Biomedical subjects

M R Bennett

Publications and source records attributed to M R Bennett.

At least 127 records · Page 7Linked to original sources

PubMed1996

Quantal transmitter release at somatic motor-nerve terminals: stochastic analysis of the subunit hypothesis.

Here we analyze the problem of determining whether experimentally measured spontaneous miniature end-plate currents (MEPCs) indicate that quanta are composed of subunits. The properties of MEPCs at end plates with or without secondary clefts at the neuromuscular junction are investigated, using both stochastic and deterministic models of the action of a quantum of transmitter. It is shown that as the amount of transmitter in a quantum is increased above about 4000 acetylcholine (ACh) molecules there is a linear increase in the size of the MEPC. It is possible to then use amplitude-frequency histograms of such MEPCs to detect a subunit structure, as there is little potentiation effect above 4000 ACh molecules. Autocorrelation and power spectral analyses of such histograms establish that their subunit structure can be detected if the coefficient of variation of the subunit size is less than about 0.12 or, if electrical noise is added, about 0.1. Positive gradients relate the rise time and half-decay times of MEPCs to their amplitude, even in the absence of potentiating effects; these gradients are shallower at motor nerve terminals that possess secondary clefts. The effect of asynchronous release of subunits is also investigated. The criteria determined by this analysis for identifying a subunit composition in the quantum are applied to an amplitude-frequency histogram of MEPCs recorded from a small group of active zones at a visualized amphibian motor-nerve terminal. This did not provide evidence for a subunit structure.

Acetylcholine↗

PubMed1996

In vitro stimulation of c-Fos protein expression in the suprachiasmatic nucleus of hypothalamic slices.

Light regulates the c-fos protooncogene in the suprachiasmatic nucleus (SCN), with increased expression after animals are exposed to light during the dark phase of a light-dark cycle or during the subjective night in constant darkness. To determine whether this phase-dependent activation of c-Fos persists in an acute in vitro preparation, we prepared horizontal slices of hamster ventral hypothalamus, electrically stimulated the still-attached optic nerves, recorded the resulting evoked potentials in the SCN, and examined c-Fos protein levels in the nucleus by immunohistochemistry. The number of SCN cells labeled for immunoreactive c-Fos was significantly increased in slices stimulated during projected night, but not during projected day, compared to matched, sham-stimulated control slices. These results imply that the phase-dependent mechanism that gates c-Fos photoinduction in vivo is intrinsic to SCN tissue, and they suggest that an in vitro slice preparation will provide a useful model for dissecting the responsible signal transduction elements.

Animals↗

PubMed1996

Autonomic neuromuscular transmission at a varicosity.

This review attempts to clarify the definition of what constitutes an autonomic neuromuscular function formed by a varicosity. Ultrastructural studies of serial sections through varicosities, partly or wholly bare of Schwann cell covering, show that areas of close apposition occur between varicosities and muscle cell membrane that vary between 20 and 150 nm, depending on the muscle considered. Consideration of the diffusion of purine transmitters and their receptor kinetics after secretion in a packet show that the number of purinergic receptor channels opened at a site of 150 nm apposition by a varicosity is about 15% of that at a site of 50 nm apposition. These results, together with the analysis of the stochastic fast component and the deterministic slow components of the rising phase of the EJP suggest that the stochastic fast component is due to varicosities that form especially close appositions (20-50 nm), whereas the deterministic slow component is due to the large number of varicosities at distances up to about 150 nm. Varicosities forming appositions of 20-150 nm with muscle cells several hundred micrometers long possess junctional receptor types distinct from extrajunctional receptors. According to this argument, then, there are two different classes of varicosities: one that gives rise to a relatively large junctional current and another that is responsible for a very small junctional current. Present evidence suggests that two subclasses of varicosities can be discerned amongst the varicosities that generate large junctional currents. One of these subclasses of varicosity possesses relatively few post-junctional receptors compared with the amount of transmitter reaching the receptors from the varicosity, so that the junctional current generated is determined by the size of the receptor population; in this case, the size of the transmitter packages released from these varicosities is unknown and the size of the junctional current is relatively constant. The other subclass of varicosity possesses large receptor patches, sufficient to accommodate the largest amounts of transmitter released from the varicosities: in this case, the size of the transmitter packages is shown to be highly non-uniform. These speculations await confirmation by direct labelling of the receptor patches beneath varicosities, a possibility that is likely to be realized in the near future.

