Voltage sensitivity of NMDA-receptor mediated postsynaptic currents.

Patch-clamp techniques were used to record pharmacologically-isolated N-methyl-D-aspartate-mediated excitatory postsynaptic currents (NMDA-EPSCs) from dentate granule cells in thin rat hippocampal slices. Membrane voltage modulated these EPSCs in two ways. Firstly, depolarization from resting potential enhanced EPSC amplitudes, as expected for a voltage-dependent block by Mg2+ of synaptically activated NMDA receptor channels. Secondly, depolarization markedly prolonged the time course of decay of NMDA-EPSCs in normal and low extracellular Mg2+. Both mechanisms were complementary in establishing a strong dependence between membrane potential and the amount of charge, namely Ca2+, transferred through synaptically activated NMDA receptor channels, that presumably underlies induction of long-term potentiation in the hippocampus

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na+/K+ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K+ (aKe) and intracellular Na+ (aNai) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10−6 M) resulted in a membrane hyperpolarization and transient decreasesaKe andaNai which could be blocked by ouabain (3×10−4 M). In the h.p.p. muslce a continuous rise ofaNai induced by elevation ofaKe to 5.2 mM could be stopped by ADR. In addition, the intracellular K+ activity (aKi), the free intracellular Ca2+ concentration (pCai) and intracellular pH (pHi) were monitored in rat soleus muscle. During ADRaKi increased, pHi remained constant and intracellular Ca2+ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na+/K+ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle

Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord

Double-barrelled pH-sensitive micro-electrodes were used to measure changes of intracellular and extracellular pH in and around motoneurons of the isolated frog spinal cord during application of excitatory amino acids. It was found that N-methyl- -aspartate, quisqualate and kainate produced a concentration-dependent intracellular acidification. Extracellularly, triphasic pH changes (acid-alkaline-acid going pH transients) were observed during the action of these amino acids. The possible significance of such pH changes for the physiological and pathophysiological effects of excitatory amino acids are discussed

Acute anti-allodynic action of gabapentin in dorsal horn and primary somatosensory cortex: Correlation of behavioural and physiological data

Neuropathic pain is a debilitating consequence of neuronal injury or disease. Although first line treatments include the alpha-2-delta (a2d)-ligands, pregabalin and gabapentin (GBP), the mechanism of their anti-allodynic action is poorly understood. One specific paradox is that GBP relieves signs of neuropathic pain in animal models within 30min of an intraperitoneal (IP) injection yet its actions in vitro on spinal dorsal horn or primary afferent neurons take hours to develop. We found, using confocal Ca2þ imaging, that substantia gelatinosa neurons obtained ex vivo from rats subjected to sciatic chronic constriction injury (CCI) were more excitable than controls. We confirmed that GBP (100 mg/kg) attenuated mechanical allodynia in animals subject to CCI within 30min of IP injection.Substantia gelatinosa neurons obtained ex vivo from these animals no longer displayed CCI-induced increased excitability. Electrophysiological analysis of substantia gelatinosa neurons ex vivo suggest that rapidly developing in vivo anti-allodynic effects of GBP i) are mediated intracellularly, ii) involve actions on the neurotransmitter release machinery and iii) depend on decreased excitatory synaptic drive to excitatory neurons without major actions on inhibitory neurons or on intrinsic neuronal excitability. Experiments using in vivo Ca2þ imaging showed that 100 mg/kg GBP also suppressed the response of the S1 somatosensory cortex of CCI rats, but not that of control rats, to vibrotactile stimulation. Since the level of a2d1 protein is increased in primary afferent fibres after sciatic CCI, we suggest this dictates the rate of GBP action; rapidly developing actions can only be seen when a2d1 levels are elevated

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca(2+) -dependent P2Y1 receptor mechanism

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca(2+) from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca(2+) recruitment from intracellular stores leading to increases in intracellular Ca(2+) ([Ca(2+) ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca(2+) release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression

Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm

We developed a dual oscillator model to facilitate the understanding of dynamic interactions between the parafacial respiratory group (pFRG) and the preBötzinger complex (preBötC) neurons in the respiratory rhythm generation. Both neuronal groups were modeled as groups of 81 interconnected pacemaker neurons; the bursting cell model described by Butera and others [model 1 in Butera et al. (J Neurophysiol 81:382–397, 1999a)] were used to model the pacemaker neurons. We assumed (1) both pFRG and preBötC networks are rhythm generators, (2) preBötC receives excitatory inputs from pFRG, and pFRG receives inhibitory inputs from preBötC, and (3) persistent Na+ current conductance and synaptic current conductances are randomly distributed within each population. Our model could reproduce 1:1 coupling of bursting rhythms between pFRG and preBötC with the characteristic biphasic firing pattern of pFRG neurons, i.e., firings during pre-inspiratory and post-inspiratory phases. Compatible with experimental results, the model predicted the changes in firing pattern of pFRG neurons from biphasic expiratory to monophasic inspiratory, synchronous with preBötC neurons. Quantal slowing, a phenomena of prolonged respiratory period that jumps non-deterministically to integer multiples of the control period, was observed when the excitability of preBötC network decreased while strengths of synaptic connections between the two groups remained unchanged, suggesting that, in contrast to the earlier suggestions (Mellen et al., Neuron 37:821–826, 2003; Wittmeier et al., Proc Natl Acad Sci USA 105(46):18000–18005, 2008), quantal slowing could occur without suppressed or stochastic excitatory synaptic transmission. With a reduced excitability of preBötC network, the breakdown of synchronous bursting of preBötC neurons was predicted by simulation. We suggest that quantal slowing could result from a breakdown of synchronized bursting within the preBötC

Excess α-synuclein worsens disease in mice lacking ubiquitin carboxy-terminal hydrolase L1

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species

Astrocytic Mechanisms Explaining Neural-Activity-Induced Shrinkage of Extraneuronal Space

Neuronal stimulation causes ∼30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na+/K+/Cl− (NKCC1) and the Na+/HCO3− (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia–neuron interaction models for normal as well as pathophysiological situations