Ratiometric and photoconvertible fluorescent protein-based voltage indicator prototypes.

To expand the toolbox of fluorescent protein-based voltage indicators, we explored two distinct protein design strategies. Using these design strategies, we created three new voltage indicators: a red intensiometric voltage indicator (tdFlicR Δ110AR), a green/red ratiometric voltage indicator (tdFlicR-VK-ASAP), and a green to red photoconvertible voltage indicator (FlicGR1).

Methylxanthine-evoked perturbation of spontaneous and evoked activities in isolated newborn rat hippocampal networks.

Treatment of apnea of prematurity with methylxanthines like caffeine, aminophylline or theophylline can evoke hippocampal seizures. However, it is unknown at which interstitial brain concentrations methylxanthines promote such neonatal seizures or interfere with physiological 'early network oscillations' (ENOs) that are considered as pivotal for maturation of hippocampal neural networks. We studied theophylline and caffeine effects on ENOs in CA3 neurons (CA3-ENOs) and CA3 electrical stimulation-evoked monosynaptic CA1 field potentials (CA1-FPs) in sliced and intact hippocampi, respectively, from 8 to 10-days-old rats. Submillimolar doses of theophylline and caffeine, blocking adenosine receptors and phosphodiesterase-4 (PDE4), did not affect CA3-ENOs, ENO-associated cytosolic Ca(2+) transients or CA1-FPs nor did they provoke seizure-like discharges. Low millimolar doses of theophylline (⩾1mM) or caffeine (⩾5mM), blocking GABAA and glycine receptors plus sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA)-type Ca(2+) ATPases, evoked seizure-like discharges with no indication of cytosolic Ca(2+) dysregulation. Inhibiting PDE4 with rolipram or glycine receptors with strychnine had no effect on CA3-ENOs and did not occlude seizure-like events as tested with theophylline. GABAA receptor blockade induced seizure-like discharges and occluded theophylline-evoked seizure-like discharges in the slices, but not in the intact hippocampi. In summary, submillimolar methylxanthine concentrations do not acutely affect spontaneous CA3-ENOs or electrically evoked synaptic activities and low millimolar doses are needed to evoke seizure-like discharges in isolated developing hippocampal neural networks. We conclude that mechanisms of methylxanthine-related seizure-like discharges do not involve SERCA inhibition-related neuronal Ca(2+) dysregulation, PDE4 blockade or adenosine and glycine receptor inhibition, whereas GABA(A) receptor blockade may contribute partially.

Methylxanthine reversal of opioid-induced respiratory depression in the neonatal rat: mechanism and location of action.

Methylxanthines like caffeine and theophylline have long been used to treat apnea of prematurity. Despite their success in stimulating neonatal breathing, their mechanism of action remains poorly understood. Methylxanthines can act as both non-specific adenosine receptor antagonists and inhibitors of cAMP-dependent phosphodiesterases, sarcoplasmic/endoplasmic reticulum calcium ATPases or receptor-coupled anion channels, depending on the dose used. Though there is evidence for methylxanthine action at the level of the carotid body, the consensus is that methylxanthines stimulate the respiratory centers of the brainstem. Here we used the in situ neonatal rat working heart-brainstem preparation and the ex vivo neonatal rat carotid body preparation to test the hypothesis that methylxanthines act at the level of the carotid body. We conclude that although the neonatal carotid body has active adenosine receptors, the effects of methylxanthine therapy are likely mediated centrally, predominantly via inhibition of cAMP-dependent phosphodiesterase-4.

Methylxanthines do not affect rhythmogenic preBötC inspiratory network activity but impair bursting of preBötC-driven motoneurons.

Clinical stimulation of preterm infant breathing with methylxanthines like caffeine and theophylline can evoke seizures. It is unknown whether underlying neuronal hyperexcitability involves the rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) in the brainstem or preBötC-driven motor networks. Inspiratory-related preBötC interneuronal plus spinal (cervical/phrenic) or cranial hypoglossal (XII) motoneuronal bursting was studied in newborn rat en bloc brainstem-spinal cords and brainstem slices, respectively. Non-respiratory bursting perturbed inspiratory cervical nerve activity in en bloc models at >0.25mM theophylline or caffeine. Rhythm in the exposed preBötC of transected en bloc preparations was less perturbed by 10mM theophylline than cervical root bursting which was more affected than phrenic nerve activity. In the preBötC of slices, even 10mM methylxanthine did not evoke seizure-like bursting whereas >1mM masked XII rhythm via large amplitude 1-10Hz oscillations. Blocking A-type γ-aminobutyric (GABAA) receptors evoked seizure-like cervical activity whereas in slices neither XII nor preBötC rhythm was disrupted. Methylxanthines (2.5-10mM), but not blockade of adenosine receptors, phosphodiesterase-4 or the sarcoplasmatic/endoplasmatic reticulum ATPase countered inspiratory depression by muscimol-evoked GABAA receptor activation that was associated with a hyperpolarization and input resistance decrease silencing preBötC neurons in slices. The latter blockers did neither affect preBötC or cranial/spinal motor network bursting nor evoke seizure-like activity or mask corresponding methylxanthine-evoked discharges. Our findings show that methylxanthine-evoked hyperexcitability originates from motor networks, leaving preBötC activity largely unaffected, and suggest that GABAA receptors contribute to methylxanthine-evoked seizure-like perturbation of spinal motoneurons whereas non-respiratory XII motoneuron oscillations are of different origin.

Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr.

Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF-responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1(-/-)) mice displayed allodynia (p<0.05), diminished epidermalinnervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1(-/-)) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin-related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor agonist indicated that NGFacted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr's effect on DRG neurons. Overall, NGF/TrkA signaling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.

Activity and metabolism-related Ca2+ and mitochondrial dynamics in co-cultured human fetal cortical neurons and astrocytes.

Neurons and neighboring astrocytic glia are mostly studied in nervous tissues from rodents whereas less is known on their properties and interactions in the human brain. Here, confocal/multiphoton fluorescence imaging for several hours revealed that co-cultured fetal human cortical neurons and astrocytes show pronounced spontaneous rises of cytosolic Ca(2+) which last for up to several minutes without concomitant changes in either movements or membrane potential of mitochondria. Similar Ca(2+) rises were evoked mainly in neurons by bath-applied glutamate or γ-aminobutyric acid (GABA) acting via N-methyl-d-aspartate (NMDA)+AMPA/Kainate and GABAA receptors, respectively. Predominantly in astrocytes, Ca(2+) baseline was elevated by adenosine diphosphate (ADP) and adenosine triphosphate (ATP) acting via P2Y1 and P2X7 receptors, likely causing the release of glutamate and glutamine. Mainly astrocytes responded to histamine, whereas the activation of muscarinic acetylcholine (ACh) receptors raised Ca(2+) in both cell types. Evoked neuronal and astrocytic Ca(2+) rises could last for several minutes without affecting mitochondrial movements or membrane potential. In contrast, reversible depolarization of mitochondrial membrane potential accompanied neuronal Ca(2+) rises induced by cyanide-evoked chemical anoxia or the uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). During such metabolic perturbation, mitochondrial depolarization also occurred in astrocytes, whereas Ca(2+) was largely unaffected. In summary, fetal human cortical neurons and astrocytes show distinct patterns of neuro/glio-transmitter- and metabolically-evoked Ca(2+) rises and possess active mitochondria. One aspect of our discussion deals with the question of whether the functional mitochondria contribute to cellular Ca(2+) homeostasis that seems to be already well-developed in fetal human cortical brain cells.

A1 adenosine receptor modulation of chemically and electrically evoked lumbar locomotor network activity in isolated newborn rat spinal cords.

It is not well-studied how the ubiquitous neuromodulator adenosine (ADO) affects mammalian locomotor network activities. We analyzed this here with focus on roles of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)-sensitive A(1)-type ADO receptors. For this, we recorded field potentials from ventral lumbar nerve roots and electrically stimulated dorsal roots in isolated newborn rat spinal cords. At ≥ 25µM, bath-applied ADO slowed synchronous bursting upon blockade of anion-channel-mediated synaptic inhibition by bicuculline (20 µM) plus strychnine (1 µM) and this depression was countered by DPCPX (1 µM) as tested at 100 µM ADO. ADO abolished this disinhibited rhythm at ≥ 500 µM. Contrary, the single electrical pulse-evoked dorsal root reflex, which was enhanced in bicuculline/strychnine-containing solution, persisted at all ADO doses (5 µM-2 mM). In control solution, ≥ 500 µM ADO depressed this reflex and pulse train-evoked bouts of alternating fictive locomotion; this inhibition was reversed by 1 µM DPCPX. ADO (5 µM-2 mM) did not depress, but stabilize alternating fictive locomotion evoked by serotonin (10 µM) plus N-methyl-d-aspartate (4-5 µM). Addition of DPCPX (1µM) to control solution did not change either the dorsal root reflex or rhythmic activities indicating lack of endogenous A(1) receptor activity. Our findings show A(1) receptor involvement in ADO depression of the dorsal root reflex, electrically evoked fictive locomotion and spontaneous disinhibited lumbar motor bursting. Contrary, chemically evoked fictive locomotion and the enhanced dorsal root reflex in disinhibited lumbar locomotor networks are resistant to ADO. Because ADO effects in standard solution occurred at doses that are notably higher than those occurring in vivo, we hypothesize that newborn rat locomotor networks are rather insensitive to this neuromodulator.

Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems.

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven "type-I" Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The micro-opioid [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (approximately 5 mV) and decreased their resistance (approximately 30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.

Serotonergic modulation of respiratory motoneurons and interneurons in brainstem slices of perinatal rats.

Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-Bötzinger complex (pre-BötC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-BötC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-BötC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-BötC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-BötC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-BötC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.

Chemical anoxia activates ATP-sensitive and blocks Ca(2+)-dependent K(+) channels in rat dorsal vagal neurons in situ.

The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca(2+) measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca(2+) ([Ca(2+)](i)) under normoxic conditions, but did not modify the rise of [Ca(2+)](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca(2+)](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca(2+)](i) rise were abolished by Ca(2+)-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca(2+)](i) increase. Intracellular BAPTA for Ca(2+) chelation blocked the afterhyperpolarizing current and the accompanying [Ca(2+)](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca(2+)](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca(2+)](i) transient with the Ca(2+) pump blocker cyclopiazonic acid did not evoke an outward current. Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca(2+)](i) does not interfere with the activity of these K(+) channels.

ATP-independent anoxic activation of ATP-sensitive K+ channels in dorsal vagal neurons of juvenile mice in situ.

The role of ATP in anoxic activation of ATP-sensitive K+ (KATP) channels was studied in dorsal vagal neurons of mouse brainstem slices. In the whole-cell configuration, cyanide-induced chemical anoxia evoked within 10 s a 300-pA outward current that gave rise to a hyperpolarization of 24 mV. These responses were mimicked by nitrogen-aerated saline, rotenone or diazoxide and abolished by tolbutamide. The cyanide-induced hyperpolarization was due to activation of 70 pS K(ATP) channels that were half-maximally blocked by 5 microM internal ATP. Dialyzing the cells with either 1, 20 or 0 mM ATP did not, however, affect the time to onset, the kinetics or the magnitude of the cyanide-induced hyperpolarization. Impairment of ATP consumption by ouabain, vanadate or reduced temperature had no effect either. Thus, anoxia-induced activation of these KATP channels cannot be explained by a fall of cellular ATP or a concomitant rise of ADP. Anoxia-related changes of the actin cytoskeleton or the composition of the plasma membrane are also not likely to be involved, as cytochalasin D did not affect the cyanide-evoked hyperpolarization and phosphatidylinositol 4,5-bisphosphate failed to decrease the ATP sensitivity of single KATP channels. Finally, because of a lack of effects of reduced/oxidized glutathione and the oxidase blocker diphenyliodonium on the cyanide-induced hyperpolarization, cellular redox state does not appear to be involved. Our results indicate that despite a high sensitivity to ATP in excised patches, anoxic activation of KATP channels is independent of cellular ATP. Rather the ATP block seems to be removed as a consequence of impaired mitochondrial function.

Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition.

Synaptic inhibition by GABA(A) and glycine receptors, which are ligand-gated anion channels, depends on the electrochemical potential for chloride. Several potassium-chloride cotransporters can lower the intracellular chloride concentration [Cl(-)](i), including the neuronal isoform KCC2. We show that KCC2 knockout mice died immediately after birth due to severe motor deficits that also abolished respiration. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. In the spinal cord of wild-type animals, the KCC2 protein was found at inhibitory synapses. Patch-clamp measurements of embryonic day 18.5 spinal cord motoneurons demonstrated an excitatory GABA and glycine action in the absence, but not in the presence, of KCC2, revealing a crucial role of KCC2 for synaptic inhibition.

Role of bicarbonate and chloride in GABA- and glycine-induced depolarization and [Ca2+]i rise in fetal rat motoneurons in situ.