Animals↗

PubMed1996

Cell death in atherosclerotic plaques.

The investigation of the mechanisms of cell death in atherosclerosis has recently received added impetus with the realization that apoptosis is probably the predominant mechanism. This review examines the preliminary data on mechanisms of cell death in the atherosclerotic plaque.

Apoptosis↗

PubMed1996

Unexpected c-fos gene expression in the suprachiasmatic nucleus of mice entrained to a skeleton photoperiod.

Several authors have suggested that the transcriptional regulatory protein c-Fos might be part of the mechanism for photic entrainment of the circadian pacemaker in the suprachiasmatic nucleus (SCN) to environmental light:dark cycles. This hypothesis has been based on evidence gathered using single light pulses administered acutely to animals free-running in constant darkness. In order to begin to analyze SCN c-fos gene expression in animals during steady-state entrainment to photic cycles, we exposed male BALB/c mice to a skeleton photoperiod consisting of two 1-h light pulses separating a long (14 h) and a short (8 h) dark interval. The cycle was designed so that stable entrainment could be achieved in either one of two patterns (with rhythmic locomotor activity occurring during either the long or the short dark interval); SCN c-fos mRNA levels could then be measured during entrainment to light pulses at different phases of the circadian cycle, while controlling for the duration of preceding darkness. We found that c-fos was induced equally well by a light pulse that represented ZT 12 or ZT 3. The ZT 12 pulse functioned as an entraining pulse, because animals free-ran after it was removed from the lighting regimen, whereas removing the ZT 3 pulse caused little or no phase shift of activity onset. The data confirm that the expression of SCN c-Fos is not itself sufficient to reset rhythm phase, and they indicate that the role of this gene in the mechanism of photic entrainment is not yet fully understood.

Animals↗

PubMed1995

Inhibition of [3H]CGP 39653 binding to NMDA receptors by a P2 antagonist, suramin.

Suramin, which has been used for more than 50 years as an anthelmintic in humans, has recently been shown to inhibit P2 purinoceptors in the CNS and to block glutamate-evoked excitatory potentials in hippocampus. We now report that suramin inhibits (IC50 approximately 100 microM) the binding of [3H]CGP 39653, a ligand specific for NMDA-type glutamate receptors in brain. Consequently, suramin at concentrations used sometimes in physiological experiments may act as both P2x and NMDA antagonist. Since both NMDA receptors and P2x-subtype of purinoceptors form Ca(2+)-selective channels, suramin may block Ca2+ entry into neurones depolarized by either ATP, L-glutamate or any other neurotransmitter or drug acting via P2x or NMDA receptors.

2-Amino-5-phosphonovalerate↗

PubMed1995

Calcium in the nerve terminals of chick ciliary ganglia during facilitation, augmentation and potentiation.

1. The calyciform nerve terminals of chick ciliary ganglia were loaded with the calcium indicators calcium green 1 or fura-2. These were used to determine the change in calcium concentration in the terminal, [Ca2+]t, following short (10 impulses) and long (600 impulses) trains of high-frequency (30 Hz) stimulation. 2. Following a single impulse or a short train, the elevated [Ca2+]t declined along two exponentials with time constants similar to slow (F2) facilitation (0.52 s) and augmentation (4.0 s). After a long train elevated [Ca2+]t declined eventually along a single exponential with the time constant of post-tetanic potentiation (162 s). [Ca2+]t was not elevated through long-term potentiation. 3. Addition of Ba2+ (0.75 mM) to the extracellular solution slowed only the decline of [Ca2+]t associated with augmentation. The addition of the nitric oxide donor sodium nitroprusside did not affect [Ca2+]t following short or long trains. 4. Removal of extracellular calcium (buffered with EGTA) and the blockade of calcium channels with Cd2+ completely prevented the changes in [Ca2+]t. 5. The soma of ciliary ganglion cells were loaded with calcium green and the postganglionic nerves stimulated with a single impulse or a short train of impulses. Following stimuli, the elevated [Ca2+]t declined along a single exponential with a time constant similar to F2 facilitation with no augmentation component evident. 6. The results are discussed in terms of the hypothesis that each impulse in a train gives an equal increment of residual Ca2+ to a compartment for secretion and that Ca2+ is removed from the compartment by three first-order kinetics processes associated with F2 facilitation, augmentation and post-tetanic potentiation.

