Stefan Boehm

Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d‐ and l‐amphetamine as well as substrate‐induced release of preloaded N‐methyl‐4‐[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium‐dependent and was inhibited by ouabain and cocaine, which also prevented substrate‐induced release of MPP+. Patch‐clamp recordings revealed that all four substrates elicited voltage‐dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch‐clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d‐amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate‐induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.

Somatostatin Inhibits Excitatory Transmission at Rat Hippocampal Synapses via Presynaptic Receptors

Somatostatin is one of the major peptides in interneurons of the hippocampus. It is believed to play a role in memory formation and to reduce the susceptibility of the hippocampus to seizure-like activity. However, at the cellular level, the actions of somatostatin on hippocampal neurons are still controversial, ranging from inhibition to excitation. In the present study, we measured autaptic currents of hippocampal neurons isolated in single-neuron microcultures. Somatostatin and the analogous peptides seglitide and octreotide reduced glutamatergic, but not GABAergic, autaptic currents via pertussis toxin-sensitive G-proteins. This effect was observed whether autaptic currents were mediated by NMDA or non-NMDA glutamate receptors. Furthermore, somatostatin did not affect currents evoked by the direct application of glutamate, but reduced the frequency of spontaneously occurring excitatory autaptic currents. These results show that presynaptic somatostatin receptors of the SRIF1family inhibit glutamate release at hippocampal synapses. Somatostatin, seglitide, and octreotide also reduced the frequency of miniature excitatory postsynaptic currents in mass cultures without affecting their amplitudes. In addition, all three agonists inhibited voltage-activated Ca2+ currents at neuronal somata, but failed to alter K+ currents, effects that were also abolished by pertussis toxin. Thus, presynaptic somatostatin receptors in the hippocampus selectively inhibit excitatory transmission via G-proteins of the Gi/Go family and through at least two separate mechanisms, the modulation of Ca2+channels and an effect downstream of Ca2+ entry. This presynaptic inhibition by somatostatin may provide a basis for its reportedly anticonvulsive action.

Presynaptic α2‐adrenoceptors control excitatory, but not inhibitory, transmission at rat hippocampal synapses

1 The effects of noradrenaline on neurotransmission at rat hippocampal synapses were investigated by recording autaptic currents in single neurons isolated on glial microislands. Noradrenaline reduced excitatory, but not inhibitory, autaptic currents in a pertussis toxin‐sensitive manner, but the amine did not affect glutamate‐evoked currents. 2 The inhibition of excitatory autaptic currents by noradrenaline was half‐maximal at 0.11 ± 0.06 μm. The α2‐adrenoceptor agonists UK 14 304 and clonidine were equipotent to noradrenaline in reducing these currents, whereas the α1‐adrenoceptor agonist methoxamine and the β‐adrenoceptor agonist isoprenaline (isoproterenol) were ineffective. The reduction of excitatory autaptic currents by noradrenaline was not altered by the α1‐adrenergic antagonist urapidil or the β‐antagonist propranolol, but reduced by the α2‐antagonist yohimbine. The subtype‐preferring antagonists rauwolscine and phentolamine (both at 0.3 μm) caused 9‐fold and 36‐fold rightward shifts in the concentration‐response curve for the noradrenaline‐dependent reduction of excitatory autaptic currents, respectively. Prazosine (1 μm) did not affect this concentration‐response curve. 3 Noradrenaline reduced voltage‐activated Ca2+ currents in excitatory, but not in inhibitory, microisland neurons. For comparison, the GABAB agonist baclofen reduced both excitatory and inhibitory autaptic currents and diminished voltage‐activated Ca2+ currents in both types of neurons. The inhibition of Ca2+ currents by noradrenaline was half‐maximal at 0.17 ± 0.05 μm, and UK 14 304 and clonidine were equipotent to noradrenaline in reducing these currents. The noradrenaline‐induced reduction of Ca2+ currents was antagonized by yohimbine, but not by urapidil or propranolol; the subtype‐preferring α2‐adrenergic antagonists displayed the following rank order of activity: phentolamine > rauwolscine > prazosine. 4 Noradrenaline did not affect K+ currents and failed to alter the frequency of miniature excitatory postsynaptic currents measured in mass cultures of hippocampal neurons. 5 These results show that noradrenaline regulates transmission at glutamatergic, but not at GABAergic, hippocampal synapses via presynaptic α2‐adrenoceptors of the α2A/D subtype. This inhibitory action involves an inhibition of voltage‐activated Ca2+ currents, but no modulation of spontaneous vesicle exocytosis or of voltage‐activated K+ currents.

