Positronium Laser Cooling via the 1

We report on laser cooling of a large fraction of positronium (Ps) in free flight by strongly saturating the 1^{3}S-2^{3}P transition with a broadband, long-pulsed 243 nm alexandrite laser. The ground state Ps cloud is produced in a magnetic and electric field-free environment. We observe two different laser-induced effects. The first effect is an increase in the number of atoms in the ground state after the time Ps has spent in the long-lived 2^{3}P states. The second effect is one-dimensional Doppler cooling of Ps, reducing the cloud's temperature from 380(20) to 170(20) K. We demonstrate a 58(9)% increase in the fraction of Ps atoms with v_{1D}<3.7×10^{4} ms^{-1}.

Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). EXPERIMENTAL APPROACH: The habenula and IPN were loaded with [(3) H]-choline or [(3) H]-NA and placed in superfusion chambers. [(3) H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, ß2, or ß4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. KEY RESULTS: Nicotine induced release of [(3) H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [(3) H]-ACh release was abolished in ß4-knockout mice but was unaffected in ß2- and α5-knockout mice. In contrast, nicotine-induced release of [(3) H]-NA in the IPN and habenula was blocked by TTX and reduced in both ß2-knockout and ß4-knockout mice, and dose-response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [(3) H]-ACh release required more pulses delivered at a higher frequency. CONCLUSIONS AND IMPLICATIONS: Our results confirm previous findings that ß4-containing nACh receptors are critical for [(3) H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [(3) H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism.

Visualisation of the zona incerta for deep brain stimulation at 3.0 Tesla.

PURPOSE: Deep-brain stimulation (DBS) of the zona incerta (ZI) has shown promising results for medication-refractory neurological disorders including Parkinson's disease (PD) and essential tremor (ET). The success of the intervention is indispensably dependent on the reliable visualisation of the ZI. The aim of the study was to evaluate different promising new magnetic resonance imaging (MRI) methods at 3.0 Tesla for pre-stereotactic visualisation of the ZI using a standard installation the protocol. METHODS: MRI of nine healthy volunteers was acquired (T1-MPRAGE, T2-FLAIR, T2*-FLASH2D, T2-SPACE and susceptibility-weighted imaging (SWI). Image quality and visualisation of the ZI for each sequence were analysed independently by two neuroradiologists using a 6-point scale. For T2*-FLASH2D the axial, coronal and sagittal planes were compared. The delineation of the ZI versus the internal capsule, the subthalamic nucleus and the pallidofugal fibres was evaluated in all sequences and compared to T2-FLAIR using a paired t-test. Inter-rater reliability, contrast-to-noise ratios (CNR), and signal-to-noise ratios (SNR) for the ZI were computed. For illustration, coronal T2*-FLASH2D images were co-registered with the corresponding section schema of the Schaltenbrand-Wahren stereotactic atlas. RESULTS: Only the rostral part of the ZI (rZI) could be identified. The rZI was best and reliably visualised in T2*-FLASH2D (particularly coronal orientation; p < 0.05). No major artifacts in the rZI were observed in any of the sequences. SWI, T2-SPACE, and T2*-FLASH imaging offered significant higher CNR values for the rZI compared to T2-FLAIR imaging using standard parameters. The co-registration of the coronal T2*-FLASH2D images projected the ZI clearly into the boundaries of the anatomical sections. CONCLUSIONS: The delineation of the rZI is best possible in T2*-FLASH2D (particularly coronal view) using a standard installation protocol at 3.0 T. The caudal ZI could not be discerned in any of the sequences.

Arterial spin labeling at 3.0 Tesla in subacute ischemia: comparison to dynamic susceptibility perfusion.

PURPOSE: Arterial spin labeling (ASL) is a promising but clinically not established non-invasive method to assess cerebral perfusion. The purpose of this study was to compare perfusion imaging with pulsed ASL (pASL) to conventional dynamic susceptibility contrast (DSC) perfusion-weighted imaging (PWL) using commercially available equipment and postprocessing (3.0 Tesla, 32-channel head coil) in patients with subacute ischemia. METHODS: The pASL and DSC-PWI techniques were compared in 15 patients with subacute ischemia (age 49-88 years, 6 females and 9 males, time from onset to scan 4-161 h). Image inhomogeneity was assessed with the non-uniformity index. Image quality, delineation of hypoperfusion and degree of hypoperfusion were rated by two readers using a 5-scale grading system. The volume of hypoperfusion was quantified planimetrically. RESULTS: Image quality and image inhomogeneity were superior in DSC time-to-peak (TTP) compared to pASL cerebral brain flow (CBF; both p < 0.05). The delineation of hypoperfusion was better in DSC-TTP (p < 0.05) and the hypoperfusion was graded as more severe in DSC-TTP (p < 0.05). The volume of hypoperfusion did not differ between pASL-CBF and DSC-TTP, however, in pASL-CBF five cases with small infarctions (lacunar and pontine) were false negative compared to DSC-relative CBF. The mismatch frequency was lower in pASL (13%) than in DSC-rCBF (20%) and DSC-TTP (47%). CONCLUSIONS: Using a commercially available sequence and a 32-channel head coil at 3.0 Tesla pASL-CBF is feasible but limited compared to DSC-PWI in the assessment of ischemic stroke. In its present form pASL has a reserve role in clinical practice for situations when gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) is contraindicated.