Animals↗

PubMed1995

Statistics of quantal secretion during long trains of sympathetic nerve impulses in mouse vas deferens.

1. A statistical analysis has been made of the occurrence of excitatory junctional currents (EJCs) of similar amplitude recorded with an extracellular electrode during long trains of nerve impulses to the mouse vas deferens. 2. The number of EJCs of similar amplitude that occurred in consecutive impulses during trains of 500-1000 impulses at 0.5-2.0 Hz increased with the number of EJCs evoked during the train. 3. There was no evidence of significant dependence between consecutive EJCs of similar amplitude in sixteen out of eighteen trains in eighteen preparations. 4. The time course of clusters of EJCs of similar amplitude was examined by determining the standard deviation of different groups of EJCs within a cluster throughout their time course. Most EJCs within a cluster could be grouped with a coefficient of variation < 0.1 throughout their time course. 5. The observations on EJCs of similar amplitude leave open the possibility that secretion from single varicosities is, in general, multiquantal.

Animals↗

PubMed1995

NADPH diaphorase expression in the rat retina after axotomy--a supportive role for nitric oxide.

The large majority of mammalian retinal ganglion cells degenerate following section of their axons in the optic nerve. It has been suggested that some axotomized retina ganglion cells die because of toxic agents produced within their immediate environment. Our hypothesis was that nitric oxide might be one of the toxic factors implicated in the death of adult retinal ganglion cells post-axotomy. In the first instance, we determined whether there were any changes in the retinal expression of NADPH diaphorase both 3 and 14 days following intraorbital section of the optic nerve in adult rats. Secondly, if nitric oxide was indeed implicated in the death of ganglion cells, then trophic factors which rescue these neurons might do so by decreasing the expression of nitric oxide synthase. Recently, we found that a collicular proteoglycan purified from the major target of retinal ganglion cells, the superior colliculus, rescued a greater proportion of adult ganglion cells from axotomy-induced death than most other known trophic factors. We thus injected this proteoglycan intraocularly after section of the optic nerve and examined its effect on the expression of NADPH diaphorase in the retina. Thirdly, an inhibitor of nitric oxide synthetase was repeatedly injected into the eye following the section of the optic nerve in order to determine if such a treatment might improve the survival of retinal ganglion cells. The present results indicate that section of the optic nerve does not alter the overall levels of NADPH diaphorase within the adult rat retina. Intraocular injections of the collicular proteoglycan actually increased the number of neurons expressing NADPH diaphorase, particularly in the ganglion cell layer. Finally, inhibition of nitric oxide synthetase following axotomy resulted in increased loss of retinal ganglion cells over a 2 week period when compared with controls. Our findings indicate that, rather than being toxic, small amounts of nitric oxide may be important for the survival of a proportion of injured retina ganglion cells.

Animals↗

PubMed1995

Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan.

The release of nitric oxide and stimulation of glutamate receptors by excitatory amino acids has been linked to neuronal degeneration and toxicity. In the rat retina approximately 60% of retinal ganglion cells (RGCs) die during the first postnatal week. In this study we examined the effects of nitric oxide synthase blockers and glutamate on the survival of neonatal RGCs in vitro over a 16 h assay period. Less than 10% of P1 RGCs survived in serum free defined media alone (control), however survival was increased, in a dose-dependent manner, when L-glutamate (10 microM-10 mM) was added to the media; a maximum of 70% of RGCs could be maintained with the addition of 5 mM glutamate. This effect was blocked by the NMDA and non-NMDA receptor blockers APV and DNQX and was age dependent; the survival of RGCs from P5 but not P7 rats was enhanced by the addition of glutamate even in high calcium concentrations (10 mM). When the nitric oxide synthase blockers L-NAME (5 mM) or haemoglobin (25 microM) were added to the culture media, up to 61% of P1 RGCs survived. The addition of the 480 kDa chondroitin sulfate proteoglycan (SCCP) previously shown to enhance RGC survival in vivo and in vitro, potentiated the action of glutamate and L-NAME and increased RGC survival to over 90% with almost all RGCs expressing a profusion of processes. These results suggest that the release of nitric oxide and glutamate by cells within the retina may contribute to the regulation of RGC numbers in vivo during development.