Ca(2+) imaging and (perforated) patch recording were used to analyze the mechanism of GABA- and glycine-induced depolarizations in lumbar motoneurons of spinal cord slices from fetal rats. In fura-2 ester-loaded cells, the agonist-induced depolarizations increased [Ca(2+)](i) by up to 100 nm. The GABA- and glycine-evoked [Ca(2+)](i) transients were suppressed by bicuculline and strychnine, respectively. Their magnitude decreased by approximately 50% between embryonic days 15.5 and 19.5. The [Ca(2+)](i) increases were abolished by Ca(2+)-free superfusate and attenuated by approximately 65% by nifedipine, showing that the responses were mediated by voltage-activated Ca(2+) channels. The [Ca(2+)](i) rises were potentiated by >300% immediately after removal of Cl(-) from the superfusate but recovered to values of 50-200% of control during repeated agonist administration in Cl(-)-free saline. Bumetanide gradually suppressed the [Ca(2+)](i) increases by >75%. Subsequent removal of Cl(-) reconstituted the responses and increased, upon repeated agonist application, the peak [Ca(2+)](i) rises to values above control. Removal of HCO(3)(-) from the Cl(-)-free (bumetanide-containing) superfusate reversibly abolished both the agonist-induced [Ca(2+)](i) rises and depolarizations that were reestablished by formate anions. In Cl(-)-containing superfusate, removal of HCO(3)(-) decreased both the peak and duration of the agonist-evoked membrane depolarization and [Ca(2+)](i) response. Our findings show that HCO(3)(-) efflux has a major contribution to depolarizations mediated by GABA(A) and glycine receptor-coupled anion channels in prenatal neurons. We hypothesize that the HCO(3)(-)-dependent depolarizing component, which is likely to produce an intracellular acidosis, might play an important role during the early postnatal period when the Cl(-)-dependent component gradually shifts to hyperpolarization.

Molecular determinants of Ca2+-dependent K+ channel function in rat dorsal vagal neurones.

Using in situ hybridisation histochemistry in combination with patch-clamp recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+-dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. In situ hybridisation analysis at cellular resolution revealed the presence of 'big'-conductance Ca2+- and voltage-activated K+ (BK) channel alpha-subunit mRNA, and of only one 'small'-conductance Ca2+-activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. The SK channel-mediated after-hyperpolarising current (IAHP) was blocked by apamin with a half-maximal inhibitory concentration of approximately 2.2 nM. This is consistent with homomultimeric SK3 channels mediating IAHP in DVNs. IAHP was also blocked by scyllatoxin (20-30 nM) and curare (100-200 microM). Application of apamin (100 nM) or scyllatoxin (20 nM) invariably caused a substantial increase to 146.1 +/- 10.4 and 181.8 +/- 12.9 % of control, respectively, in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. The selective BK channel blocker iberiotoxin (50 nM) increased action potential duration by 22.5 +/- 7.3 %, as did low concentrations of tetraethylammonium (0.5 mM; 99.3 +/- 16.4 %) and the Ca2+ channel blocker Cd2+ (100 microM; 49.5 +/- 20.9 %). BK channel blockade did not significantly affect the firing rate of DVNs. These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones.

Ischemia but not anoxia evokes vesicular and Ca(2+)-independent glutamate release in the dorsal vagal complex in vitro.

Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca(2+)](i) rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1, 3-dicarboxylic acid nor affected by Ca(2+)-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca(2+)](i) increase that turned after several minutes into a prominent Ca(2+) transient (0.9 microM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca(2+)](i) rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca(2+)](i) rise by several minutes and attenuated their magnitudes by up to 40%. Ca(2+)-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca(2+) increase but not the initial moderate [Ca(2+)](i) rise. Cyclopiazonic acid or arachidonic acid in Ca(2+)-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl(-) channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca(2+) entry via N-methyl-D-aspartate receptors and voltage-activated Ca(2+) channels. An early Ca(2+)-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl(-) channels. The results also show that glycolysis prevents glutamate release during anoxia.

Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ.

In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (DeltaPsi) changes and intracellular Ca(2+) ([Ca(2+)](i)) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca(2+)](i) rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by

Anticonvulsant A(1) receptor-mediated adenosine action on neuronal networks in the brainstem-spinal cord of newborn rats.

