Excitatory Junctional Currents Research Articles

Triose-phosphate isomerase deficiency is associated with a dysregulation of synaptic vesicle recycling in Drosophila melanogaster.

Numerous neurodegenerative diseases are associated with neuronal dysfunction caused by increased redox stress, often linked to aberrant production of redox-active molecules such as nitric oxide (NO) or oxygen free radicals. One such protein affected by redox-mediated changes is the glycolytic enzyme triose-phosphate isomerase (TPI), which has been shown to undergo 3-nitrotyrosination (a NO-mediated post-translational modification) rendering it inactive. The resulting neuronal changes caused by this modification are not well understood. However, associated glycation-induced cytotoxicity has been reported, thus potentially causing neuronal and synaptic dysfunction via compromising synaptic vesicle recycling. This work uses Drosophila melanogaster to identify the impacts of altered TPI activity on neuronal physiology, linking aberrant TPI function and redox stress to neuronal defects. We used Drosophila mutants expressing a missense allele of the TPI protein, M81T, identified in a previous screen and resulting in an inactive mutant of the TPI protein (TPIM81T , wstd1). We assessed synaptic physiology at the glutamatergic Drosophila neuromuscular junction (NMJ), synapse morphology and behavioural phenotypes, as well as impacts on longevity. Electrophysiological recordings of evoked and spontaneous excitatory junctional currents, alongside high frequency train stimulations and recovery protocols, were applied to investigate synaptic depletion and subsequent recovery. Single synaptic currents were unaltered in the presence of the wstd1 mutation, but frequencies of spontaneous events were reduced. Wstd1 larvae also showed enhanced vesicle depletion rates at higher frequency stimulation, and subsequent recovery times for evoked synaptic responses were prolonged. A computational model showed that TPI mutant larvae exhibited a significant decline in activity-dependent vesicle recycling, which manifests itself as increased recovery times for the readily-releasable vesicle pool. Confocal images of NMJs showed no morphological or developmental differences between wild-type and wstd1 but TPI mutants exhibited learning impairments as assessed by olfactory associative learning assays. Our data suggests that the wstd1 phenotype is partially due to altered vesicle dynamics, involving a reduced vesicle pool replenishment, and altered endo/exocytosis processes. This may result in learning and memory impairments and neuronal dysfunction potentially also presenting a contributing factor to other reported neuronal phenotypes.

Alterations of RNA-binding protein found in neurons in Drosophila neurons and glia influence synaptic transmission and lifespan.

The found in neurons (fne), a paralog of the RNA-binding protein ELAV gene family in Drosophila, is required for post-transcriptional regulation of neuronal development and differentiation. Previous explorations into the functions of the FNE protein have been limited to neurons. The function of fne in Drosophila glia remains unclear. We induced the knockdown or overexpression of fne in Drosophila neurons and glia to determine how fne affects different types of behaviors, neuronal transmission and the lifespan. Our data indicate that changes in fne expression impair associative learning, thermal nociception, and phototransduction. Examination of synaptic transmission at presynaptic and postsynaptic terminals of the larval neuromuscular junction (NMJ) revealed that loss of fne in motor neurons and glia significantly decreased excitatory junction currents (EJCs) and quantal content, while flies with glial fne knockdown facilitated short-term synaptic plasticity. In muscle cells, overexpression of fne reduced both EJC and quantal content and increased short-term synaptic facilitation. In both genders, the lifespan could be extended by the knockdown of fne in neurons and glia; the overexpression of fne shortened the lifespan. Our results demonstrate that disturbances of fne in neurons and glia influence the function of the Drosophila nervous system. Further explorations into the physiological and molecular mechanisms underlying neuronal and glial fne and elucidation of how fne affects neuronal activity may clarify certain brain functions.

