Auto-inhibition of rat parallel fibre–Purkinje cell synapses by activity-dependent adenosine release

Adenosine is an important signalling molecule involved in a large number of physiological functions. In the brain these processes are as diverse as sleep, memory, locomotion and neuroprotection during episodes of ischaemia and hypoxia. Although the actions of adenosine, through cell surface G-protein-coupled receptors, are well characterized, in many cases the sources of adenosine and mechanisms of release have not been defined. Here we demonstrate the activity-dependent release of adenosine in the cerebellum using a combination of electrophysiology and biosensors. Short trains of electrical stimuli delivered to the molecular layer in vitro , release adenosine via a process that is both TTX and Ca²⁺ sensitive. As ATP release cannot be detected, adenosine must either be released directly or rapidly produced by highly localized and efficient extracellular ATP breakdown. Since adenosine release can be modulated by receptors that act on parallel fibre–Purkinje cell synapses, we suggest that the parallel fibres release adenosine. This activity-dependent adenosine release exerts feedback inhibition of parallel fibre–Purkinje cell transmission. Spike-mediated adenosine release from parallel fibres will thus powerfully regulate cerebellar circuit output.     

Regulation of extracellular adenosine in rat hippocampal slices is temperature dependent: role of adenosine transporters

While a great deal is known about stimuli that can induce the release of adenosine from brain tissue, relatively little is known about the regulation of the basal extracellular concentration of adenosine that is present in the absence of stimulation. Under normal conditions, enough adenosine is present to tonically activate a significant portion of the high-affinity adenosine A1 receptors. The present experiments demonstrated that the estimated basal concentration of extracellular adenosine in rat hippocampal slices maintained at 21 degrees C (430 nM) is approximately twice that at 32 degrees C (220 nM). The sensitivity of presynaptic modulatory adenosine A1 receptors was not significantly different at 21 degrees C or at 32 degrees C. Slices maintained at 21 degrees C also showed a reduced ability to inactivate extracellular adenosine, which reflects a reduction in adenosine transport across cell membranes. This effect appears to be primarily due to a reduction in the function of the equilibrative, dipyridamole-sensitive (ei) adenosine transporter; the nitrobenzylthioinosine-sensitive equilibrative transporter (es transporter) appears to be relatively less affected by temperature than is the ei transporter. These experiments demonstrate that extracellular concentrations of adenosine in the brain are sensitive to temperature, and suggest that some of the neurological effects of hypothermia might be mediated via increased concentrations of adenosine in the extracellular space.     

Convergent control of synaptic GABA release from rat dorsal horn neurones by adenosine and GABA autoreceptors

Perforated patch clamp recordings were performed on cultured superficial neonatal rat dorsal horn (DH) spinal cord neurones in order to study the presynaptic modulation of GABA release at unitary synaptic connections. Since ATP can be coreleased with GABA at about two-thirds of GABAergic synapses between DH neurones, and can be rapidly metabolized to adenosine in the extracellular space, we investigated the potential role of A1 adenosine receptors and GABA_B receptors which might function as inhibitory autoreceptors. Adenosine and GABA_B receptor agonists reduced the amplitude of electrically evoked GABAergic inhibitory postsynaptic currents (eIPSCs) as well as the frequency of GABAergic miniature IPSCs, suggesting a presynaptic action of these substances. The actions of adenosine were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The effects of adenosine and GABA_B agonists were occlusive, indicating a functional convergence of the signalling pathways engaged by A1 and GABA_B receptors. A1 and GABA_B antagonists increased the amplitude of eIPSCs in a supra-additive manner, suggesting a tonic activation of these receptors by ambient adenosine and GABA. Moreover, using trains of electrical stimulations, we were able to unravel a phasic (activity-dependent) activation of presynaptic A1 and GABA_B autoreceptors only in the case of neurones coreleasing ATP and GABA, despite the presence of functional presynaptic A1 and GABA_B receptors on all GABAergic DH neurones. This selective, convergent and activity-dependent inhibition of GABA release by A1 and GABA_B autoreceptors might modulate the integrative properties of postsynaptic DH neurones under physiological conditions and/or during the development of pathological pain states.     

