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H R Brenner

Publications and source records attributed to H R Brenner.

At least 37 records · Page 2Linked to original sources
PubMed1992

Normal development of nerve-muscle synapses in mice lacking the prion protein gene.

The expression of acetylcholine receptors (AChR) at neuromuscular synapses in skeletal muscle is regulated by innervation. Recent evidence suggests that the neurotrophic factors involved in the expression of AChR subunit genes may be related to the prion protein (PrPc), a protein of unknown function expressed primarily in neurons which, in its modified form, PrPSc, is thought to have a role in the pathogenesis of transmissible spongiform encephalopathies. We have tested for an involvement of PrPc in the neurotrophic regulation of synaptic AChRs in muscle by comparing the contents of AChR epsilon- and gamma-subunit mRNAs by Northern blot analysis and by in situ hybridization in mice with normal and with deleted PrP genes. At the protein level, AChR expression was assessed electrophysiologically. No difference was found between muscles from the two types of animals, suggesting that the neural regulation of AChR subunit expression in skeletal muscle can be mediated by factors that are not derived from the PrP gene.

Animals↗
PubMed1992

The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA.

A full-length cDNA coding for a homolog of the human Myf-5 was isolated from a BC3H-1 mouse library and characterized. The clone codes for a protein of 255 amino acids that is 89%, 88% and 68% identical to the human, bovine and Xenopus myf-5, respectively. The mouse Myf-5 cDNA (mmyf-5), as well as sequences coding for MyoD, myogenin and Mrf-4, were used to probe Northern blots to analyze the effects of innervation on the expression of the MyoD family of myogenic factors. Mouse myf-5, MyoD and myogenin mRNAs levels were found to decline in hind limb muscles of mice between embryonic day 15 (E15) and the first postnatal week, a period that coincides with innervation. In contrast, Mrf-4 transcripts increase during this period and reach steady-state levels by 1-week after birth. To distinguish if the changes in myogenic factor expression are due to a developmental program or to innervation, mRNA levels were analyzed at different times after muscle denervation. Mmyf-5 transcripts begin to accumulate 2 days postdenervation; after 1 week levels are 7-fold higher than in innervated muscle. Mrf-4, MyoD and myogenin transcripts begin to accumulate as soon as 8h after denervation, and attain levels that are 8-, 15- and 40-fold higher than found in innervated skeletal muscle, respectively. The accumulation of these three mRNAs precedes the increase of nicotinic acetylcholine receptor alpha subunit transcripts, a gene that is transcriptionally regulated by MyoD-related factors in vitro. Using extracellular electrodes to directly stimulate in situ the soleus muscle of rats, we found that 'electrical activity' per se, in absence of the nerve, represses the increases of myogenic factor mRNAs associated with denervation.

Amino Acid Sequence↗
PubMed1992

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.

Animals↗
PubMed1991

Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity.

Myogenin and MyoD are proteins that bind to the regulatory regions of a battery of skeletal muscle genes and can activate their transcription during muscle differentiation. We have recently found that both proteins interact with the enhancer of the nicotinic acetylcholine receptor (nAChR) alpha subunit, a gene that is regulated by innervation. This observation prompted us to study if myogenin and MyoD transcript levels are also regulated by skeletal muscle innervation. Using Northern blot analysis, we found that MyoD and myogenin mRNA levels begin to decline at embryonic day 17 and attain adult levels in muscle of newborn and 3-week-old mice, respectively. In contrast, nAChR mRNAs are highest in newborn and 1-week-old mouse muscle and decline thereafter to reach adult levels in 3-week-old mice. To determine if the down-regulation of myogenin and MyoD mRNA levels during development is due to innervation, we quantitated message levels in adult calf muscles after denervation. We found that in denervated muscle myogenin and MyoD mRNAs reach levels that are approximately 40- and 15-fold higher than those found in innervated muscle. Myogenin mRNAs begin to accumulate rapidly between 8 and 16 hr after denervation, and MyoD transcripts levels begin to increase sharply between 16 hr and 1 day after denervation. The increases in myogenin and MyoD mRNA levels precede the rapid accumulation of nAChR alpha-subunit transcripts; receptor mRNAs begin to accumulate significantly after 1 day of denervation. The effects of denervation are specific because skeletal alpha-actin mRNA levels are not affected by denervation. In addition, we found that the repression of myogenin and MyoD expression by innervation is due, at least in part, to "electrical activity." Direct stimulation of soleus muscle with extracellular electrodes repressed the increase of myogenin and MyoD transcripts after denervation by 4- to 3-fold, respectively. In view of these results, it is interesting to speculate that myogenin and/or MyoD may regulate a repertoire of skeletal muscle genes that are down-regulated by electrical activity.

Aging↗
PubMed1991

Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity.

During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.

Animals↗
PubMed1991

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.

Animals↗
PubMed1990

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses.