Modulation of Smooth Muscle Relaxation by Short and Long Carbon Based Chemicals

Introduction: Short and long-chained based carbon chemicals derived from plants are found in many household products and are widely used in complementary medicine. It has been suggested anecdotally that some of these chemicals have the ability to relax smooth muscle tissues, and to test this concept, this study examines the effects of 1,8-cineole, 1-hepta-nol, α-pinene, cis-3-hexen-1-ol and trans-2-hexenal on neurotransmission and contraction of mouse and rat vas deferens. Methods: Focal extracel-lular recordings from the surface of mouse vas deferens of electrically-evoked nerve terminal impulses (NTIs) and electrically-evoked excitatory junctional currents (EJCs) were examined in both the presence and the absence of each chemical. Likewise, noradrenaline-evoked contractile forces of rat vas deferens were examined in both the presence and the absence of each chemical. Results: Relative to the respective controls, 1,8-cineole, 1-heptanol and trans-2-hexenal each statistically significantly decreased both smooth muscle contractile forces and EJC amplitudes, with 1-heptanol and trans-2-hexenal also statistically significantly decreasing NTI amplitudes. On NTI and EJC amplitudes the effects of 1,8-cineole and 1-heptanol were reversible whereas the effects of trans-2-hexenal were irreversible. Conclusion: Our results demonstrate that 1,8-cineole and trans-2-hexenal decrease smooth muscle neurotransmission and contraction and thereby cause smooth muscle relaxation, thus suggesting that these chemicals may have clinical applications and health benefits.

Effects of Lavender and Linalool on Neurotransmission and Contraction of Smooth Muscle

Introduction: Lavender (Lavandula angustifolia) is commonly used in household products, perfumes, aromatherapy and complementary medicines. This study assesses the effects of lavender and its component linalool on neurotransmission and contraction of smooth muscle. Methods: The concentration-dependent effects of lavender (0.001% to 0.05%) and linalool (0.001% to 0.05%) on electrically evoked nerve terminal impulse (NTI) and excitatory junction current (EJC) amplitudes were assessed, while the effects of lavender (0.03%) and linalool (0.03%) on 5-hydroxytryptamine, acetylcholine, histamine, noradrenaline and oxytocin evoked responses were examined. Reversibility of lavender (0.03%) and linalool (0.03%) effects on electrically evoked NTI and EJC amplitudes, as well as on acetylcholine evoked contractile responses, were also analysed. Results: Lavender and linalool caused concentration-dependent decreases of electrically evoked NTI and EJC amplitudes, and attenuated the contractile responses towards 5-hydroxytryptamine, acetylcholine, histamine, noradrenaline and oxytocin. Repeated washing of tissues treated with lavender following pre-treatment with acetylcholine reversed the inhibitory effects of lavender, whereas linalool’s effects were not readily reversible. Conclusion: Lavender and linalool may cause inhibition of smooth muscle presynaptic action potential propagation and postsynaptic G-protein coupled receptor evoked responses.

Drosophila neuroligin 1 regulates synaptic growth and function in response to activity and phosphoinositide-3-kinase

Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca(2+) sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required Drosophila neurexin 1 (dnrx1) function, suggesting that increased DNlg1/DNrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms.

Extracellular Protons Reduce Quantal Content and Prolong Synaptic Currents at the Drosophila Larval Neuromuscular Junction

: Fluctuations in extracellular pH occur in the nervous system in response to a number of physiological and pathological processes, such as ischemia, hypercapnea, and high-frequency activity. Using the Drosophila larval neuromuscular junction, the author has examined acute effects of low and high pH on excitability and synaptic transmission. Acidification rapidly and reversibly reduces the size of electrically evoked excitatory junctional currents (EJCs) in a concentration-dependent manner, with transmission nearly abolished at pH 5.0. Conversely, raising pH to 7.8 increases EJC amplitude significantly. Further elevation to pH 8.5 causes an initial increase in amplitude, followed by profound, long-lasting depression of the synapse. Amplitudes of spontaneous miniature EJCs (mEJCs) are modestly, but significantly reduced at pH 5.0. It is therefore the number of quanta released per action potential, rather than the size of individual quanta, that is most strongly affected. Decay times of both EJCs and mEJCs are dramatically lengthened at low pH, suggesting that glutamate remains in the synaptic cleft for much longer than normal. Presynaptic excitability is also reduced, as indicated by increased latency between nerve shock and EJC onset. The response to low pH was not altered by mutations in genes encoding Transient Receptor Potential, Mucolipin subfamily (TRPML) and Slowpoke ion channels, which had previously been implicated as possible targets of extracellular protons. The author concludes that extracellular protons have strong effects on the release of glutamate and the time course of synaptic currents. These phenotypes can be exploited to study the mechanisms of acid-mediated changes in neuronal function, and to pursue the way in which pH modulates synaptic function in normal and pathophysiological conditions.