Modulation of visual inputs to accessory optic system by theophylline during hypoxia

Neural tissues from fresh water turtles have been electrophysiologically studied in vitro due to their significant resistance to hypoxia. Such neurons have resting membrane potentials that are similar to intact animals and receive similar synaptic inputs evoked by sensory stimuli. One mechanism to reduce the brain’s metabolic requirement in the absence of oxygenated blood flow was investigated by blocking adenosine receptors before and during hypoxia. Extracellular and whole-cell patch recordings were made from the basal optic nucleus, whose neurons respond to visual stimuli in vitro. While the addition of the adenosine antagonist theophylline to oxygenated superfusate had minimal effect on the neural activity, theophylline in superfusate bubbled with nitrogen strongly increased activity compared to either oxygenated theophylline or control superfusate bubbled with nitrogen. The increase in spontaneous activity was due to increases to both amplitude and frequency of excitatory synaptic events. Even during these increases, the neurons continued to exhibit their direction-sensitive responses. These results indicate that adenosine may play a role in protecting the viability of the brainstem during hypoxia without reducing visually mediated brainstem reflex control.     

Hypothesis: Adenosine mediates hemodynamic changes in renal failure

The decreased filtration fraction and glomerular filtration rate characteristic of renal failure could be produced, at least in part, by increased concentration of an endogenous substance which constricts afferent and dilates efferent arterioles. Adenosine satisfies several criteria: (a) exogenous adenosine and its precursors decrease filtration fraction and glomerular filtration rate; (b) the kidneys produce and release adenosine, and production and release are augmented during conditions associated with the induction of renal failure—hypoxia, ischemia, and renal vasoconstriction; (c) several substances known to antagonize adenosine-uptake processes in some cells, which could thereby increase extracellular adenosine concentration, not only have “adenosine-like” effects on the kidney but also induce and/or potentiate existing renal failure. A corollary of this hypothesis is that adenosine-receptor antagonists, such as the methylxanthines, should counteract the hemodynamic changes characteristic of renal failure. It has been known for several years that aminophylline (1,3-dimethylxanthine ethylenediamine) increases the filtration fraction and the glomerular filtration rate.     

Release of autonomic neuromediators by local ventricular electrical stimulation

Trains of electrical stimuli were applied to the ventricular epicardium in pentobarbital-anesthetized dogs. The trains, delivered during the absolute refractory period via Walton-Brodie strain gauge arches, resulted in a highly localized potentiation of ventricular contractile force. The magnitude of the potentiation was directly related to the intensity of the trains. The positive inotropic response of either ventricle to trains of stimuli was abolished following chronic cardiac denervation or beta-adrenergic blockade with tolamolol, indicating that the response was probably mediated by the excitation of local sympathetic nerve fibers. Following the elimination of adrenergic influences, a slight negative inotropic response to trains of stimuli was observed in some animals. Administration of atropine blocked the negative inotropic response. When the adrenergic system was intact the administration of atropine resulted in a significant augmentation of the positive inotropic response of both ventricles to trains of stimuli. Thus in addition to norepinephrine release, it also appeared that electrical stimulation of the ventricles resulted in activation of local parasympathetic fibers with subsequent release of acetylcholine.     

利多卡因的脑保护机制

利多卡因具有脑保护作用，其机制与下列因素有关：阻断膜Na+-K+交换,ATP的消耗减少,因而减少自由基的产生,同时抑制缺血脑细胞Ｋ+外流及游离脂肪酸释放；抑制膜上电压依赖性Ca2+通道，减轻H2O2诱导的脂质过氧化反应；有效减轻缺血再灌注时神经细胞离子的紊乱,阻止细胞内Na+浓度的升高,降低突触前的谷氨酸释放；抑制线粒体的有氧代谢,降低线粒体内能量物质代谢速率,趋缓乳酸水平的升高,从而减轻细胞内乳酸的堆积,提高细胞对缺氧的耐受力；抑制缺血脑灌注后脑型肌酸激酶的释放,从而使脑缺血时的神经膜保持稳定；改善细胞渗透压和ATP的利用及Ca2+的清除等,从而起到保护神经的作用。     

Adenosine release in nucleus tractus solitarii does not appear to mediate hypoxia-induced respiratory depression in rats

