Calcium Transients and Transmitter Secretion in Different Parts of Frog Nerve Endings in Different Conditions of Calcium Ion Influx

Neuroscience and Behavioral Physiology - Experiments on frog neuromuscular preparations were performed to study the characteristics of the calcium response and the quantum secretion of...     

Oral treatment with alpha-lipoic acid improves symptomatic diabetic polyneuropathy: the SYDNEY 2 trial

OBJECTIVE: The aim of this trial was to evaluate the effects of alpha-lipoic acid (ALA) on positive sensory symptoms and neuropathic deficits in diabetic patients with distal symmetric polyneuropathy (DSP). RESEARCH DESIGN AND METHODS: In this multicenter, randomized, double-blind, placebo-controlled trial, 181 diabetic patients in Russia and Israel received once-daily oral doses of 600 mg (n = 45) (ALA600), 1,200 mg (n = 47) (ALA1200), and 1,800 mg (ALA1800) of ALA (n = 46) or placebo (n = 43) for 5 weeks after a 1-week placebo run-in period. The primary outcome measure was the change from baseline of the Total Symptom Score (TSS), including stabbing pain, burning pain, paresthesia, and asleep numbness of the feet. Secondary end points included individual symptoms of TSS, Neuropathy Symptoms and Change (NSC) score, Neuropathy Impairment Score (NIS), and patients' global assessment of efficacy. RESULTS: Mean TSS did not differ significantly at baseline among the treatment groups and on average decreased by 4.9 points (51%) in ALA600, 4.5 (48%) in ALA1200, and 4.7 (52%) in ALA1800 compared with 2.9 points (32%) in the placebo group (all P < 0.05 vs. placebo). The corresponding response rates (>/=50% reduction in TSS) were 62, 50, 56, and 26%, respectively. Significant improvements favoring all three ALA groups were also noted for stabbing and burning pain, the NSC score, and the patients' global assessment of efficacy. The NIS was numerically reduced. Safety analysis showed a dose-dependent increase in nausea, vomiting, and vertigo. CONCLUSIONS: Oral treatment with ALA for 5 weeks improved neuropathic symptoms and deficits in patients with DSP. An oral dose of 600 mg once daily appears to provide the optimum risk-to-benefit ratio.     

The Effects of Carbachol on the Proximal and Distal Parts of Frog Motor Nerve Endings

Cholinomimetics not only activate postsynaptic cholinoreceptors in neuromuscular synapses, but also alter the process of acetylcholine secretion from nerve endings. However, the mechanism of action of cholinomimetics on the secretory process remains unidentified. We approached the question of the mechanism of the presynaptic action of cholinomimetics in the present study by investigating the effects of the n,m-cholinomimetic carbachol on nerve ending currents and postsynaptic membrane currents. Carbachol induced decreases in the postsynaptic response, without affecting the duration and amplitude of the nerve ending current in both the central and distal part of the nerve ending. However, carbachol increased the time between the arrival of the presynaptic action potential and the start of transmitter secretion. This effect on synaptic delay was more marked in the distal parts of the ending. The action of another potential modulator, extracellular potassium, was accompanied by decreases in presynaptic currents and also by increases in synaptic delay. These data provide evidence for the suppressive effect of carbachol on acetylcholine secretion acting via presynaptic metabotropic cholinoreceptors which control the level and time course of secretion of neurotransmitter quanta.     