Loss of Skywalker Reveals Synaptic Endosomes as Sorting Stations for Synaptic Vesicle Proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.

Mutations in Wnt2 Alter Presynaptic Motor Neuron Morphology and Presynaptic Protein Localization at the Drosophila Neuromuscular Junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins.

Electrophysiological Recording in the <em>Drosophila</em> Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of <8 hours (at 25°C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the &lt;em&gt;Drosophila&lt;/em&gt; Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of <8 hours (at 25°C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Role of rut Adenylyl Cyclase in the Ensemble Regulation of Presynaptic Terminal Excitability: Reduced Synaptic Strength and Pprecision in a Drosophila Memory Mutant

: Although modulation of presynaptic terminal excitability can profoundly affect transmission efficacy, how excitability along axonal terminal branches is regulated requires further investigations. We performed focal patch recording in Drosophila larval neuromuscular junctions (NMJs) to monitor the activity of individual synaptic boutons along the presynaptic terminal. Analysis of the learning mutant rutabaga (rut) suggests a tight regulation of presynaptic terminal excitability by rut adenylyl cyclase (AC) that is responsible for Ca2+/calmodulin-dependent cAMP synthesis. Focal excitatory junctional currents (ejcs) demonstrated that disrupted cAMP metabolism in rut mutant boutons leads to decreased transmitter release, coupled with temporal dispersion and amplitude fluctuation of ejcs during repetitive activity. Strikingly, rut motor terminals displayed greatly increased variability among corresponding terminal branches of identified NMJs in different preparations. However, boutons throughout single terminal branches were relatively uniform in either WT or rut mutant larvae. The use of electrotonic depolarization to directly evoke transmitter release from axonal terminals revealed that variability in neurotransmission originated from varying degrees of weakened excitability in rut terminals. Pharmacological treatments and axonal action potential recordings raised the possibility that defective rut AC resulted in reduced Ca2+ currents in the nerve terminal. Thus, our data indicate that rut AC not only affects transmitter release machinery, but also plays a previously unsuspected role in local excitability control, both contributing to transmission level and precision along the entire axonal terminal.

The ubiquitin–proteasome system postsynaptically regulates glutamatergic synaptic function

The ubiquitin–proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS' roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome β2 and β6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.

Analysis of Conditional Paralytic Mutants in Drosophila Sarco-Endoplasmic Reticulum Calcium ATPase Reveals Novel Mechanisms for Regulating Membrane Excitability

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.

Isoflurane depresses glutamate release by reducing neuronal excitability at the Drosophila neuromuscular junction.

The mechanisms through which volatile general anaesthetics exert their behavioural effects remain unclear. The accessibility of the Drosophila larval neuromuscular junction to genetic and neurophysiological analysis has made it an attractive model system for identification of anaesthetic targets. This study provides a mechanistic basis for the genetic analysis of anaesthetic action, by analysing the neurophysiological effects of the volatile anaesthetic isoflurane on axonal and synaptic function in the Drosophila larva. The most robust effect of isoflurane was a reversible decrease in the amplitude and area of glutamatergic excitatory junctional currents (EJCs) evoked at the neuromuscular junction. Isoflurane did not affect postsynaptic glutamate receptor function detectably, in that the amplitudes, areas and decay times of spontaneous miniature EJCs were unchanged at any concentration. Therefore, decreased EJC amplitude resulted from reduction of neurotransmitter release. Reduced neurotransmitter release was associated with decreased presynaptic excitability, measured as increased delay to EJC onset and reduced axonal conduction velocity. EJC amplitude was rescued to control levels by direct electrotonic stimulation of the synapse in the presence of tetrodotoxin, indicating that isoflurane inhibits neurotransmitter release by reducing presynaptic excitability. In addition, isoflurane reduced release probability, measured as increased paired-pulse facilitation. The EC(50) for suppression of larval locomotion was similar to that for reduction of transmitter release, indicating that the axonal and synaptic effects were occurring in a behaviourally relevant range. These results provide a cellular context for ongoing genetic and neurophysiological analyses of volatile anaesthetic action in Drosophila, and suggest candidate anaesthetic target molecules.