The time course of adenosine release in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) during acute systemic hypoxia was investigated in the anaesthetised rat by means of amperometric enzymatic sensors. It was found that acute hypoxia induced a significant delayed increase in adenosine level (reaching levels as high as 5 μM) in the NTS and that hypoxia-induced release of adenosine was similar at various regions of the NTS along its rostro-caudal axis. Significantly smaller or no increases in adenosine levels at all in response to hypoxia were observed in the VLM. The increase in adenosine level in the NTS occurred during reoxygenation after the termination of the hypoxic challenge and was accompanied by a smaller increase in inosine concentration. At the dorsal surface of the brainstem, only release of inosine was detected following acute hypoxia. Addition of the ecto-5'-nucleotidase inhibitor α,β-methylene ADP (200 μM) to the dorsal surface of the brainstem completely abolished the signal evoked by hypoxia, suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. This study indicates that following systemic hypoxia, adenosine levels in the NTS increase to a significantly greater extent than in the VLM. However, the increase in adenosine concentration in the NTS occurs too late to be responsible for the hypoxia-induced depression of the respiratory activity.     

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

ABSTRACT: BACKGROUND: Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development. Methods: We performed a genetic analysis of wild type and CD73-/- (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling. Results: We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73-/- mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS. Conclusions: We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.     

Brain site-specificity of extracellular adenosine concentration changes during sleep deprivation and spontaneous sleep: an in vivo microdialysis study

Previous data suggested that increases in extracellular adenosine in the basal forebrain mediated the sleep-inducing effects of prolonged wakefulness. The present study sought to determine if the state-related changes found in basal forebrain adenosine levels occurred uniformly throughout the brain. In vivo microdialysis sample collection coupled to microbore high-performance liquid chromatography measured extracellular adenosine levels in six brain regions of the cat: basal forebrain, cerebral cortex, thalamus, preoptic area of hypothalamus, dorsal raphe nucleus and pedunculopontine tegmental nucleus. In all these brain regions extracellular adenosine levels showed a similar decline of 15 to 20% during episodes of spontaneous sleep relative to wakefulness. Adenosine levels during non-rapid eye movement sleep did not differ from rapid eye movement sleep. In the course of 6h of sleep deprivation, adenosine levels increased significantly in the cholinergic region of the basal forebrain (to 140% of baseline) and, to a lesser extent in the cortex, but not in the other regions. Following sleep deprivation, basal forebrain adenosine levels declined very slowly, remaining significantly elevated throughout a 3-h period of recovery sleep, but elsewhere levels were either similar to, or lower than, baseline.The site-specific accumulation of adenosine during sleep deprivation suggests a differential regulation of adenosine levels by as yet unidentified mechanisms. Moreover, the unique pattern of sleep-related changes in basal forebrain adenosine level lends strong support to the hypothesis that the sleep-promoting effects of adenosine, as well as the sleepiness associated with prolonged wakefulness, are both mediated by adenosinergic inhibition of a cortically projecting basal forebrain arousal system.     

Extracellular adenosine in the human brain during sleep and sleep deprivation: an in vivo microdialysis study

STUDY OBJECTIVES: To examine the pattern of extracellular adenosine in the human brain during sleep deprivation, sleep, and normal wake. DESIGN: Following recovery from implantation of clinical depth electrodes, epilepsy patients remained awake for 40 continuous hours, followed by a recovery sleep episode. SETTING: Neurology ward at UCLA Medical Center. PATIENTS OR PARTICIPANTS: Seven male epilepsy patients undergoing depth electrode localization of pharmacologically refractory seizures. INTERVENTIONS: All subjects were implanted with depth electrodes, a subset of which were customized to contain microdialysis probes. Microdialysis samples were collected during normal sleep, sleep deprivation, and recovery sleep from human amygdalae (n = 8), hippocampus (n = 1), and cortex (n = 1). MEASUREMENTS AND RESULTS: In none of the probes did we observe an increase in extracellular adenosine during the sleep deprivation. There was a significant, though very small, diurnal oscillation (2.5%) in 5 of the 8 amygdalae. There was no effect of epileptogenicity on the pattern of extracellular adenosine. CONCLUSIONS: Our observations, along with those in animal studies, indicate that the role of extracellular adenosine in regulating sleep pressure is not a global brain phenomenon but is likely limited to specific basal forebrain areas. Thus, if energy homeostasis is a function of sleep, an increased rate of adenosine release into the extracellular milieu of the amygdala, cortex, or hippocampus is unlikely to be a marker of such a process.     

Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings

Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 n m ) and adenosine (50 µ m ) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release ( m ) = np . We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n ; neither agent affected the parameter p . PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles.     