Animals↗

PubMed1995

Antisense therapy for angioplasty restenosis. Some critical considerations.

The high affinity of even relatively short sequences of DNA for their target mRNA suggests that antisense agents represent an ideal method of suppressing specific gene products both in vitro and in vivo. In experiments performed thus far, an effect on the target mRNA in cultured vascular cells and in the vessel wall can be documented. The in vitro activity, toxicity, and pharmacokinetic data of antisense oligonucleotides are encouraging, and the in vivo animal experiments demonstrating suppression of neointimal formation are very promising. If animals trials presently under way show continued suppression not only of intimal formation but also of loss of lumen caliber after a single application, then effective delivery of antisense oligonucleotides is a realistic possibility. Nevertheless, some words of caution regarding the use of antisense oligonucleotides are warranted. Potential nonspecific effects of antisense oligonucleotides should be carefully considered in studies in which antisense agents are used to define biological functions of specific genes. In particular, demonstration that the target mRNA has been suppressed does not prove that other sequences within the mRNA pool have not also been suppressed. Critical control measures include adding back the target mRNA or protein and demonstrating similar biological effects with antisense sequences, which also suppress target gene expression directed at different regions of the target mRNA. At the clinical level, the systemic effects of antisense oligonucleotides, the dosage required, the timing of administration compared with mechanical intervention, and the toxicity of breakdown products all need to be established. In addition, the most appropriate targets for antisense use in restenosis remain largely obscure. Indiscriminate suppression of cell-cycle genes or proto-oncogenes may be as acutely toxic as current anticancer chemotherapy if the site delivery is not completely localized. Furthermore, much of the clinical evidence suggests that restenosis is a chronic process, continuing to develop weeks to months after the procedure. If this is the case, then the current approaches that rely on a transient, local application of an antisense agent may fail. If, however, a target gene is identified that is specific to vascular tissue, then repeated administration of an antisense agent may be tolerated via a systemic route. This approach has proved successful in targeting mutated genes with little suppression of closely related genes and with minimal systemic toxicity. An alternative approach is to transfect the target tissue with a gene that makes it susceptible to systemic delivery of a drug that is not normally toxic to mammalian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Angioplasty, Balloon↗

PubMed1995

Probabilistic secretion of quanta at somatic motor-nerve terminals: the fusion-pore model, quantal detection and autoinhibition.

The probability of detecting first, second, and later quanta secreted at release sites of a motor-nerve terminal during the early release period following a nerve impulse has been addressed. The possibility that early quantal release autoinhibits later quantal release during this period has also been ascertained. In this investigation, a model for the secretion of a quantum at a release site is developed in which, following the influx and diffusion of calcium ions to a release site protein associated with synaptic vesicles, kappa steps of association of the ions with the protein then occur at rate alpha. The release site protein then undergoes a conformational change which may not go on to completion if calcium ions dissociate from the protein at rate gamma. If this process does reach completion then a fusion-pore between the vesicle and the presynaptic membrane is created; this happens at rate delta. Key assumptions of this fusion-pore model are that the quantal secretions from each site are independent of each other, and that there is a large number of vesicles, each with a small probability of secretion, so that the number of secretions is Poisson in nature. These assumptions allow analytical expressions to be obtained for predicting the times at which first, second and later quanta are secreted during the early release period following an impulse. To test the model, experiments were performed in which the times of first, second and later quantal releases were determined at discrete regions along the length of visualized motor-terminal branches in toad (Bufo marinus) muscles. Estimates of model rate constants and of kappa from the times for first quantal secretions failed to give satisfactory predictions of the observed times of later secretions. Therefore, either the model fails, or the procedure used for detecting later quantal events as a consequence of their being masked by earlier quantal events is inadequate. To solve this detection problem, a two-dimensional analysis of the spread of charge following the secretion of a quantum at a random site on the motor-terminal branch has been done. This allows determination of the probability that later quanta will be detected following secretion of earlier quanta. The detection model was then incorporated into the fusion-pore model to predict the times at which second and later quanta occur during the early release period, based on the estimates of the model parameters derived from the analysis of first quantal releases. Good estimates were now obtained for the observed times of second and later quantal releases, indicating that appropriate procedures must be adopted for adequate detection of quantal secretions. Furthermore, the experiments provide support for the fusion-pore model. It has been suggested that the binomial nature of quantal release from the entire motor-nerve terminal may be explained if early quantal release inhibits later quantal release during the early quantal release phase (M. R. Bennett & J. Robinson 1990, Proc. R. Soc. Lond. B 239, 329-358). Although the fusion-pore detection error model gave good predictions of the observed times of first, second and later quantal releases, these may be improved if a model for autoinhibition is included. In this model the first quantum was taken as giving rise to an inhibition of secretion that propagates to surrounding release sites with a constant velocity, v. A combined model incorporating the fusion-pore detection error model and that for autoinhibition was then used to predict second and later quantal latencies, by using the first quantal latencies to determine the estimates for the parameters in the combined model. When this analysis was done on the times for quantal secretion at sites on thirteen different motor-nerve terminals, the value of v was estimated as zero in each case, so that no autoinhibitory effect was observed.