Differential sensitivity to calcium and osmotic pressure of fast and slow ATP currents at sympathetic varicosities in mouse vas deferens

Secretion of noradrenaline from large dense-core vesicles in chromaffin cells involves both rapid and slow components of exocytosis which are differentially sensitive to changes in external calcium, osmotic pressure and interruption of the interacting SNARE proteins. Electrical signs of secretion of ATP from sympathetic nerve terminals of mouse vas deferens, the excitatory junctional currents (EJCs), also indicate both rapid and slow mechanisms of exocytosis, which might also show such differential sensitivity. We report here that the large and fast EJCs are highly sensitive to changes in extracellular calcium ions whereas the small and slow EJCs are not. Furthermore, the frequency of fast EJCs is accelerated by hypotonic solutions whereas the slow EJCs are accelerated by hypertonic solution. Fast EJCs, but not slow EJCs, are blocked by peptide fragments of α-SNAP and syntaxin whereas slow EJCs are not. These observations point to two classes of exocytosis from sympathetic nerve terminals that parallel those of exocytosis from chromaffin cells.

Regional differences in sympathetic purinergic transmission along the length of the mouse vas deferens.

Contraction of the smooth muscle in the mouse vas deferens is elicited by sympathetic nerves releasing at least two neurotransmitters, adenosine triphosphate (ATP) and noradrenaline (NA). Several studies have indicated the presence of regional variation in the purinergic and noradrenergic contributions to sympathetic nerve-evoked contractions in rodent vasa deferentia. We examined the relative contribution of ATP and NA to neurotransmission and contraction at the prostatic and epididymal ends of the mouse vas deferens. The success rate of recording excitatory junction currents (EJCs, extracellular indication of ATP release) from varicosities at the prostatic end of the vas deferens was eight times greater than for varicosities located at the epididymal end. Both regions of the vas deferens responded similarly to focal application of NA and ATP. Furthermore, the relative density and distribution of P2X(1)-receptor mRNA and anti-P2X(1) immunostaining did not differ between the two regions. Our results suggest that most varicosities located at the epididymal end of the vas deferens are releasing an insufficient amount of ATP to evoke detectable EJCs.

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability.

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice.

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.

GABA Enhances Transmission at an Excitatory Glutamatergic Synapse

GABA mediates both presynaptic and postsynaptic inhibition at many synapses. In contrast, we show that GABA enhances transmission at excitatory synapses between the lateral gastric and medial gastric motor neurons and the gastric mill 6a and 9 (gm6a, gm9) muscles and between the lateral pyloric motor neuron and pyloric 1 (p1) muscles in the stomach of the lobster Homarus americanus. Two-electrode current-clamp or voltage-clamp techniques were used to record from muscle fibers. The innervating nerves were stimulated to evoke excitatory junctional potentials (EJPs) or excitatory junctional currents. Bath application of GABA first decreased the amplitude of evoked EJPs in gm6a and gm9 muscles, but not the p1 muscle, by activating a postjunctional conductance increase that was blocked by picrotoxin. After longer GABA applications (5-15 min), the amplitudes of evoked EJPs increased in all three muscles. This increase persisted in the presence of picrotoxin. beta-(Aminomethyl)-4-chlorobenzenepropanoic acid (baclofen) was an effective agonist for the GABA-evoked enhancement but did not increase the postjunctional conductance. Muscimol activated a rapid postsynaptic conductance but did not enhance the amplitude of the nerve-evoked EJPs. GABA had no effect on iontophoretic responses to glutamate and decreased the coefficient of variation of nerve-evoked EJPs. In the presence or absence of tetrodotoxin, GABA increased the frequency but not the amplitude of miniature endplate potentials. These data suggest that GABA acts presynaptically via a GABA(B)-like receptor to increase the release of neurotransmitter.

Spatial distribution and developmental appearance of postjunctional P2X1 receptors on smooth muscle cells of the mouse vas deferens.

P2X1-type purinoceptors have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta-MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X1 receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X1 staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X1 mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X1 were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X1 receptors, distributed diffusely across the smooth muscle cell surface.