Adenosine in the spinal cord and periphery: release and regulation of pain

In the central nervous system (CNS), adenosine is an important neuromodulator and regulates neuronal and non-neuronal cellular function (e.g. microglia) by actions on extracellular adenosine A(1), A(2A), A(2B) and A(3) receptors. Extracellular levels of adenosine are regulated by synthesis, metabolism, release and uptake of adenosine. Adenosine also regulates pain transmission in the spinal cord and in the periphery, and a number of agents can alter the extracellular availability of adenosine and subsequently modulate pain transmission, particularly by activation of adenosine A(1) receptors. The use of capsaicin (which activates receptors selectively expressed on C-fibre afferent neurons and produces neurotoxic actions in certain paradigms) allows for an interpretation of C-fibre involvement in such processes. In the spinal cord, adenosine availability/release is enhanced by depolarization (K(+), capsaicin, substance P, N-methyl-D-aspartate (NMDA)), by inhibition of metabolism or uptake (inhibitors of adenosine kinase (AK), adenosine deaminase (AD), equilibrative transporters), and by receptor-operated mechanisms (opioids, 5-hydroxytryptamine (5-HT), noradrenaline (NA)). Some of these agents release adenosine via an equilibrative transporter indicating production of adenosine inside the cell (K(+), morphine), while others release nucleotide which is converted extracellularly to adenosine by ecto-5'-nucleotidase (capsaicin, 5-HT). Release can be capsaicin-sensitive, Ca(2+)-dependent and involve G-proteins, and this suggests that within C-fibres, Ca(2+)-dependent intracellular processes regulate production and release of adenosine. In the periphery, adenosine is released from both neuronal and non-neuronal sources. Neuronal release from capsaicin-sensitive afferents is induced by glutamate and by neurogenic inflammation (capsaicin, low concentration of formalin), while that from sympathetic postganglionic neurons (probably as adenosine 5'-triphosphate (ATP) with NA) occurs following more generalized inflammation. Such release is modified differentially by inhibitors of AK and AD. Following nerve injury, there is an alteration in capsaicin-sensitive adenosine release, as spinal release now is less responsive to opioids, while peripheral release is less responsive to inhibitors of metabolism. Following inflammation, adenosine is released from a variety of cell types in addition to neurons (e.g. endothelial cells, neutrophils, mast cells, fibroblasts). ATP is released both spinally and peripherally following inflammation or injury, and may be converted to adenosine by ecto-5'-nucleotidase contributing an additional source of adenosine. Release of adenosine from both spinal and peripheral compartments has inhibitory effects on pain transmission, as methylxanthine adenosine receptor antagonists reduce analgesia produced by agents which augment extracellular levels of adenosine spinally (morphine, 5-HT, substance P, AK inhibitors) and peripherally (AK inhibitors, AD inhibitors). Increases in extracellular adenosine availability also may contribute to antiinflammatory effects of certain agents (methotrexate, sulfasalazine, salicylates, AK inhibitors), and this could have secondary effects on pain signalling in chronic inflammation. The purpose of the present review is to consider: (a). the factors that regulate the extracellular availability of adenosine in the spinal cord and at peripheral sites; and (b). the extent to which this adenosine affects pain signalling in these two distinct compartments.     

Adenosine, 'pertussis-sensitive' G-proteins, and K+ conductance in central mammalian neurones under energy deprivation

There is a striking similarity between the effects of adenosine and of hypoxia or glucose depletion on membrane potential and conductance of hippocampal neurones in tissue slices of rat brain. Both induce a membrane hyperpolarization by an increase in potassium conductance. It seemed likely, therefore, that a rise in extracellular adenosine concentration during energy deprivation may link neuronal metabolism with membrane K+ conductance. To test this hypothesis, we have now investigated the effects of hypoxia/glucose deprivation on hippocampal neurones from pertussis toxin-treated rats. In such slices adenosine had no effect on postsynaptic membrane potential and input resistance. Nevertheless, hypoxia or glucose depletion were as effective as in controls. These data provide evidence against adenosine as the main mediator between cell metabolism and potassium conductance.     