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.

Animals↗
PubMed1990

Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity.

The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed.

Animals↗
PubMed1989

On the effect of muscle activity on the end-plate membrane in denervated mouse muscle.

1. Mouse soleus muscles were denervated and some of them were chronically stimulated. Sixteen to twenty-one days later, the number of junctional acetylcholine receptors (AChR) and their metabolic stability were examined by measuring binding of 125I-alpha-bungarotoxin, their gating properties by analysis of acetylcholine-induced current fluctuations and the ultrastructure of the end-plate membrane by electron microscopy. 2. In agreement with other studies on inactive muscles, no effect of denervation on junctional AChR number could be resolved. However, some of the fast-gating 'adult' AChR channels had been replaced by slowly gating fetal AChRs, their half-life was lowered to 38 h and the folding of the end-plate membrane was reduced. 3. These changes were prevented in denervated but stimulated active muscles: the junctional AChR population remained homogeneously 'adult', the half-life of junctional AChRs was 13 days and folding of the end-plate membrane remained comparable to that in control muscles. 4. The significance of these results is discussed with respect to the role of muscle activity in end-plate development.

Animals↗
PubMed1988

Dependence of acetylcholine receptor channel conversion on muscle activity at denervated neonatal rat endplates.

During the development of the motor endplate, the apparent mean open time of the junctional acetylcholine (AChR) channels decreases during the first 3 weeks of postnatal life from about 4 to about 1 ms. This decrease is prevented by early denervation of the muscle, suggesting a neural control of subsynaptic AChR channel properties. To further examine the nature of this neural influence, neonatal rat soleus muscles were now denervated at an early stage of endplate development and the muscles were kept active by chronic stimulations in vivo for 4 days. The gating properties of AChR channels at the denervated endplates and at control endplates of similar age were then examined by analysis of acetylcholine-induced endplate current fluctuations and of the time course of miniature endplate currents, respectively. The results indicate, that stimulation restores the maturation of 'adult' AChR channel properties even in the absence of the nerve, suggesting an important role of muscle activity in synapse development.

Action Potentials↗
PubMed1988

Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity.

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.

Animals↗
PubMed1987

Control of end-plate channel properties by neurotrophic effects and by muscle activity in rat.

1. The formation of ectopic neuromuscular synapses was induced in rat soleus muscle by implantation of the fibular nerve into the proximal part of the muscle and subsequent sectioning of the soleus nerve. The gating properties of acetylcholine (ACh) receptors at the newly formed end-plates were examined by analysis of acetylcholine-induced membrane current fluctuations. 2. In agreement with earlier studies, the apparent mean open time of end-plate channels decreased during synaptic development from about 4 ms to about 1 ms (-60 mV membrane potential, 22 degrees C) within 7-18 days after the soleus nerve had been cut. 3. When the fibular nerve was cut at an early stage of end-plate development, fast-gating channels with apparent mean open times of 1 ms characteristic of mature end-plates did not develop within the next 10-14 days. 4. When the fibular nerve was cut at an early stage of end-plate development and the soleus muscle was then stimulated chronically via implanted electrodes, fast-gating channels did develop in the absence of the nerve terminals within 4-6 days. 5. When impulse conduction in the transplanted fibular nerve was blocked chronically at the time of soleus nerve section such that ectopic end-plates formed in inactive muscle, fast-gating channels developed within 12-14 days. 6. The results show that motoneurones control the conversion from slow-gating fetal to fast-gating adult-type ACh receptor channels at ectopic end-plates in rat soleus muscles. The conversion occurs in the absence of impulse activity provided the nerve continues to be present. However, it also occurs in the absence of the nerve provided the muscle is active and had received an early priming influence from the nerve. Thus, nerve-evoked muscle activity and nerve-released trophic influences complement each other in controlling the gating properties of junctional ACh receptor channels.

Action Potentials↗
PubMed1985

On the neurotrophic control of acetylcholine receptors at frog end-plates reinnervated by the vagus nerve.

To test whether the properties of subsynaptic acetylcholine (ACh) receptors in skeletal muscle fibre are influenced by the type of the innervating neurone some pharmacological properties of ACh receptor in normal end-plates and in denervated end-plates reinnervated by the vagus nerve in the frog were compared. Blockade of nerve-evoked synaptic currents by 200 microM-hexamethonium was stronger at vagus-reinnervated than at normal end-plates. Blockade at both types of junctions was voltage dependent. The effect of hexamethonium on equilibrium currents induced by bath-applied ACh and carbamylcholine was similar at the two types of junctions. At both normal and vagus-reinnervated junctions, decamethonium had similar partial agonist properties. Following a step in membrane potential, the relaxations of ACh-induced conductance changes at the two types of junctions were affected in a similar fashion by hexamethonium: hyperpolarization first produced a fast decrease and then a slow exponential increase in conductance. Upon depolarization, a fast increase was followed by an exponential decline to its original level. The time constant of the slow relaxation was slightly prolonged compared to control. These findings are consistent with a fast blocking action of open channels by hexamethonium. The effectiveness of hexamethonium in blocking end-plate currents was reduced in the presence of (+)-tubocurarine, indicating that hexamethonium has a competitive blocking action on the receptors. These results do not indicate that the pharmacological properties of the ACh receptors are changed after an end-plate is reinnervated by a preganglionic neurone. The differential effect of hexamethonium on transmission at normal and vagus-reinnervated end-plates is discussed as a consequence of different transmitter release characteristics at the two types of junctions.