Impact of A-Site Cation Deficiency on Charge Transport in La0.5−xSr0.5FeO3−δ

The electrical conductivity of La_0.5−xSr_0.5FeO_3−δ, investigated as a function of the nominal cation deficiency in the A-sublattice, x, varying from 0 to 0.02, has demonstrated a nonlinear dependence. An increase in the x value from 0 to 0.01 resulted in a considerable increase in electrical conductivity, which was shown to be attributed mainly to an increase in the mobility of the charge carriers. A combined analysis of the defect equilibrium and the charge transport in La_0.5−xSr_0.5FeO_3−δ revealed the increase in the mobility of oxygen ions, electrons, and holes by factors of ~1.5, 1.3, and 1.7, respectively. The observed effect is assumed to be conditioned by a variation in the oxide structure under the action of the cationic vacancy formation. It was found that the cation deficiency limit in La_0.5−xSr_0.5FeO_3−δ did not exceed 0.01. A small overstep of this limit was shown to result in the formation of (Sr,La)Fe₁₂O₁₉ impurity, which even in undetectable amounts reduced the conductivity of the material. The presence of (Sr,La)Fe₁₂O₁₉ impurity was revealed by X-ray diffraction on the ceramic surface after heat treatment at 1300 °C. It is most likely that the formation of traces of the liquid phase under these conditions is responsible for the impurity migration to the ceramic surface. The introduction of a cation deficiency of 0.01 into the A-sublattice of La_0.5−xSr_0.5FeO_3−δ can be recommended as an effective means to enhance both the oxygen ion and the electron conductivity and improve ceramic sinterability.     

Regulation of transmitter release by synapsin II in mouse motor terminals

We investigated quantal release and ultrastructure in the neuromuscular junctions of synapsin II knockout (Syn II KO) mice. Synaptic responses were recorded focally from the diaphragm synapses during electrical stimulation of the phrenic nerve. We found that synapsin II affects transmitter release in a Ca²⁺-dependent manner. At reduced extracellular Ca²⁺ (0.5 m m ), Syn II KO mice demonstrated a significant increase in evoked and spontaneous quantal release, while at the physiological Ca²⁺ concentration (2 m m ), quantal release in Syn II KO synapses was unaffected. Protein kinase inhibitor H7 (100 μ m ) suppressed quantal release significantly stronger in Syn II KO synapses than in wild type (WT), indicating that Syn II KO synapses may compensate for the lack of synapsin II via a phosphorylation-dependent pathway. Electron microscopy analysis demonstrated that the lack of synapsin II results in an approximately 40% decrease in the density of synaptic vesicles in the reserve pool, while the number of vesicles docked to the presynaptic membrane remained unchanged. Synaptic depression in Syn II KO synapses was slightly increased, which is consistent with the depleted vesicle store in these synapses. At reduced Ca²⁺ frequency facilitation of synchronous release was significantly increased in Syn II KO, while facilitation of asynchronous release was unaffected. Thus, at the reduced Ca²⁺ concentration, synapsin II suppressed transmitter release and facilitation. These results demonstrate that synapsin II can regulate vesicle clustering, transmitter release, and facilitation.     

Efficacy and safety of antioxidant treatment with α-lipoic acid over 4 years in diabetic polyneuropathy: the NATHAN 1 trial

OBJECTIVE

To evaluate the efficacy and safety of α-lipoic acid (ALA) over 4 years in mild-to-moderate diabetic distal symmetric sensorimotor polyneuropathy (DSPN).

RESEARCH DESIGN AND METHODS

In a multicenter randomized double-blind parallel-group trial, 460 diabetic patients with mild-to-moderate DSPN were randomly assigned to oral treatment with 600 mg ALA once daily (n = 233) or placebo (n = 227) for 4 years. Primary end point was a composite score (Neuropathy Impairment Score [NIS]–Lower Limbs [NIS-LL] and seven neurophysiologic tests). Secondary outcome measures included NIS, NIS-LL, nerve conduction, and quantitative sensory tests (QSTs).

RESULTS

Change in primary end point from baseline to 4 years showed no significant difference between treatment groups (P = 0.105). Change from baseline was significantly better with ALA than placebo for NIS (P = 0.028), NIS-LL (P = 0.05), and NIS-LL muscular weakness subscore (P = 0.045). More patients showed a clinically meaningful improvement and fewer showed progression of NIS (P = 0.013) and NIS-LL (P = 0.025) with ALA than with placebo. Nerve conduction and QST results did not significantly worsen with placebo. Global assessment of treatment tolerability and discontinuations due to lack of tolerability did not differ between the groups. The rates of serious adverse events were higher on ALA (38.1%) than on placebo (28.0%).