Potassium accumulation as dynamic modulator of neurohypophysial excitability

Activity-dependent modulation of excitable responses from neurohypophysial axons and their secretory swellings has long been recognized as an important regulator of arginine vasopressin and oxytocin release during patterned stimulation. Various activity-dependent mechanisms, including action potential broadening, potassium accumulation, and autocrine or paracrine feedback, have been proposed as underlying mechanisms. However, the relevance of any specific mechanism on net excitability in the intact preparation, during different levels of overall activation, and during realistic stimulation with trains of action potentials has remained largely undetermined. Using high-speed optical recordings and potentiometric dyes, we have quantified the dynamics of global excitability under physiologically more realistic conditions, that is in the intact neurohypophysis during trains of stimuli at varying frequencies and levels of overall activity. Net excitability facilitated during stimulation at low frequencies or at low activity. During persistent high-intensity or high-frequency stimulation, net excitability became severely depressed. Depression of excitable responses was strongly affected by manipulations of extracellular potassium levels, including changes to resting [K⁺]_out, increases of interstitial spaces with hypertonic solutions and inhibition of Na⁺/K⁺ ATPase activity. Application of the GABA_A receptor blocker bicuculline or manipulations of Ca²⁺ influx showed little effect. Numerical simulation of K⁺ accumulation on action potentials of individual axons reproduced optically recorded population responses, including the overall depression of action potential (AP) amplitudes, modest AP broadening and the prominent loss of hyperpolarizing undershoots. Hence, extracellular potassium accumulation dominates activity-dependent depression of neurohypophysial excitability under elevated stimulation conditions. The intricate dependence on the short-term stimulation history and its resulting feedback on neurohypophysial excitability renders [K⁺]_out accumulation a surprisingly complex mechanism for regulating axonal excitability and subsequent neuroendocrine release.     

Modulation of the ischemia-induced taurine release by adenosine receptors in the developing and adult mouse hippocampus

The release of the inhibitory amino acid taurine is markedly enhanced under ischemic conditions in both adult and developing hippocampus, together with a pronounced increase in the release of excitatory amino acids and the neuromodulator adenosine. We studied the effects of adenosine receptor agonists and antagonists as well as adenosine transport inhibitors on hippocampal [(3)H]taurine release in normoxia and ischemia, using a superfusion system. Under standard conditions the adenosine A(1) receptor agonists N(6)-cyclohexyladenosine and R(-)N(6)-(2-phenylisopropyl)adenosine potentiated basal taurine release in developing mice and depressed the release in adults in a receptor-mediated manner. Adenosine A(2) receptor compounds had only minor effects on the basal release and the K(+)-stimulated release was not affected by these drugs. The adenosine uptake inhibitor dipyridamole enhanced basal taurine release in the developing hippocampus and reduced it in the adult. In ischemia the adenosine compounds had no marked effects on taurine release in immature animals, whereas A(1) receptor activation was still able to evoke taurine release in adults by a receptor-mediated mechanism. The results show that the basal release of taurine is modulated by A(1) receptors in both mature and immature hippocampus, whereas in ischemia these receptors potentiate taurine release only in adults.The elevated taurine levels together with the depression of excitatory amino acid release by adenosine receptor activation could be beneficial under ischemic conditions, protecting neural cells against excitotoxicity and hyperexcitation.     

Inhibition of acetylcholine release in guinea pig ileum by adenosine.

The effect of adenosine on cholinergic neuroeffector transmission was studied in the isolated guinea pig ileum. Adenosine caused a dose-dependent and inverse frequency-dependent inhibition of contraction responses to transmural nerve stimulation. Blockade of adrenergic neurotransmission did not alter the inhibitory effect of adenosine. Adenosine also inhibited contraction responses to serotonin, angiotensin and high potassium, but not the responses to acetylcholine, histamine or direct electrical stimulation of the smooth muscle cells. Adenosine had little effect on basal outflow of acetylcholine but inhibited markedly and reversibly the release of acetylcholine induced by nerve stimulation. Acetylcholine was determined with gas chromatography-mass spectrometry. The results provide direct evidence that adenosine inhibits cholinergic neuroeffector transmission in the gut by a prejunctional action on acetylcholine release. This may be of functional importance since adenine compounds are released during stimulation of intestinal nerves.     

Mechanisms associated with hypoxia- and contraction-mediated glucose transport in muscle are fibre-dependent.