Animals↗

PubMed1995

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

Monte Carlo methods are used to analyze the stochastic interaction between ATP, released in a packet at a bouton, and the underlying patch of purinoceptors. The time-course of the average quantal current recorded with an intracellular electrode in the peripheral and central nervous systems is reconstructed, given the geometry of the synapse and the known kinetics of ATP action. This leads to certain restrictions on the possible numbers of ATP molecules in a quantum and the density of purinoceptors at the synapses. The addition of an ectoenzyme into the synaptic cleft with the known kinetics of ATPase gives rise to a quantal current of appropriate time-course if the number of ATP molecules in a quantum is increased over that in the absence of the ATPase. The stochastic variability in the quantal current is less than 0.1 for a given size quantum.

Adenosine Triphosphate↗

PubMed1995

Opioids acting on delta-receptors modulate Ca2+ currents in cultured postganglionic neurones of avian ciliary ganglia.

Opioid agonists inhibited Ba2+ currents in cultured postganglionic neurons of 3-15-day-old embryonic avian ciliary ganglia. Leucine-enkephalin (LENK, 10-20 microM) inhibited transient Ba2+ currents by 50% and sustained currents by 21%. The delta-opioid receptor agonist, DPDPE (0.02-10 microM), also inhibited Ba2+ currents, but kappa- (U50488H) and mu- (DAMGO) receptor agonists had no effect. Inhibition of Ba2+ currents showed profound desensitisation during prolonged application of agonists, being reduced to approximately 25% of the original response after 4 min superfusion with leucine-enkephalin (10-20 microM).

Analgesics↗

PubMed1995

Quantal secretion recorded from visualized boutons.

A method is described for recording the electrical signs of transmission at single boutons. Monopolar rat pelvic ganglion cells are electrically compact and receive an innervation from either single hypogastric or pelvic nerves that consists of a set of boutons which innervate the soma of the cells; each bouton possesses an active zone and can be observed with the confocal microscope after dextran-rhodamine orthograde labelling of the hypogastric nerves. Extracellular electrodes of about 8 microns diameter can be positioned in the loose-patch configuration over visualized boutons following their fluorescence with the vital dye, 3,3-diethyloxardicarbocyanine iodide (DiOC2(5)). Excitatory post-synaptic currents (EPSCs), due to stimulation of the hypogastric nerve, can be recorded from boutons in this way, provided that [Ca2+]o is lowered sufficiently to ensure failure of the initiation of the soma action potential by the EPSCs.

Animals↗

PubMed1995

Location of nitric oxide synthase in the developing avian ciliary ganglion.