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of β-actin mRNA and fewer dendritic β-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Serotonin-transporter mediated efflux: A pharmacological analysis of amphetamines and non-amphetamines

The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.

UTP‐ and ATP‐triggered transmitter release from rat sympathetic neurones via separate receptors

In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca2+‐dependent and tetrodotoxin‐sensitive [3H]‐noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 μmol 1−1, the concentration used in all subsequent experiments. Pre‐exposure of the neurones to 100 μmol 1−1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre‐exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 μmol 1−1 pyridoxal‐5″‐phosphate or 30 μmol 1−1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 μmol 1−1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.

RECEPTORS CONTROLLING TRANSMITTER RELEASE FROM SYMPATHETIC NEURONS IN VITRO

Primary cultures of postganglionic sympathetic neurons were established more than 30 years ago. More recently, these cultures have been used to characterize various neurotransmitter receptors that govern sympathetic transmitter release. These receptors may be categorized into at least three groups: (1) receptors which evoke transmitter release: (2) receptors which facilitate; (3) receptors which inhibit, depolarization-evoked release. Group (1) comprises nicotinic and muscarinic acetylcholine receptors, P2X purinoceptors and pyrimidinoceptors. Group (2) currently harbours beta-adrenoceptors, P2 purinoceptors, receptors for PACAP and VIP, as well as prostanoid EP1 receptors. In group (3), muscarinic cholinoceptors, alpha 2- and beta-adrenoceptors, P2 purinoceptors, and receptors for the neuropeptides NPY, somatostatin (SRIF1) and LHRH, as well as opioid (delta and kappa) receptors can be found. Receptors which regulate transmitter release from neurons in cell culture may be located either at the somatodendritic region or at the sites of exocytosis, i.e. the presynaptic specializations of axons. Most of the receptors that evoke release are located at the soma. There ionotropic receptors cause depolarizations to generate action potentials which then trigger Ca(2+)-dependent exocytosis at axon terminals. The signalling mechanisms of metabotropic receptors which evoke release still remain to be identified. Receptors which facilitate depolarization-evoked release appear to be located preferentially at presynaptic sites and presumably act via an increase in cyclic AMP. Receptors which inhibit stimulation evoked release are also presynaptic origin and most commonly rely on a G protein-mediated blockade of voltage-gated Ca2+ channels. Results obtained with primary cell cultures of postganglionic sympathetic neurons have now supplemented previous data about neurotransmitter receptors involved in the regulation of ganglionic as well as sympatho-effector transmission. In the future, this technique may prove useful to identify yet unrecognized receptors which control the output of the sympathetic nervous system and to elucidate underlying signalling mechanisms.

The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters

Background: Ibogaine is a noncompetitive inhibitor of SERT that stabilizes the transporter in an inward-open conformation. Results: Ibogaine binds to a site accessible from the cell exterior that does not overlap with the substrate-binding site. Conclusion: Ibogaine binds to a novel binding site on SERT and DAT. Significance: This study provides a mechanistic understanding of an unique inhibitor of SERT and DAT. Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.

