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Biomedical subjects

M R Bennett

Publications and source records attributed to M R Bennett.

At least 145 records · Page 8Linked to original sources
PubMed1995

Trophic effect of collicular proteoglycan on neonatal rat retinal ganglion cells in situ.

Naturally occurring neuronal death is widespread in the central nervous system of mammals. To date, the causes and mechanisms of such death are poorly understood. A major hypothesis is that developing neurons compete for limited amounts of trophic factor(s) released from their target centres as in the case of the peripheral nervous system and nerve growth factor. The present study aims to test this 'trophic hypothesis' in the mammalian central nervous system. In the rat, more than 50% of retinal ganglion cells die in the early post-natal period. Schulz and coworkers [57] purified a potential trophic agent from their major target, the superior colliculus, which was identified as a 480 kDa chondroitin sulfate proteoglycan. This proteoglycan or control solutions were injected into the eyes of rat pups during the post-natal part of the period of naturally occurring ganglion cell death. It was found that the collicular proteoglycan prevented the death of a significant number of the ganglion cells that would normally have been lost over a post-injection period of one or two days. The effect of the proteoglycan was dose- and time-dependent. These results support the notion that trophic interactions are a determining factor in the survival of retinal ganglion cells during the period of naturally occurring cell death. It is also the first time that a proteoglycan has been shown to possess neurotrophic properties in situ.

Animals↗
PubMed1995

Effect of collicular proteoglycan on the survival of adult rat retinal ganglion cells following axotomy.

Consistent with numerous previous studies, we have found that in adult rats 29% of cells retrogradely prelabelled by injections into retino-recipient nuclei are lost 1 week after intraorbital section of the optic nerve. This figure increases to 76% 2 weeks after axotomy. Intraocular injections of 150 ng of 480 kDa chondroitin sulphate proteoglycan purified from the superior colliculi of neonatal rats were performed every third day after axotomy. This procedure resulted in the loss of only 3 and 28% of the axotomized retinal ganglion cells 7 and 14 days respectively after optic nerve section. Intraocular injections of chondroitin sulphate type C, one of the sugar types present on the collicular proteoglycan, also resulted in a significant saving of axotomized ganglion cells (with the loss of only 48% 14 days after optic nerve lesion). These findings suggest that the collicular proteoglycan, and to a lesser extent its sugar moieties, substantially slows down the degeneration of adult retinal ganglion cells following axotomy.

Animals↗
PubMed1995

Electrotonic coupling between two CA3 hippocampal pyramidal neurons: a distributed cable model with somatic gap-junction.

A model of a pair of electrotonically coupled CA3 hippocampal pyramidal neurons is presented. Each neuron is represented by a tapered equivalent cable attached to an isopotential soma. The synaptic potential in a neuron soma is determined as a consequence of electrical coupling to another soma that receives a synaptic input on its dendritic tree. Estimates of the coupling resistances, soma input resistances and soma-to-dendritic tree conductance ratio show that a substantial current may arise in a neuron as a consequence of synaptic activity in a neuron coupled to it. The small increase in decay time due to coupling in the model indicates that actual coupling is between more than just pairs of neurons.

Animals↗
PubMed1995

The origin of Gaussian distributions of synaptic potentials.

