Evoked Excitatory Junction Potentials Research Articles

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a "ceiling effect," because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.

Bacterial lipopolysaccharide hyperpolarizes the membrane potential and is antagonized by the K2p channel blocker doxapram

Exposure of Drosophila skeletal muscle to bacterial lipopolysaccharides (LPS) rapidly and transiently hyperpolarizes membrane potential. However, the mechanism responsible for hyperpolarization remains unclear. The resting membrane potential of the cells is maintained through multiple mechanisms. This study investigated the possibility of LPS activating calcium-activated potassium channels (KCa) and/or K2p channels. 2-Aminoethyl diphenylborinate (2-APB), blocks uptake of Ca2+ into the endoplasmic reticulum (ER); thus, limiting release from ryanodine-sensitive internal stores to reduce the function of KCa channels. Exposure to 2-APB produces waves of hyperpolarization even during desensitization of the response to LPS and in the presence of doxapram. This finding in this study suggests that doxapram blocked the acid-sensitive K2p tandem-pore channel subtype known in mammals. Doxapram blocked LPS-induced hyperpolarization and depolarized the muscles as well as induced motor neurons to produce evoked excitatory junction potentials (EJPs). This was induced by depolarizing motor neurons, similar to the increase in extracellular K+ concentration. The hyperpolarizing effect of LPS was not blocked by decreased extracellular Ca2+or the presence of Cd2+. LPS appears to transiently activate doxapram sensitive K2p channels independently of KCa channels in hyperpolarizing the muscle. Septicemia induced by gram-negative bacteria results in an increase in inflammatory cytokines, primarily induced by bacterial LPS. Currently, blockers of LPS receptors in mammals are unknown; further research on doxapram and other K2p channels is warranted. (220 words).

The effects of levetiracetam on glutamatergic synaptic transmission: Crayfish and Drosophila NMJs

Epilepsy is a neurological disorder characterized by recurring, unpredictable seizures. Its disease burden is high, seeing as it ranks fourth in the world's neurological disorders burden following: tension-type headaches, migraines, and Alzheimer's disease. The commonly used anti-epileptic drug levetiracetam (Keppra) reduces epileptic seizures, but the exact mechanism through which it works is not known. Some studies suggest sodium and/or potassium ionic channels are directly altered reducing membrane excitability, while others suggest the drug interacts with SV2 protein to alter synaptotagmin (a calcium sensor protein) action in the presynaptic nerve terminal (reducing excitability). The glutamatergic synapses at crayfish and larval Drosophila neuromuscular junctions (NMJs) were used to assess the drug's action. The evoked excitatory junction potentials (EJPs) of the crayfish NMJ were enhanced through exposure to 1 mM of the drug, but not lower doses, with 20 minutes of stimulation after 10 minutes of static incubation. However, no significant alterations were noted in the amplitude of the EJPs at the Drosophila NMJ during the 20 minutes of stimulation at 5 Hz. The crayfish model effects of levetiracetam were then used as an authentic curriculum undergraduate research experience (ACURE) in a neurophysiology teaching laboratory with 16 students. It appears levetiracetam acts differently in different animal models, or varied experimental conditions are required to note its effects.

Insight into the Mode of Action of Haedoxan A from Phryma leptostachya.

Haedoxan A (HA) is a major active ingredient in the herbaceous perennial plant lopseed (Phryma leptostachya L.), which is used as a natural insecticide against insect pests in East Asia. Here, we report that HA delayed the decay rate of evoked excitatory junctional potentials (EJPs) and increased the frequency of miniature EJPs (mEJPs) on the Drosophila neuromuscular junction. HA also caused a significant hyperpolarizing shift of the voltage dependence of fast inactivation of insect sodium channels expressed in Xenopus oocytes. Our results suggest that HA acts on both axonal conduction and synaptic transmission, which can serve as a basis for elucidating the mode of action of HA for further designing and developing new effective insecticides.

Electrical properties of purinergic transmission in smooth muscle of the guinea-pig prostate.