The purpose of this study was to examine the effects of hypoxia and muscle contractions on rates of 2-deoxyglucose (2-DG) transport in red and white portions of the gastrocnemius muscle of the rat. 2-DG transport was measured during the last 10 min of a 60-min hindlimb perfusion in male Wistar rats ( approximately 300 g), with or without muscle contractions of one limb. The medium was gassed with either 95% oxygen and 5% carbon dioxide or 95% nitrogen and 5% carbon dioxide to achieve normal or hypoxic conditions, respectively. Muscle contractions began after 30 min of perfusion and consisted of isometric muscle actions (200-ms trains, 100 Hz; one train per second) for two sets of 5 min, with 1-min rest between sets. 2-DG transport in white gastrocnemius was higher (P < 0.05) than basal during hypoxia (4.8-fold) and following contractions using oxygenated or hypoxic medium (4.6-fold and 5.4-fold, respectively; n=6 for each group). 2-DG transport was not different (P > 0.05) between these stimulated conditions. Similarly, 2-DG transport in red gastrocnemius was 5.1- and 4.8-fold higher (P < 0.05) than basal during hypoxia and following contractions in oxygenated medium, respectively. However, 2-DG transport following contractions during hypoxic conditions in red gastrocnemius was, unlike white gastrocnemius, higher (8.9-fold over basal; P < 0.05) than in all other conditions. These results suggest that mechanisms associated with hypoxia- and muscle contraction-mediated glucose transport are fibre type-dependent, with additive effects of the two stimuli in fast-twitch, oxidative fibres.     

Regulation by Rab3A of an endogenous modulator of neurotransmitter release at mouse motor nerve endings

Rab3A, a small GTP-binding protein attached to synaptic vesicles, has been implicated in several stages in the process of neurosecretion, including a late stage occurring just prior to the actual release of neurotransmitter. The inhibitory neuromodulator adenosine also targets a late step in the neurosecretory pathway. We thus compared neuromuscular junctions from adult Rab3A ^−/- mutant mice with those from wild-type mice with respect to: (a) the basic electrophysiological correlates of neurotransmitter release at different stimulation frequencies, and (b) the actions of exogenous and endogenous adenosine on neurotransmitter release in normal calcium solutions. Neither the spontaneous quantal release of acetylcholine (ACh) nor basal evoked ACh release (0.05 Hz) differed between the mutant and wild-type mice. At 50-100 Hz stimulation (10-19 stimuli), facilitation of release was observed in the mutant mice but not in wild-type, followed by a depression of ACh release in both strains. ACh release at the end of the stimulus train in the mutant mouse was approximately double that of the wild-type mouse. The threshold concentration for inhibition of ACh release by exogenous adenosine was over 20-fold lower in the mutant mouse than in the wild-type mouse. The adenosine A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) increased ACh release (0.05-1 Hz stimulation) in the mutant mouse under conditions in which it had no effect in the wild-type mouse. CPT had no effect on the pattern of responses recorded during repetitive stimulation in either strain. The results suggest that Rab3A reduces the potency of adenosine as an endogenous mediator of neuromuscular depression.     

Extracellular stimulation of mammalian neurons through repetitive activation of Na+ channels by weak capacitive currents on a silicon chip

Reliable extracellular stimulation of neuronal activity is the prerequisite for electrical interfacing of cultured networks and brain slices, as well as for neural implants. Safe stimulation must be achieved without damage to the cells. With respect to a future application of highly integrated semiconductor chips, we present an electrophysiological study of capacitive stimulation of mammalian cells in the geometry of adhesion on an insulated titanium dioxide/silicon electrode. We used HEK293 cells with overexpressed Na(V)1.4 channels and neurons from rat hippocampus. Weak biphasic stimuli of falling and rising voltage ramps were applied in the absence of Faradaic current and electroporation. We recorded the response of the intra- and extracellular voltage and evaluated the concomitant polarization of the attached and free cell membranes. Falling ramps efficiently depolarized the central area of the attached membrane. A transient sodium inward current was activated that gave rise to a weak depolarization of the cell on the order of 1 mV. The depolarization could be enhanced step by step by a train of biphasic stimuli until self-excitation of sodium channels set in. We applied the same protocol to cultured rat neurons and found that pulse trains of weak capacitive stimuli were able to elicit action potentials. Our results provide a basis for safe extracellular stimulation not only for cultured neurons on insulated semiconductor electrodes, but also more generally for metal electrodes in cell culture and brain tissue.