Multiple glacial refugia and complex postglacial range shifts of the obligatory woodland plant Polygonatum verticillatum (Convallariaceae).

The phylogeography of typical alpine plant species is well understood in Europe. However, the genetic patterns of boreo-montane species are mostly unstudied. Therefore, we analysed the AFLPs of 198 individuals of Polygonatum verticillatum over a major part of its European distribution. We obtained a total of 402 reproducible fragments, of which 96.8% were polymorphic. The average Phi(ST) over all samples was high (73.0%). The highest number of private fragments was observed in the Cantabrian Mountains; the highest genetic diversities of the populations were detected in populations from the Alps. BAPS, Principal Coordinates and Cluster analyses revealed a deep split between the Cantabrian population and all other samples. The latter further distinguished two major groups in western and eastern Europe. These results suggest a complex biogeographical history of P. verticillatum. The Cantabrian population was most probably isolated for the longest time. Furthermore, putative glacial survival centres might have existed in the western group around the glaciated Alps and in the eastern group in the foothills of the Carpathian and Balkan mountain systems. The origin of the Scandinavian populations is still unresolved, but an origin from the southeastern Alps or the western Balkans appears the most likely scenario.

The influence of membrane lipids in Staphylococcus aureus gamma-hemolysins pore formation.

The natural target of Staphylococcus aureus bicomponent gamma-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that gamma-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142-2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.

Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genes.

Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits alpha5, alpha7, or beta2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, alpha3, which is expressed in all SCG nAChRs, mRNA levels of beta4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, alpha4 declined sharply after birth and was barely detectable in adult animals. alpha5, alpha7, and beta2 subunit message levels also declined, though more slowly and less completely than alpha4. The subunits alpha6 and beta3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas alpha2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation.

S-phase progression stimulates both the mutagenic KU-independent pathway and mutagenic processing of KU-dependent intermediates, for nonhomologous end joining.

We used intrachromosomal substrates to directly monitor the effect of the cell cycle on the efficiency and the accuracy of nonhomologous end joining (NHEJ) in mammalian cells. We show that both KU and KU-independent (KU-alt) pathways are efficient when maintaining cells in G1/S, in G2/M or during dynamic progression through S phase. In addition, the accuracy of NHEJ is barely altered when the cells are blocked in G1/S or in G2/M. However, progression through S phase increases the frequency of deletions, which is a hallmark of the KU-alt pathway. Moreover, we show that the intermediates that are generated by the KU-dependent end joining of non-fully complementary ends, and which contain mismatches, nicks or gap intermediates, are less accurately processed when the cells progress through S phase. In conclusion, both KU and KU-alt processes are active throughout the cell cycle, but the repair is more error prone during S phase, both by increasing the mutagenic KU-alt pathway and decreasing the accuracy of the repair of the intermediates generated by the KU-dependent pathway.

Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation.

BACKGROUND AND PURPOSE: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. EXPERIMENTAL APPROACH: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. KEY RESULTS: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. CONCLUSIONS AND IMPLICATIONS: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.

Multicenter evaluation of a commercial PCR-enzyme-linked immunosorbent assay diagnostic kit (Onychodiag) for diagnosis of dermatophytic onychomycosis.