Prostatic smooth muscle develops spontaneous myogenic tone which is modulated by autonomic neuromuscular transmission. This study aimed to investigate the role of purinergic transmission in regulating electrical activity of prostate smooth muscle and whether its contribution may be altered with age. Intracellular recordings were simultaneously made with isometric tension recordings in smooth muscle preparations of the guinea-pig prostate. Immunostaining for P2X1 receptors on whole mount preparations was also performed. In prostate preparations which generated spontaneous slow waves, electrical field stimulation (EFS)-evoked excitatory junction potentials (EJPs) which were abolished by guanethidine (10 μM), α-β-methylene ATP (10 μM) or pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS, 10 μM) but not phentolamine (1 μM). Consistently, immunostaining revealed the expression of P2X1 receptors on prostatic smooth muscle. EJPs themselves did not cause contractions, but EJPs could sum to trigger a slow wave and associated contraction. Yohimbine (1 μM) and 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM) but not propranolol (1 μM) potentiated EJPs. Although properties of EJPs were not different between young and aging guinea-pig prostates, ectoATPase inhibitor ARL 67156 (100 μM) augmented EJP amplitudes by 64.2 ± 29.6% in aging animals, compared to 22.1 ± 19.9% in young animals. These results suggest that ATP released from sympathetic nerves acts on P2X1 purinoceptors located on prostate smooth muscle to evoke EJPs, while pre-junctional α2-adrenergic and adenosine A2 receptors may play a role in preventing excessive transmitter release. Age-related up-regulation of enzymatic ATP breakdown may be a compensatory mechanism for the enhanced purinergic transmission which would cause hypercontractility arising from increased ATP release in older animals.

Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction.

ABSTRACTChanges in UBE3A expression levels in neurons can cause neurogenetic disorders ranging from Angelman syndrome (AS) (decreased levels) to autism (increased levels). Here we investigated the effects on neuronal function of varying UBE3A levels using the Drosophila neuromuscular junction as a model for both of these neurogenetic disorders. Stimulations that evoked excitatory junction potentials (EJPs) at 1 Hz intermittently failed to evoke EJPs at 15 Hz in a significantly higher proportion of Dube3a over-expressors using the pan neuronal GAL4 driver C155-GAL4 (C155-GAL4>UAS-Dube3a) relative to controls (C155>+ alone). However, in the Dube3a over-expressing larval neurons with no failures, there was no difference in EJP amplitude at the beginning of the train, or the rate of decrease in EJP amplitude over the course of the train compared to controls. In the absence of tetrodotoxin (TTX), spontaneous EJPs were observed in significantly more C155-GAL4>UAS-Dube3a larva compared to controls. In the presence of TTX, spontaneous and evoked EJPs were completely blocked and mEJP amplitude and frequency did not differ among genotypes. These data suggest that over-expression of wild type Dube3a, but not a ubiquitination defective Dube3a-C/A protein, compromises the ability of motor neuron axons to support closely spaced trains of action potentials, while at the same time increasing excitability. EJPs evoked at 15 Hz in the absence of Dube3a (Dube3a15b homozygous mutant larvae) decayed more rapidly over the course of 30 stimulations compared to w1118 controls, and Dube3a15b larval muscles had significantly more negative resting membrane potentials (RMP). However, these results could not be recapitulated using RNAi knockdown of Dube3a in muscle or neurons alone, suggesting more global developmental defects contribute to this phenotype. These data suggest that reduced UBE3A expression levels may cause global changes that affect RMP and neurotransmitter release from motorneurons at the neuromuscular junction. Similar affects of under- and over-expression of UBE3A on membrane potential and synaptic transmission may underlie the synaptic plasticity defects observed in both AS and autism.

Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction.

Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.

Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na(+) channel blocker tetrodotoxin and the Ca(2+) channel blocker Cd(2+). Both EJPs and sEJPs were abolished by the application of the P2X(1) antagonist 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism.

Sodium nitroprusside enhances contractions of the guinea-pig isolated vas deferens.