Membrane potential of ventral respiratory group neurons as well as inspiratory-related cranial (hypoglossal) and spinal (C(1)-Th(4)) nerve activities were analysed in brainstem-spinal cord preparations from neonatal rats. Block of Cl(-)-mediated inhibition with bicuculline (plus strychnine) affected neither rhythmic depolarizations nor spike discharge in 23 of 30 ventral respiratory group cells. In the other seven neurons, block of inhibitory postsynaptic potentials evoked pronounced depolarizations and spike discharge that was synchronous with seizure-like spinal nerve activity. Respiratory hypoglossal nerve activity persisted after transection at the spinomedullary junction, whereas spinal rhythm was blocked. After transection, the moderate bicuculline-evoked seizure-like perturbation of hypoglossal nerve activity was abolished and rhythmic ventral respiratory group neuron activity was not disturbed, whereas epileptiform discharge persisted in spinal nerves. The seizure-like nerve activity and depolarization of the minor subpopulation of perturbed ventral respiratory group neurons were reversed by either adenosine or the A(1) adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine. The A(2) receptor agonist CGS 21860 had no effect. In control preparations, inspiratory nerve activity and membrane potential fluctuations (29 of 35 cells) were not changed by adenosine, 2-chloro-N(6)-cyclopentyladenosine or CGS 21860. In the other six cells, adenosine evoked a hyperpolarization (<10 mV) with no major change in input resistance. The anticonvulsant effects of adenosine and 2-chloro-N(6)-cyclopentyladenosine were antagonized by the A(1) adenosine receptor blocker 8-cyclopentyl-1,3-dipropylxanthine. After pre-incubation with 8-cyclopentyl-1,3-dipropylxanthine, bicuculline also evoked seizure-like discharge in the hypoglossal nerve. The results indicate that seizure-like spinal motor output of the respiratory network upon block of Cl(-)-mediated inhibition is caused by disinhibition of spinal neuronal networks with afferent connections to the ventral respiratory group. Presynaptic A(1) adenosine receptors exert an anticonvulsant action on the disinhibited spinal motor network, but have no depressing effect per se on the isolated medullary respiratory network.

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ.

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.

Neuron-glia signaling via alpha(1) adrenoceptor-mediated Ca(2+) release in Bergmann glial cells in situ.

Adrenoceptors were among the first neurotransmitter receptors identified in glial cells, but it is not known whether these receptors meditate glial responses during neuronal activity. We show that repetitive nerve activity evoked a rise of intracellular calcium in Bergmann glia and neighboring Purkinje neurons of cerebellar slices of mice. The glial but not the neuronal calcium transient persisted during block of ionotropic and metabotropic glutamate receptors. In contrast, the glial calcium response was abolished by cyclopiazonic acid and prazosin; however, prazosin affected neither the inward current nor the resulting depolarization that accompanied the stimulus-induced glial calcium transients. The glial depolarization was attenuated by 38% by the mixture of glutamate receptor blockers, which abolished the evoked neuronal depolarization and afterhyperpolarization. Ba(2+) reduced the glial currents by 66% without affecting the concomitant calcium transients. In the presence of Ba(2+), the mixture of glutamate receptor blockers exerted no effect on the glial inward current or calcium rise. Furthermore, Ba(2+) greatly potentiated both the activity-related Purkinje cell inward current and the accompanying neuronal calcium rises. The results indicate that release of noradrenaline from afferent fibers activates a glial alpha(1) adrenoceptor that promotes calcium release from intracellular stores. Glial calcium rises are known to stimulate a diversity of processes such as transmitter release, energy metabolism, or proliferation. Thus the adrenoceptor-mediated mechanism described here is well suited for feedback modulation of neuronal function that is independent of glutamate.

Respiratory network function in the isolated brainstem-spinal cord of newborn rats.

The in vitro brainstem-spinal cord preparation of newborn rats is an established model for the analysis of respiratory network functions. Respiratory activity is generated by interneurons, bilaterally distributed in the ventrolateral medulla. In particular non-NMDA type glutamate receptors constitute excitatory synaptic connectivity between respiratory neurons. Respiratory activity is modulated by a diversity of neuroactive substances such as serotonin, adenosine or norepinephrine. Cl(-)-mediated IPSPs provide a characteristic pattern of membrane potential fluctuations and elevation of the interstitial concentration of (endogenous) GABA or glycine leads to hyperpolarisation-related suppression of respiratory activity. Respiratory rhythm is not blocked upon inhibition of IPSPs with bicuculline, strychnine and saclofen. This indicates that GABA- and glycine-mediated mutual synaptic inhibition is not crucial for in vitro respiratory activity. The primary oscillatory activity is generated by neurons of a respiratory rhythm generator. In these cells, a set of intrinsic conductances such as P-type Ca2+ channels, persistent Na+ channels and G(i/o) protein-coupled K+ conductances mediates conditional bursting. The respiratory rhythm generator shapes the activity of an inspiratory pattern generator that provides the motor output recorded from cranial and spinal nerve rootlets in the preparation. Burst activity appears to be maintained by an excitatory drive due to tonic synaptic activity in concert with chemostimulation by H+. Evoked anoxia leads to a sustained decrease of respiratory frequency, related to K+ channel-mediated hyperpolarisation, whereas opiates or prostaglandins cause longlasting apnea due to a fall of cellular cAMP. The latter observations show that this in vitro model is also suited for analysis of clinically relevant disturbances of respiratory network function.