CONCLUSIONS

Four-year treatment with ALA in mild-to-moderate DSPN did not influence the primary composite end point but resulted in a clinically meaningful improvement and prevention of progression of neuropathic impairments and was well tolerated. Because the primary composite end point did not deteriorate significantly in placebo-treated subjects, secondary prevention of its progression by ALA according to the trial design was not feasible.Diabetic distal symmetric sensorimotor polyneuropathy (DSPN) is a chronic progressive disease affecting around one-third of the diabetic population and accounts for considerable morbidity, increased mortality, and reduced quality of life (1,2). Recent long-term studies in type 2 diabetic patients indicate that the current strategies of intensive diabetes therapy or multifactorial cardiovascular risk intervention are not sufficient to slow the progression of DSPN (3–5). Thus, effective treatment of DSPN remains challenging for the physician (1,6).Based on the pathogenetic mechanisms of DSPN, potential disease-modifying therapeutic approaches have been developed including antioxidants such as α-lipoic acid (ALA) (7–9) to diminish increased oxidative stress (10). Other potential modalities include the aldose reductase inhibitors (11), growth factors (12), and the protein kinase C-β inhibitor ruboxistaurin (13). These drugs have been designed to favorably influence the underlying pathophysiology of the disorder rather than for symptomatic pain relief. However, several problems have been encountered previously in designing appropriate clinical trials in DSPN. Among these, the most important are as follows: 1) the lack of homogeneity of patients studied with respect to both the form of neuropathy and the degree of glycemic control; 2) different pathogenetic pathways, the relative importance of which may vary intraindividually; 3) stages of neuropathy that are too advanced; 4) the use of end points with rather large variability between individuals and between centers; 5) the unknown relevance of end points used; and 6) study durations too short to allow for a favorable functional or structural effect (6,14,15).Treatment with ALA administered intravenously or orally for several weeks or months improves neuropathic symptoms and deficits (7–9). However, based on the assumption that a therapeutic agent may prevent worsening of DSPN but not cause improvement, clinical trials should be conducted for a minimum period of 3 years to achieve a clinically meaningful change of two Neuropathy Impairment Score (NIS) points (16,17). Therefore, we assessed the efficacy and safety of oral treatment with 600 mg ALA once daily in mild-to-moderate DSPN.     

The Role of Intracellular cAMP in Mediating the Synchronizing Action of Noradrenaline on the Evoked Release of Quanta of Mediator in the Frog Synapse

Experiments on frog neuromuscular junction preparations with extracellular recording of action currents in nerve endings and single-quantum currents from endplates were used to assess the time course of evoked quantum mediator secretion by analyzing histograms showing the distribution of true synaptic delays. Studies using the cyclic AMP analog dibutyryl-cAMP (db-cAMP), the adenylate cyclase activator forskolin, and the nucleotide-dependent phosphodiesterase inhibitor isobutylmethylxanthine, showed that these agents, like noradrenaline, altered the kinetics of secretion of quanta, leading to synchronization of the release of mediator. After preliminary treatment of the neuromuscular preparation with db-cAMP, forskolin, or isobutylmethylxanthine, noradrenaline did not induce the synchronization of mediator release in quanta. It was concluded that the action of noradrenaline on the time course of secretion is mediated by activation of presynaptic receptors, increased adenylate cyclase activity, and increases in intracellular cAMP levels.     

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 × 10⁻⁴ m ), carbachol (CCh, 1 × 10⁻⁵ m ) or nicotine (5 × 10⁻⁶ m ) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90–120 μm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 × 10⁻⁷ m (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m _o (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m _o and dispersed the release (ACh, CCh and nicotine). The effects on m _o were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.