We prospectively evaluated a new PCR-enzyme-linked immunosorbent assay kit (Onychodiag; BioAdvance, France) for the diagnosis of dermatophytic onychomycosis by testing nail samples from 438 patients with suspected onychomycosis and from 108 healthy controls in three independent laboratories. In two laboratories, samples were collected by trained mycologists as close as possible to the lesions (proximal samples). In one laboratory, samples were collected by other physicians. All samples were processed by conventional mycological techniques and by Onychodiag, blindly to the mycological results. An additional distal sample, collected by clipping the nail plate, was obtained from 75 patients and tested with Onychodiag alone. In patients with culture-proven dermatophytic onychomycosis, the sensitivity of Onychodiag was 83.6% (87.9% including the gray zone) and ranged from 75 to 100% according to the laboratory and the sampling conditions. The specificity was 100% when healthy subjects were considered true negative controls. Onychodiag was positive on 68 patient samples that were sterile or yielded nondermatophyte species in culture. Based on the results of Onychodiag for mycologically proven positive samples and true-negative samples, these results were considered true positives, and the poor performance of mycology on these samples was attributed to inconvenient sampling conditions or to contaminants. When tested on distal samples, Onychodiag was positive in 49/53 (92%) cases of proven dermatophytic onychomycosis. Finally, with either proximal or distal samples, Onychodiag provided a diagnosis of dermatophytic onychomycosis within 24 to 48 h after sampling, and its sensitivity was close to that of mycological techniques applied to proximal samples.

Alternate use of distinct intersubunit contacts controls GABAA receptor assembly and stoichiometry.

GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

Expression of B cell receptor-associated signaling molecules in human lupus.

B cell receptor (BcR) signaling requires a tight regulation of several protein tyrosine kinases and phosphatases, and associated co-receptors. Mounting evidence indicates that abnormal BcR signaling, such as occurs in SHP-1 and Lyn-deficient mice, results in production of pathogenic autoantibodies and lupus-like glomerulonephritis, suggesting that altered signaling thresholds could underlie the development of systemic autoimmunity. To test this hypothesis, we investigated expression of BcR-associated signaling molecules in lymphocytes from patients with systemic lupus erythematosus (SLE) during inactive phases of the disease. We found that the transmembrane regulatory protein tyrosine phosphatase CD45 is expressed at abnormal levels. Strikingly, this reduction persisted during four months of follow-up. By contrast, despite its potent role as a regulator of thymus-independent immune responses and of B cell life span, the CD22 co-receptor is expressed at normal levels in B lymphocytes isolated ex vivo from SLE patients. We also noted unusual levels of the cytosolic protein tyrosine kinase Lyn and the protein tyrosine phosphatase SHP-1 in the lymphocytes of the patients. Since in normal B cells Lyn and SHP-1 act in concert within a common negative pathway in which CD45 counteracts SHP-I regulatory role, we propose that this feedback regulatory pathway is crippled to different degrees in human SLE B cells. Break of the balance between positive and negative signaling molecules likely modifies the BcR signaling thresholds. Such alterations, together with other factors, may contribute to the disruption of self-tolerance in this disease.

Health education groups for caregivers in an HMO.

The short-term effectiveness of a Health Education Group (HEP) intervention program for spouses of frail older adults was compared to the usual care (UC) offered to the spouses of frail older persons in a staff model health maintenance organization. HEP is a multicomponent group program offered in eight weekly, two-hour group sessions, and ten monthly, two-hour follow-up group sessions. It includes emotion-focused and problem-focused coping strategies, education, and support. One-hundred and five spouses were recruited and randomly assigned to HEP (n = 58) or UC (n = 47). Spouse caregivers and care recipients were assessed within two weeks of intervention and within two weeks after the completion of the eight weekly group meetings. The results indicate that, for caregivers, HEP was more effective than UC in reducing depression, maintaining social integration, increasing effectiveness in solving pressing problems, increasing knowledge of community services and how to access them, changing caregivers' feelings of competence, and the way they respond to the care giving situation. No significant differences, however, were found between care recipients in the two arms of the study on any of the outcome measures.

Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells.

Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.

The implementation of the EverCare demonstration project.

EverCare represents a creative approach to providing medical services to long-stay nursing home patients. It offers a capitated package of Medicare-covered services with more intensive primary care provided by nurse practitioners. The program's underlying premise is that better primary care will result in reduced hospital use. This work examines the implementation of the program in six locations. It identifies some of the issues that must be addressed if the program is to succeed both operationally and financially.

Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis.