Spontaneous synaptic potentials were identified at the motor endplate 40 years ago. These were shown to possess amplitudes that could be described by a Gaussian distribution as could the amplitudes of evoked synaptic potentials under conditions of very low probability for secretion. As these Gaussians were identical, the idea of a unit or quantum of transmission was conceived. The failure to obtain similar Gaussian distributions for both spontaneous and low-probability evoked potentials during development of endplates indicated that a unit of transmission was not operating. However both the spontaneous and very low-probability evoked potentials could each be described by mixtures of Gaussians indicating a subunit of transmission might be operative. There are no ganglionic or central synapses at which comparisons have been made between spontaneous and low-probability evoked potentials that show each can be described by a Gaussian distribution, let alone that these are the same indicating a unit of transmission as originally conceived. There is some evidence that mixtures of Gaussians can be used to describe both spontaneous and very low-probability evoked synaptic potential amplitudes, opening up the possibility for a subunit of transmission at these synapses. The vesicle hypothesis, that the quantum of transmission at the endplate is due to the exocytosis of the contents of a synaptic vesicle, was also enunciated nearly 40 years ago. The existence of subunits of transmission has required reconsideration of this hypothesis. Three alternatives are considered: in one, the calcium-transient hypothesis, the subunit of secretion is due to the release of calcium from one of several calcium stores in the nerve terminal, so that several subunits are released when a number of these calcium stores are engaged in a regenerative response to the terminal action potential; a second alternative, the mediatophore hypothesis, is that a subunit of secretion occurs when a single transmitter transport protein channels transmitter across the terminal membrane, several such mediatophore proteins acting in concert then give multiple subunit release; finally, there is the vesicle fusion-pore hypothesis, in which individual transient openings of a fusion-pore channel joining a synaptic vesicle to the terminal membrane are responsible for secretion of a transmitter subunit, with multiple transients giving several subunits. Perhaps we will have distinguished between these possibilities before the quantal hypothesis is 50 years old.

Animals↗
PubMed1995

Probabilistic secretion of quanta: spontaneous release at active zones of varicosities, boutons, and endplates.

The amplitude-frequency histogram of spontaneous miniature endplate potentials follows a Gaussian distribution at mature endplates. This distribution gives the mean and variance of the quantum of transmitter. According to the vesicle hypothesis, this quantum is due to exocytosis of the contents of a single synaptic vesicle. Multimodal amplitude-frequency histograms are observed in varying degrees at developing endplates and at peripheral and central synapses, each of which has a specific active zone structure. These multimodal histograms may be due to the near synchronous exocytosis of more than one vesicle. In the present work, a theoretical treatment is given of the rise of intraterminal calcium after the stochastic opening of a calcium channel within a particular active zone geometry. The stochastic interaction of this calcium with the vesicle-associated proteins involved in exocytosis is then used to calculate the probability of quantal secretions from one or several vesicles at each active zone type. It is shown that this procedure can account for multiquantal spontaneous release that may occur at varicosities and boutons, compared with that at the active zones of motor nerve terminals.

Animals↗
PubMed1995

Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.

Adenosine Triphosphatases↗
PubMed1995

Effect of P2-purinoceptor antagonists on glutamatergic transmission in the rat hippocampus.

1. A study has been made of the effects of P2-purinoceptor antagonists on the evoked excitatory postsynaptic currents (e.p.s.cs) generated in CA1 pyramidal cells on stimulation of Schaffer collaterals and in CA3 pyramidal cells on stimulation of mossy fibres. The effects of these antagonists on currents generated in the cells on application of glutamate has also been determined. 2. Suramin blocked the evoked e.p.s.cs with an 50% inhibition (ID50) of 62 +/- 8 microM (mean +/- s.e.mean, n = 17), spontaneous miniature e.p.s.cs and the currents induced by application of 100 microM glutamate with an ID50 = 121 +/- 36 microM (n = 15) in all the cells studied. 3. Reactive Blue 2 (RB-2) in a concentration of 200 microM decreased the e.p.s.cs by 80 +/- 10% (n = 6) and the glutamate-activated currents by 83 +/- 3% (n = 6). 4. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) in the concentration-range of 40-500 microM decreased the amplitude of the e.p.s.cs in 12 out of 13 cells studied. PPADS at 200 microM reduced the amplitude of the e.p.s.cs by 60 +/- 10% (n = 3). PPADS did not affect the glutamate-induced currents in 4 cells and produced potentiation of the current amplitude by 60 +/- 10% in 4 other cells. 5. These results suggest that both presynaptic and postsynaptic P2-purinoceptors in the hippocampus can modulate the release and action of endogenous glutamate.