Action Potentials↗
PubMed1983

Synthesis and properties of NBD-n-acylcholines, fluorescent analogs of acetylcholine.

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Neurotrophic control of channel properties at neuromuscular synapses of rat muscle.

1. Ectopic neuromuscular synapses formed when the fibular nerve was implanted into the proximal part of rat soleus muscle and the soleus nerve was cut. The gating properties of acetylcholine (ACh) receptor channels in the newly formed ectopic and in the denervated original end-plates were examined at various stages of ectopic synapse formation.2. At ectopic end-plates the apparent mean open time of ACh receptor channels changes during synaptic development. Channels in immature ectopic end-plates, examined 1 week after cutting the soleus nerve, have apparent mean open times of approximately 4 ms (at -70 mV, 22 degrees C), similar to those of the extrasynaptic ACh receptor channels of completely denervated fibres. The channel gating of mature ectopic end-plates, examined 3-7 weeks after nerve section, is characterized by a mean open time of approximately 1 ms and resembles that found in normal end-plates of adult fibres.3. The conversion of end-plate channel gating occurs during the second and third week of synapse formation. During this period two discrete classes of channels with different gating behaviour are present in the ectopic end-plate.4. Examination of ectopic end-plates in the electron microscope reveals that junctional folds begin to appear in the subsynaptic membrane during the period of channel conversion.5. At the denervated original end-plates of ectopically innervated fibres the apparent mean open time of ACh receptor channels remains similar to that of normally innervated end-plates. Original end-plates retain the normal synaptic class of channel for at least 42 days after denervation. At this time, most of the ACh receptors present originally in the membrane have been replaced by newly inserted receptors.6. At former end-plates of completely denervated fibres ACh activates two classes of channels, even when most of the ACh receptors originally present in the end-plate have been replaced by new receptors.7. The results show that during synapse formation a neurally controlled conversion of ACh receptor channels occurs about 2-3 weeks after establishment of the nerve muscle contact. Thereafter end-plate channel properties are independent of neural influences. These observations are consistent with a mechanism of channel conversion whereby the nerve modifies not the ACh receptor channel itself, but another constituent of the end-plate membrane which determines the gating properties of end-plate channels.

Acetylcholine↗
PubMed1981

Channel gating at frog neuromuscular junctions formed by different cholinergic neurones.

1. Drug-induced membrane current fluctuations were analysed at frog ectopic neuromuscular junctions formed de novo by somatic motoneurones and by preganglionic autonomic neurones of the vagus nerve, and at denervated end-plates reinnervated by the vagus nerve. 2. At ectopic motor end-plates, the mean open time of the ion channels activated by ACh is tau ACh = 1.8 +/- 0.1 msec (S.E.) at -70 to -90 mV and 15-18 degrees C. Carbachol- and suberyldicholine-induced channels have average open times tau Carb = 1.0 +/- 0.1 msec (-80 mV, 6-8 degrees C) and tau Sub = 3.3 +/- 0.1 msec (-80 mV, 16-18 degrees C). 3. The conductances gamma of single channels induced by ACh, carbachol and suberyldicholine are: gamma ACh = 21.0 +/- 1.1 pS (+/- S.E.), gamma Carb = 14.6 +/- 1.2 pS and gamma Sub = 22.1 +/- 1.2 pS. 4. The mean open times tau of the channels is prolonged as the membrane is hyperpolarized. Their voltage dependence is similar to that of normal end-plate channels. 5. The average open time of ACh-induced channels at ectopic vagus junctions is tau = 1.6 +/- 0.2 msec (S.E.) at -70 to -80 mV and 14-15 degrees C. At denervated motor end-plates reinnervated by vagal neurones, the mean channel open time is tau = 1.5 +/- 0.1 msec at -80 mV and 14-18 degrees C. 6. At all vagus junctions, the synaptic currents are blocked by alpha-bungarotoxin. 7. Somatic motoneurons and vagal neurones induce junctional folds in the muscle membrane where they contact it to form an ectopic end-plate. Staining the ACh receptors of the motor end-plates with horseradish peroxidase-alpha-bungarotoxin conjugate shows that the receptors are restricted to the junctional region of the muscle fibre membrane.

Acetylcholine↗