz it is ∼0.4 that for DC fields. Effects of fields of ≤ 16 V m⁻¹ peak-to-peak (p-p) did not outlast application. Kainic acid (100 n m ) induced coherent network oscillations in the beta and gamma bands (15–100 Hz). Applied fields of ≥ 6 V m⁻¹ p-p (2.1 V m⁻¹ r.m.s.) shifted the gamma peak in the power spectrum to centre on the applied field frequency or a subharmonic. Statistically significant effects on the timing of pyramidal cell firing within the oscillation appeared at distinct thresholds: at 50 Hz, 1 V m⁻¹ p-p (354 mV m⁻¹ r.m.s.) had statistically significant effects in 71% of slices, and 0.5 V m⁻¹ p-p (177 mV m⁻¹ r.m.s.) in 20%. These threshold fields are consistent with current environmental guidelines. They correspond to changes in somatic potential of ∼70 μV, below membrane potential noise levels for neurons, demonstrating the emergent properties of neuronal networks can be more sensitive than measurable effects in single neurons.     

Sequence variants in the 5' flanking region of the leptin gene are associated with obesity in women

Few mutations have been found in the human leptin gene and the relationship between leptin gene sequence variation and human overweight is uncertain. To determine whether sequence variation within the leptin gene and its regulatory elements contribute to extreme obesity, we screened ∼3 kb of the 5' flanking region and the three exons in 125 unrelated extremely obese (BMI ≥ 40 kg/m²) and 86 average weight women (BMI < 27 kg/m²). Within the protein coding regions only one heterozygous silent mutation was found (codon 102; AAC/AAT). Within the 5' flanking region, six frequent sequence variants were detected ( q > 0.10), and the allele frequencies of three of these variants differed between obese and average weight Caucasian women (+19, χ²= 4.46, p = 0.035; −1823, χ²= 4.36, p = 0.037; −2548, χ²= 5.73, p = 0.017). Nine infrequent sequence variants were detected ( q < 0.05) but they did not occur more often among obese women compared with those of average-weight. For extremely obese women, three polymorphisms (+19, −188, and −633) predicted the degree of obesity. Allelic variants may influence the regulation of the leptin gene and thereby influence body weight, particularly among extremely obese women. However, given the low variability in coding regions and the high variability in the 5' flanking region, discerning the functional significance of each variant is likely to be difficult.     

Slow removal of Na+ channel inactivation underlies the temporal filtering property in the teleost thalamic neurons

It has been previously shown that the 'large cell' in the corpus glomerulosum (CG) of a teleost brain has a low-pass temporal filtering property. It fires a single spike only in response to temporally sparse synaptic inputs and thus extracts temporal aspects of afferent activities. To explore the ionic mechanisms underlying this property, we quantitatively studied voltage-gated Na⁺ channels of the large cell in the CG slice preparation of the marine filefish by means of whole-cell patch clamp recordings in the voltage-clamp mode. Recorded Na⁺ current was well described using the Hodgkin-Huxley ' m³h ' model. It was revealed that the Na⁺ channels have a novel feature: remarkably slow recovery from inactivation. In other words, the time constant for the ' h ' gate was extremely large (∼100 ms at −80 to −50 mV). In order to test whether the analysed Na⁺ current serves as a mechanism for filtering, the behaviour of the membrane model incorporating the Na⁺ channel was simulated using a computer program called NEURON. In response to current injections, the membrane model displayed low-pass filtering and firing properties similar to those reported in real cells. The present results suggest that slow removal of Na⁺ channel inactivation serves as a crucial mechanism for the low-pass temporal filtering property of the large cell. The simulation study also suggested that velocity and/or amplitude of a spike propagating though an axon expressing Na⁺ channels of this type could potentially be modulated depending on the preceding activities of the cells.     

Difference in the three-dimensional structure of the sinusoids between hepatocellular carcinoma and cirrhotic liver

The purpose of the present study was to estimate the difference in three-dimensional (3-D) structure of sinusoids between hepatocellular carcinoma (HCC) and cirrhotic liver, by the use of topology. Ten surgically resected lesions of HCC and 10 lesions of liver cirrhosis (LC) were used. Computer-alded reconstruction models of HCC sinusoids and LC sinusoids were developed from 20 4μm thick serial tissue sections from each specimen. A topologlcal invariant, called the first Bettl numberp., was used to estimate the complexity degree of the 3-D sinusoidal structure. The mean p, of the sinusoidal network in the examined tissue, 200X200X80 μm³ in size, was 46.5X33.0 In 10 HCC and 84.9 ±19.1 in 10 cirrhotic livers. There was a statistically significant difference between the two values (P<0.01), while there was no significant difference in the sinusoidal volume of the same size tissue between the HCC and the cirrhotic liver. It was found, therefore, that the sinusoidal network of HCC was more sparsely and coarsely knit in 3-D space than that of the cirrhotic liver.     

Somatic Exocytosis of Serotonin Mediated by L-Type Calcium Channels in Cultured Leech Neurones

We studied somatic exocytosis of serotonin and its mediation by L-type calcium (Ca²⁺) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 m m potassium (K⁺), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca²⁺ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 m m K⁺. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ . In the presence of the L-type Ca²⁺ channel blocker nimodipine (10 μ m ), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (   P < 0.05  ). Subsequent incubation with 10 m m caffeine to induce Ca²⁺ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N-, P-, Q- or invertebrate Ca²⁺ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.