The effects of sodium nitroprusside on the electrical and mechanical properties of the smooth muscle of the guinea-pig vas deferens, and its responses to transmitter substances, have been investigated by use of the sucrose-gap technique. Isolated longitudinal segments of guinea-pig vas deferens contracted in response to electrical field stimulation (100 V, 0.04-0.1 ms, 1-5 Hz, 10 s train every 60 s) and application of ATP (1 mM) or noradrenaline (10 microM). Sodium nitroprusside (0.1 mM) did not affect resting tension but did enhance contractions evoked by electric-field stimulation but not by ATP or noradrenaline. The sodium nitroprusside-induced enhancement was unaffected by the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (0.1 mM). Conversely, electrically evoked contractions were unaffected by the nitric oxide precursor L-arginine (1 mM) or the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) (0.1 mM). The amplitudes of electrically evoked excitatory junction potentials (EJPs) were not affected by application of sodium nitroprusside, although it caused a small depolarization of 0.7+/-0.3 mV. Similarly, the depolarization caused by exogenous application of ATP or noradrenaline was unaffected by the presence of sodium nitroprusside. L-NAME, L-arginine and SNAP did not affect EJP amplitude or baseline membrane potential. It is concluded that sodium nitroprusside enhances electrically evoked contractions of the guinea-pig vas deferens by reducing the threshold voltage for action potential firing in smooth-muscle cells.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Although intracellular recording is particularly valuable in revealing membrane potential changes, it has several limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body-wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of minis (spontaneous miniature synaptic potentials) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only at low release conditions when nonlinear summation is not a major issue. The voltage-clamp technique can overcome these limitations by using negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval NMJ preparation.

Electrical coupling and release of K+ from endothelial cells co‐mediate ACh‐induced smooth muscle hyperpolarization in guinea‐pig inner ear artery

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b) activation of inward rectifier K(+) channels (K(ir)) and Na(+)-K(+) pump current by elevated interstitial K(+) released from the endothelial cells, these being responsible for about 60% and 40% of the hyperpolarization, respectively. The role ratio of K(ir) and pump current activation is at 8 : 1 or less.

Studies on the mechanism of enhancement of purinergic transmission by caffeine in the guinea-pig isolated vas deferens.

1. Purinergic transmission from sympathetic nerves in the guinea-pig vas deferens was monitored using intracellular recording techniques. Stimulation of the hypogastric nerve with trains of 15 pulses at 1 Hz evoked excitatory junction potentials (EJPs) which increased in amplitude from the first pulse and reached a maximum after 6-8 pulses. 2. Caffeine (3 and 10 mm), depolarized cells by 5-10 mV and increased the amplitude of the first few EJPs in each train but reduced the maximum amplitude of EJPs late in the train. 3. The adenosine receptor antagonist 8-p-sulphophenyl-theophylline (8-SPT; 30 microm) had no effect on either the resting membrane potential or the EJP amplitude; however, at 100 microm it reduced the amplitude of all EJPs by 5-10%. 4. Adenosine (10 and 30 microm) reduced the amplitude of EJPs in a concentration-dependent manner. The inhibitory effect of adenosine on EJP amplitude was prevented by pretreatment with either caffeine (3 mm) or 8-SPT (30 microm). 5. Ryanodine (30 microm) did not alter EJP amplitude and did not inhibit the enhancement of the first EJP by caffeine (3 mm). Incubation of the tissue with the cell permeable calcium chelator 1-2-bis(o-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPT-AM) resulted in a depression of EJP amplitude and a longer time to reach maximum amplitude. In cells that had been exposed to BAPT-AM, caffeine 3 mm still increased amplitude of EJP early in the train. 6. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 500 microm), hyperpolarized cells and increased the amplitude of EJP throughout the train of stimulation. In the presence of IBMX, caffeine 3 mm still depolarized the cells and enhanced the EJP early in the train of stimulation. 7. The findings in this study confirm that caffeine and 8-SPT are effective inhibitors of the actions of adenosine. However, caffeine has an additional action to enhance EJP early during a train of stimulation, which cannot be attributed to blockade of adenosine receptors, but which may be related to inhibition of phosphodiesterase.

ATP released from perivascular nerves hyperpolarizes smooth muscle cells by releasing an endothelium-derived factor in hamster mesenteric arteries.