A study has been made of the distribution of nitric oxide synthase (NOS) in the developing avian ciliary ganglion. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity first appeared in ciliary neurones at embryonic day 10 (E10). The number of NADPH-d positive neurones appeared maximal at this age and thereafter declined; at post hatched day 4 (P4) these neurones were found predominately in the periphery of the ganglion. At the light microscope level the NADPH-d stain appeared throughout the cell soma of the ciliary neurones. This was confirmed using tissue culture techniques. Ultrastructural delineation of horseradish peroxidase-labelled NOS antibodies was also found in the calyx where it was bound to the membranes of the endoplasmic reticulum as well as to the outer membranes of mitochondria. This distribution of NOS in the soma and calyx is consistent with the physiological role of NO as a co-transmitter and retrograde messenger that regulates the quantal secretion of the principal transmitter, acetylcholine, from the calyx.

Aging↗

PubMed1995

On the contribution of quantal secretion from close-contact and loose-contact varicosities to the synaptic potentials in the vas deferens.

A bidomain model of the smooth muscle syncytium has been used to analyse the sources of transmitter secretion that give rise to the excitatory junction potential (EJP) in the guinea-pig vas deferens. The timecourse of the spontaneous excitatory junction potential (SEJP) has been taken to be the same as the time course of action of a quantum of transmitter. The amplitude of the SEJP is dependent on both the size of the quantum secreted and the distance away of the source of the quantum from the muscle cells. Two such sources are considered, one identified as the close-contact varicosities (CCVS) about 50 nm from the muscle and the other as loose-contact varicosities (LCVS) at greater distances. It is shown that in order for the syncytium to reach equipotential by the time the EJP has declined to about 80% of its peak, each muscle cell must receive a quantum of transmitter. The relatively low density of innervation of muscle cells by CCVS so far reported, together with the extremely low probability for secretion from these, indicates that many LCVS surrounding each muscle cell contribute to the EJP. The rising phase of the EJP contains components that indicate the sources of the transmitter responsible for its generation, and these components have been made explicit by differentiating the EJP to give the DEJP. This always has a smooth and relatively slow component that lasts for about 80 to 100 ms and occasionally has fast components superimposed on it. These latter are shown to be almost certainly due to secretion of quanta from the CCVS. It is known that there is a distribution of action potential velocities in the sympathetic nerves to the vas deferens. To account for this, the secretion of quanta from different CCVS on a set of muscle cells in the syncytium were given different delays so that the DEJP consisted of a slow wave form that extended over 80 ms, composed of clearly discernible components arising from the CCVS. This wave form could be smoothed by allowing each cell in the syncytium to receive a quantum of transmitter from a CCV, a condition that did not then allow for the appearance of fast components in the DEJP. A model that generated both the non-intermittent slow component of the DEJP and the intermittent fast component consisted of each cell in the syncytium receiving an innervation from a single CCV as well as from a large number of LCVS. In this case, all the varicosities could secrete a quantum of transmitter with a particular probability after a delay characteristic for that varicosity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic Fibers↗

PubMed1995

Evidence for a critical role of nitric oxide in the tonic excitation of rabbit renal sympathetic preganglionic neurones.

1. A large proportion of sympathetic preganglionic neurones contain nitric oxide synthase. The purpose of this study was to determine the effects of facilitation and inhibition of nitric oxide synthesis within the lower thoracic spinal cord (which contains the majority of renal preganglionic neurones) on renal sympathetic nerve activity (rSNA). 2. In anaesthetized rabbits, rSNA was recorded before and after intrathecal injection (50 microliters of 0.5 M solution) of either L-arginine, a precursor of nitric oxide, or N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, into the lower thoracic spinal cord. Spinal cord sections were also stained for the presence of NADPH diaphorase, a marker of nitric oxide synthesizing neurones. 3. A high density of NADPH diaphorase-containing neurones was found within the intermediolateral cell column of the lower thoracic spinal cord. 4. Intrathecal injection of L-arginine and L-NAME resulted in a large increase (113 +/- 25%) and decrease (43 +/- 8%), respectively, in rSNA. In contrast, injection of the inactive isomers D-arginine and D-NAME had no significant effect on rSNA. 5. The results indicate that endogenous nitric oxide in the lower thoracic spinal cord (1) has a potent excitatory action on renal sympathetic preganglionic neurones, and (2) helps to maintain the tonic activity of renal sympathetic nerves under resting conditions.

Adrenergic Fibers↗