Animals↗
PubMed1995

Autoradiography of P2x ATP receptors in the rat brain.

1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosine triphosphate (2-methyl-S-ATP) and 2-chloro-adenosine triphosphate (2-C1-ATP) nor 2-methylthio-adenosine diphosphate (2-methyl-S-ADP) a ligand for the P2 receptor on blood platelets ('P2T' type) produced strong inhibitions except for P1,P4-di(adenosine-5')tetraphosphate (Ap4A). 3. Inhibitors of Na+,K(+)-dependent adenosine triphosphatase (ATPase) ouabain, P1-ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4. The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation.

Adenosine Diphosphate↗
PubMed1995

Apoptosis of rat vascular smooth muscle cells is regulated by p53-dependent and -independent pathways.

Apoptosis of vascular smooth muscle cells has recently been described in culture and also in remodeling of the artery after birth. However, the genes that regulate apoptosis in smooth muscle cells are mostly unknown. We studied the regulation of apoptosis in rat smooth muscle cells stably infected with retrovirus constructs containing c-myc, adenovirus E1A, bcl-2, and a temperature-sensitive mutant of the tumor suppressor gene p53. Apoptosis was verified by electron microscopy and quantified by time-lapse videomicroscopy. Death was induced by c-myc and E1A when cells were deprived of serum survival factors, bcl-2 suppressed apoptosis of cells infected with c-myc and E1A and also normal smooth muscle cells. Overexpression of wild-type p53 induced apoptosis of cells infected with E1A and c-myc but not normal cells. In contrast, expression of mutant p53, which blocks wild-type p53 function, suppressed apoptosis of cells infected with E1A or c-myc but not normal cells. Both adenovirus E1A and c-myc increased the expression of endogenous p53 protein but not p53 mRNA. Although bcl-2 suppressed apoptosis induced by E1A and c-myc, upregulation of p53 protein induced by these agents was unaffected. We conclude that apoptosis of vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Death induced by c-myc and E1A is mediated by, and dependent on, p53. However, the suppression of apoptosis by bcl-2 is not mediated by changes in p53 expression, and the low level of apoptosis seen in normal VSMCs upon removal of survival factors is independent of p53.

Analysis of Variance↗
PubMed1995

Binding and phagocytosis of apoptotic vascular smooth muscle cells is mediated in part by exposure of phosphatidylserine.

Apoptosis of vascular smooth muscle cells has recently been demonstrated to occur in vitro and in vivo. Uptake of apoptotic cells into adjacent normal cells appears to be rapid and specific. We have investigated binding and phagocytosis of apoptotic vascular smooth muscle cells by normal smooth muscle cell monolayers. Vascular smooth muscle cells were infected with the proto-oncogene c-myc or the adenovirus E1A gene, induced to undergo apoptosis in low-serum conditions, and then incubated with normal smooth muscle cells. Apoptosis was accompanied by a marked increase in exposure of phosphatidylserine on the outer surface of the cell, which was recognized by binding to annexin V. Liposomes containing phosphatidylserine but not phosphatidylinositol inhibited uptake of apoptotic cells in a dose-dependent manner to a maximum of 50% inhibition; annexin V also inhibited the uptake of apoptotic cells in a dose-dependent and calcium-dependent manner. Binding of apoptotic bodies did not appear to be mediated by endogenous annexin V, as evidenced by the inability of an antibody to annexin V to inhibit uptake. Smooth muscle cells were also able to recognize exposed phosphatidylserine on other cell types, as judged by their ability to bind erythrocytes having a high degree of exposed phosphatidylserine. We conclude that smooth muscle cells express phosphatidylserine during apoptosis, and this exposure partly mediates binding and phagocytosis of dead cells. This mechanism may be important in promoting rapid cell removal in the vessel wall.

Analysis of Variance↗
PubMed1995

Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques.