1. The interaction between perivascular nerves and endothelium was investigated by measuring the changes in smooth muscle membrane potentials using intracellular microelectrode techniques in hamster mesenteric thin (100-150 microm) and thick (300-350 microm) arteries. 2. In both arteries, nerve stimulation evoked excitatory junction potentials (EJPs) which were strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (0.5-5 microM). This result indicated that the EJPs were induced by the activation of P2X receptors. 3. Transient hyperpolarizations were evoked by trains of pulses at 20 Hz in PPADS (5 microM)-pre-treated thin arteries, but not in the thick arteries. ATP (100 microM) applied to adventitial surfaces mimicked the hyperpolarizations. Both the ATP- and nerve stimulation-induced hyperpolarizations were blocked by cibacron blue F3GA (2-100 microM) and were also abolished after endothelium removal, indicating that the neurally released ATP evoked transient hyperpolarization through the activation of P2Y receptors located on the endothelium. 4. In endothelium-intact preparations, intimal application of uridine 5'-triphosphate (UTP 100 microM), a P2Y2-like receptor agonist, but not 2-methylthio ATP (7 microM), hyperpolarized the smooth muscle. The UTP-induced hyperpolarization was significantly inhibited by cibacron blue F3GA and was abolished after endothelium removal. 5. These results suggest that ATP released from the perivascular nerves may reach the endothelium and activate P2Y2-like receptors to induce the release of an endothelium-derived hyperpolarizing factor in thin arteries.

Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.

1. Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea-pig urethra using conventional microelectrode techniques. 2. Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3. STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4. When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5. Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either alpha,beta-methylene-ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co-application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6. In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+-activated chloride channels. STDs sum to activate L-type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.

Regional differences in sympathetic neurotransmission to cutaneous arteries in the guinea-pig isolated ear

The effects of sympathetic nerve stimulation on different cutaneous arteries were examined in arteries isolated from guinea-pig ears, by measuring membrane potential changes in smooth muscle cells in response to electrical field stimulation. Resting membrane potential (RMP) was similar in proximal (main ear artery) and distal (3rd or 4th branch order) cutaneous arteries (mean −71 mV). Single stimuli evoked excitatory junction potentials (EJPs) in all arteries. The EJPs in proximal arteries were twice the amplitude, and the time constant of EJP decay was almost half the value, compared with distal cutaneous arteries. EJP amplitude was reduced by >90% by suramin (30 μM) or α,β,methylene-ATP (α,β,m-ATP) (1 μM) in all proximal, and most distal arteries. Residual responses in distal arteries were resistant to tetrodotoxin. The N-type calcium channel blocker, ω-conotoxin GVIA (30 nM), reduced EJP amplitude by 70–100% in both proximal and distal arteries. Successive EJPs evoked by trains of stimuli at 1 to 5 Hz were depressed in amplitude in proximal arteries, but showed facilitation in distal arteries. EJP depression in proximal arteries was reversed to facilitation by the α 2-adrenoceptor antagonist, yohimbine (30 nM). Trains of stimuli delivered at 10–20 Hz produced summation of EJPs and active membrane responses in 30% of proximal arteries. Active responses were never detected in distal arteries. Slow depolarizations following the EJPs were detected in most arteries after trains of stimuli, and were abolished by prazosin (0.3 μM) or ω-conotoxin GVIA (30 nM). The density of the perivascular plexus of axons innervating proximal arteries, demonstrated with catecholamine fluorescence histochemistry, was twice that in distal cutaneous arteries. These regional differences in sympathetic neurotransmission suggest that cutaneous vasoconstriction in response to thermoregulatory stimuli, which occurs predominantly in distal cutaneous segments, is likely to be qualitatively different from cutaneous vasoconstriction of proximal arteries in response to other physiological stimuli.