Systemic lupus erythematosus (SLE) is characterized by an accelerated apoptosis of peripheral lymphocytes and an impairment of the clearance of apoptotic cells. Since changes in DNA methylation and in deoxycytosine and deoxyguanine (GC) content have been shown to enhance the potential of DNA to activate murine and human B lymphocytes, we tested the capacity of lymphocytes undergoing apoptosis (under conditions that mimic the deletion of self-reactive cells after antigen receptor engagement) to generate nucleosomes with a particular base composition. Using two cell culture systems and four apoptosis triggers, we found an increase of deoxymethylcytosine in fragmented chromosomal DNA of apoptotic B and T lymphocytes. However, this increase was not associated with modulation of DNA (cytosine-5) methyltransferase, the enzyme that methylates eukaryotic DNA, which suggests that the changes in DNA methylation patterns are not linked to the process of de novo DNA methylation during cell death. In addition, we could not detect a unique methylation pattern in highly repetitive Alu sequences present in the human genome of SLE subjects, as compared with controls. However, the abnormal DNA methylation of apoptotic nucleosomes was associated with an unusual pattern of nuclease-resistant, GC-rich regions in these DNA fragments. We propose that the combination of an accelerated apoptosis with a defect in the clearance of apoptotic cells results in release of increased amounts of nucleosomes with abnormally methylated, GC-rich DNA and provides an autologous stimulation that could bypass tolerance to self in systemic autoimmune diseases. These findings support the concept that the structure and dynamics of nucleosomes are critical in determining their immunogenicity in SLE.

Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences.

In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented.

Modulation of spontaneous and stimulation-evoked transmitter release from rat sympathetic neurons by the cognition enhancer linopirdine: insights into its mechanisms of action.

The mechanisms by which the cognition enhancer linopirdine may affect transmitter release were investigated in cultures of rat superior cervical ganglion neurons. Overflow of previously incorporated [3H]noradrenaline evoked by 10 microM UTP or 0.1 microM bradykinin was enhanced by linopirdine at > or =3 microM, overflow evoked by 25 mM K(-), 100 microM nicotine, or 300 microM ATP was enhanced by linopirdine at > or =10 microM, and overflow due to 40 mM K+ or electrical field stimulation was not altered by linopirdine. Ba2+ (0.3 mM) augmented the same types of stimulation-evoked overflow to a similar extent as linopirdine. K+ (25 mM), nicotine (100 microM), and ATP (300 microM) triggered transmitter release in a partially tetrodotoxin-resistant manner, and the release-enhancing action of linopirdine was lost in the presence of tetrodotoxin (1 microM). Linopirdine (10 microM) raised spontaneous tritium outflow and reduced currents through muscarinic K+ (K(M)) channels with a similar time course. The secretagogue action of linopirdine was concentration- and Ca2(+)-dependent and abolished by tetrodotoxin (1 microM) or Cd2+ (100 microM). Linopirdine (10 microM) added to the partial inhibition of K(M) channels by 1 or 3 mM Ba2(+) but not to the complete inhibition by 10 mM Ba2(+). Likewise, the secretagogue action of 1 and 3 mM, but not that of 10 mM, Ba2+ was enhanced by linopirdine. These results indicate that linopirdine facilitates and triggers transmitter release via blockade of K(M) channels and suggest that these K+ channels are located at neuronal somata rather than at presynaptic sites.

Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles.

1. The release of [3H]-noradrenaline ([3H]-NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist-induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2. [3H]-NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage-gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX- or Cd2+-insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 microM (DMPP) to 110 microM (ACh). 3. [3H]-NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX- or Cd2+-sensitive release). TTX-sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine approximately nicotine approximately ACh, which due to its similarity to TTX-insensitive release indicates that it might also be triggered by prejunctional-type nAChRs. The EC50 values for TTX (Cd2+)-sensitive release were less than 10 microM for all four agonists. 4. By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine approximately DMPP > ACh. EC50 values for the induction of currents exceeded 20 microM for all four agonists. 5. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. alpha-Bungarotoxin (alpha-BuTX) was, on the other hand, without significant effect on nicotine-induced TTX-insensitive release. The competitive antagonist dihydro-beta-erythroidine (DHbetaE) caused rightward shifts of the dose-response curves for both TTX-sensitive and TTX-insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. 6. Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.

Presynaptic inhibition by concanavalin A: are alpha-latrotoxin receptors involved in action potential-dependent transmitter release?

Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [3H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 microM. Concanavalin A also reduced the release triggered by extracellular Ca2+ in neurons depolarized by 25 mM K or rendered Ca2+-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the alpha2-adrenergic agonist UK 14,304. Inactivation of Gs and Gi/Go type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K+ and Ca2+ currents. In contrast, the lectin efficiently blocked both the Ca2+-dependent and -independent alpha-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as alpha-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl alpha-D-mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to alpha-latrotoxin, concanavalin A failed to alter K+-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of alpha-latrotoxin receptors may reduce action potential-dependent transmitter release.