We studied death of human vascular smooth muscle cells derived from coronary plaques and normal coronary arteries and aorta. Cells from normal arteries underwent death only upon removal of serum growth factors. In contrast, plaque-derived cells died even in high serum conditions, and death increased after serum withdrawal. Death was characteristically by apoptosis in both normal and plaque-derived cells, as determined by time-lapse videomicroscopy, electron microscopy, and DNA fragmentation patterns. IGF-1 and PDGF were identified as potent survival factors in serum, whereas EGF and basic fibroblast growth factor had little effect. Stable expression of bcl-2, a protooncogene that regulates apoptosis in other cell lines, protected smooth muscle cells from apoptosis, although there was no detectable difference in endogenous bcl-2 expression between cells from plaques or normal vessels. We conclude that apoptosis of human vascular smooth muscle cells is regulated by both specific gene products and local cytokines acting as survival factors. Apoptosis may therefore regulate cell mass in the normal arterial wall and the higher rates of apoptosis seen in plaque smooth muscle cells may ultimately contribute to plaque rupture and breakdown and thus to the clinical sequelae of atherosclerosis.

Adolescent↗
PubMed1994

Nitric oxide modulation of quantal secretion in chick ciliary ganglia.

1. Long-term potentiation of quantal secretion was studied at ciliary ganglion synapses of post-hatched birds following tetanic stimulation of the oculomotor nerve and the effects of nitric oxide (NO) on quantal secretion were determined. 2. Tetanic stimulation of the oculomotor nerve at 30 Hz for 20 s at room temperature increased the amplitude of the excitatory postsynaptic potential (EPSP) by about 100%; 1-2 min after the tetanus the EPSP declined exponentially with a time constant of about 10 min (long-term potentiation; LTP). LTP was due to an increase in the quantal content of the EPSP not to a change in quantal size. 3. A component of LTP was shown to be due to the release of NO in the ganglion, as blocking the synthesis of NO with L-arginine methyl ester decreased the potentiation by 70%. 4. Exogenous application of NO using sodium nitroprusside increased the amplitude of the EPSP by more than 30% due to an increase in the quantal content of the EPSP. 5. Both 8-bromo-cGMP and 8-bromo-cAMP increased the quantal content of the EPSP by more than 44% without changing the quantal size. 6. The results suggest that endogenous NO is involved in either the initiation or maintenance phase of LTP. This may occur through an increase in quantal secretion consequent on the action of an elevated cGMP increasing cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

The pattern of expression of a 13-kDa protein (CS-32 antigen) in the retina and its target nuclei in developing and mature rats.

A polyclonal antibody (designated CS-32) immunopurifies a 13-kDa protein from the superior colliculus (SC) of neonatal rat. There is a strikingly inverse temporal relationship in expression of the 13-kDa protein in the developing retina and its principal target nuclei, SC and the dorsal lateral geniculate nucleus. The 13-kDa is a cell-surface protein expressed exclusively by neurones and is clearly associated with synapses.

Animals↗
PubMed1994

Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.

1. The effects of calcium on the secretion of quanta recorded from single varicosities on the surface of the mouse vas deferens has been determined. 2. If recordings were made from two adjacent varicosities and the [Ca2+]o increased from a low value (1 mM), then the increase in the mean quantal content of the endplate potential (m(e)) was almost entirely due to an increase in the binomial probability for secretion (pe). At higher [Ca2+]o there was an increasing tendency for the binomial parameter (ne) to increase from 1 to 2. When ne increased there was very little change in pe, indicating that the new release site recruited from the other varicosity has a relatively low probability for secretion. 3. If recordings were restricted to single varicosities and the [Ca2+]o increased in the range from 1 to 4 mM then the increases in m(e) were almost always due to an increase in pe. The gradient relating the log of m(e) to the log of [Ca2+]o for [Ca2+]o of 1.0-1.5 mM was in the range 3.2-5.4 (mean 4.2). 4. Test impulses gave a similar proportional increase in m(e) following a conditioning impulse at all varicosities from which recordings were made. 5. Facilitation of m(e) declined exponentially with an increase in the test-conditioning interval from 0.5 to 4 s. The time constant for this decline was about 6 s. 6. The results indicate that single release sites show a similar fourth power dependency on [Ca2+]o and facilitate to about the same degree following a conditioning impulse.