Inhibition of N‐type Calcium Channels: The Only Mechanism by which Presynaptic α2‐Autoreceptors Control Sympathetic Transmitter Release

α2‐Adrenoceptors are known to inhibit voltage‐dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic α2‐adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10μM nicotine, or by adding Ca2+ to otherwise Ca2+‐free medium when cells had been made permeable by the calcium ionophore A23187 or by α‐latrotoxin. Pretreatment of neurons with the N‐type Ca2+ channel blocker ω‐conotoxin GVIA and application of the α2‐adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with α‐latrotoxin or A23187. In contrast, the L‐type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or α‐latrotoxin‐ and A23187‐permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L‐type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In ω‐conotoxin GVIA‐treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole‐cell recordings of voltage‐activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω‐conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α2‐adrenergic effects. Our results indicate that the α2‐autoreceptor‐mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω‐conotoxin GVIA‐sensitive N‐type Ca2+ channels. Mechanisms downstream of these channels and voltage‐sensitive Ca2+ channels other than N‐type appear not to be important.

Glycine Receptors in Cultured Chick Sympathetic Neurons are Excitatory and Trigger Neurotransmitter Release

1 Total RNA isolated from embryonic chick paravertebral sympathetic ganglia was used in a reverse transcription‐polymerase chain reaction (RT‐PCR) assay with a pair of degenerate oligonucleotide primers deduced from conserved regions of mammalian glycine receptor α‐subunits. Three classes of cDNA were identified which encode portions of the chicken homologues of the mammalian glycine receptor αl, α2 and α3 subunits. 2 The presence of functional glycine receptors was investigated in the whole‐cell configuration of the patch‐clamp technique in neurons dissociated from the ganglia and kept in culture for 7–8 days. In cells voltage clamped to −70 mV, glycine consistently induced inward currents in a concentration‐dependent manner and elicited half‐maximal peak current amplitudes at 43 μm. 3 The steady‐state current–voltage relation for glycine‐induced currents was linear between ±80 and −60 mV, but showed outward rectification at more hyperpolarized potentials. Reversal potentials of these currents shifted with changes in intracellular chloride concentrations and matched the calculated Nernst potentials for chloride. 4 β‐Alanine and taurine were significantly less potent than glycine in triggering inward currents, with half‐maximal responses at 79 and 86 μm, respectively. At maximally active concentrations, β‐alanine‐evoked currents were identical in amplitude to those induced by glycine. Taurine‐evoked currents, in contrast, never reached the same amplitude as glycine‐induced currents. 5 The classical glycine receptor antagonist strychnine reversibly reduced glycine‐induced currents, with half‐maximal inhibition occurring at 62 nm. Two more recently characterized glycine receptor antagonists, isonipecotic acid (half‐maximal inhibition at 2 mm) and 7‐trifluoromethyl‐4‐hydroxyquinoline‐3‐carboxylic acid (half‐maximal inhibition at 67 μm), also blocked glycine‐evoked currents in a reversible manner. The chloride channel blocker picrotoxin reduced glycine‐evoked currents, with half‐maximal effects at 348 μm. Inhibition by the glycine receptor channel blocker cyanotriphenylborate was half‐maximal at 4 μm. 6 Apart from evoking inward currents, glycine occasionally triggered short (< 100 ms) spike‐like currents which were abolished by hexamethonium and thus reflected synaptic release of endogenous acetylcholine. In addition, glycine caused Ca2+‐dependent and tetrodotoxin‐sensitive tritium overflow from neurons previously labelled with [3H] noradrenaline. This stimulatory action of glycine was reduced in the presence of strychnine and after treatment with the chloride uptake inhibitor furosemide (frusemide). 7 In 65% of neurons loaded with the Ca2+ indicator fura‐2 acetoxymethyl ester, glycine increased the ratio of the fluorescence signal obtained with excitation wavelengths of 340 and 380 nm, respectively, which indicates a rise in intracellular Ca2+ concentration. 8 The results show that sympathetic neurons contain transcripts for different glycine receptor α‐subunits and carry functional heteromeric glycine receptors which depolarize the majority of neurons to trigger transmitter release.