Animals↗
PubMed1994

Down-regulation of the c-myc proto-oncogene in inhibition of vascular smooth-muscle cell proliferation: a signal for growth arrest?

Vascular smooth muscle (VSM) cell proliferation contributes to the pathogenesis of atherosclerosis, restenosis after angioplasty and vein graft disease. The regulation of genes involved in VSM cell proliferation, particularly by naturally occurring inhibitors, is therefore of some importance. We have investigated the role of the c-myc proto-oncogene in growth arrest of exponentially proliferating rat VSM cells, following mitogen withdrawal, treatment with heparin (50 micrograms/ml), interferon-gamma (IFN-gamma) (100 i.u./ml), or the cyclic nucleotide analogues, 8-bromo-adenosine-3'5'-cyclic monophosphate (8-Br-cAMP; 0.1 mM) and 8-bromoguanosine-3'5'-cyclic monophosphate (8-Br-cGMP; 0.1 mM). Growth arrest was accompanied by down-regulation of c-Myc protein and mRNA following treatment with all inhibitors. Serum withdrawal or IFN-gamma treatment suppressed c-myc expression by more than 50% within 2 h, and this occurred throughout the cell cycle. Platelet-derived growth factor, epidermal growth factor and basic fibroblast growth factor all contributed independently to the maintenance of c-myc expression. Heparin, 8-Br-cAMP or 8-Br-cGMP also suppressed c-myc, but this occurred later, after 24-48 h, and was also observed following arrest by metabolic block. We conclude that c-myc expression is linked to VSM cell growth arrest in response to endogenous regulators and metabolic block. Down-regulation of c-myc expression may thus be an essential part of the arrest programme in VSM cells induced by many pharmacological agents.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Are glia among the cells that express immunoreactive c-Fos in the suprachiasmatic nucleus?

Light physiologically regulates the levels of c-Fos in the suprachiasmatic nucleus (SCN), but the identity of most of the cells that express this DNA-binding protein is unknown. To determine whether SCN c-Fos expression is restricted to neurons, we performed light microscopic double-label immunohistochemistry on sections of rat SCN using two c-Fos antibodies in conjunction with antibodies to neurofilament and S-100 proteins (as markers of neuronal and astrocytic cell bodies, respectively). In addition to c-Fos labeled nuclei in neurons, we found a small population of labeled nuclei in glial cell bodies. These observations suggest that c-Fos is expressed in a heterogeneous cell population in the SCN and that its function in circadian clock mechanisms may be cell specific.

Animals↗
PubMed1994

Effect of yohimbine on the contractile response of the mouse vas deferens to short, low frequency trains of nerve impulses.

A study was undertaken to ascertain why in the mouse vas deferens excitatory junction potentials facilitate whereas contractions depress during low frequency stimulation. In a set of contraction studies, where a conditioning stimulus was followed 2 s later by a test stimulus, depression in the first phase of contraction was observed only at high stimulus strengths when many nerves are activated leading to a large secretion. Low stimulus strengths did not produce depression, suggesting that the autoinhibitory effect due to released noradrenaline is absent. At supramaximal stimulus strengths short, low frequency (0.1-0.5 Hz) trains gave varying degrees of depression of the first phase at each stimulus frequency. This depression was reversed by yohimbine (10 microM), indicating that the level of autoinhibition depends on the total amount of noradrenaline secreted. In the absence of autoinhibition, facilitation could be predicted in terms of the residual Ca2+ hypothesis.

Adrenergic alpha-2 Receptor Antagonists↗