Differential effects of a molting hormone, 20-hydroxyecdysone, on the neuromuscular junctions of the claw opener and abdominal flexor muscles of the American lobster

Intracellular recordings at the neuromuscular junction of the dactyl opener muscle and the abdominal phasic flexor muscle of intermolt lobsters were made in the presence and absence of the steroid hormone, 20-hydroxyecdysone (20-HE). Evoked excitatory junctional potentials (EJPs) were recorded from both muscles; spontaneous miniature excitatory junctional potentials (MEJPs) were recorded from the claw. In the opener muscle, 20-HE caused an increase in EJP amplitudes and MEJP frequency. In the abdomen 20-HE caused EJP amplitudes to become significantly smaller than controls. These results are consistent with changes in the relative activity of the two muscles over the molt cycle. In premolt lobsters where the titers of 20-HE are highest, there is an increased tendency towards aggressive behaviors (which include the meral spread) and a corresponding reduction of escape swimming. The results also support our earlier findings that one or more molt-related blood-borne factors can modulate peripheral synaptic transmission.

Electrical properties of smooth muscle in the guinea-pig urinary bladder.

1. The effects of transmural nerve stimulation were examined on preparations of detrusor smooth muscle from guinea-pig urinary bladder using intracellular recording techniques. Most recordings were made from preparations in which spontaneous and evoked action potentials had been inhibited by nifedipine (10 microM), a dihydropyridine that blocks L-type Ca2+ channels. 2. Supramaximal stimuli evoked excitatory junction potentials (EJPs) which could be divided into three basic types. Type 1 EJPs had short latencies (< 30 ms) and fast rise times (< 60 ms). Type 2 EJPs consisted of two components: a small depolarization that was followed by a second depolarization with a faster rise time. In a third type of cell, at high strengths of stimulation, EJPs resembled type 1 EJPs but at lower strengths of stimulation were similar in time course to type 2 EJPs. 3. All EJPs were abolished by tetrodotoxin (1 microM) and reduced by omega-conotoxin (0.1 microM), but were unaffected by hexamethonium (0.1 mM), suggesting that they result from the release of transmitter from post-ganglionic nerve fibres. All responses persisted in the presence of atropine (1 microM) but were abolished following the desensitization of P2-purinoceptors with alpha, beta-methylene ATP (m-ATP; 10 microM). 4. Spontaneous excitatory junction potentials (SEJPs) were also recorded from most cells. SEJPs were similar in appearance to fast single-component EJPs; however, in general they had a briefer time course. SEJPs persisted in the presence of tetrodotoxin (1 microM). 5. The electrical properties of urinary bladder smooth muscle were also examined. Voltage changes induced by point current injection into cells had fast rates of rise and decay (time constant, 5-20 ms). The input resistance of cells ranged between 12 and 108 M omega. When recordings were taken from cells near the point of current injection, resultant electrotonic potentials could be detected in only a small proportions of cells. 6. The results are discussed in relation to the idea that transmural nerve stimulation in the guinea-pig urinary bladder causes the activation of at least two different membrane conductances. Cells appear to be electrically coupled with one another. However, it is likely that coupling exists within discrete bundles of the smooth muscle.

Regional difference in the distribution of L‐NAME‐sensitive and ‐insensitive NANC relaxations in cat airway.

1. To investigate the distribution profile of functional inhibitory non-adrenergic non-cholinergic (i-NANC) nerves and the contribution of NO to the NANC relaxation in the cat, we studied the effects of N omega-nitro-L-arginine methyl ester (L-NAME) on NANC relaxation elicited by electrical field stimulation (EFS) in the trachea, bronchus and bronchiole. 2. EFS applied to the tracheal smooth muscle during contraction induced by 5-HT (10(-5) M) in the presence of atropine (10(-6) M) and guanethidine (10(-6) M) elicited a monophasic NANC relaxation. By contrast, NANC relaxation elicited in the peripheral airway was biphasic, comprising an initial fast followed by a second slow component and L-NAME (10(-5) M) selectively abolished the first component without affecting the second one. In the trachea, L-NAME (10(-5) M) completely suppressed the monophasic NANC relaxation when single or short repetitive stimuli (< 5) with 1 ms pulse duration were applied. However, at higher repetitive stimuli (> 10) with 1 or 4 ms pulse duration, suppression of NANC relaxation was incomplete. 3. In the small bronchi obtained from L-NAME-pretreated cats, EFS applied during contraction induced by 5-HT (10(-5) M) elicited only the slow component of NANC relaxation which is sensitive to tetrodotoxin. 4. In the peripheral airway, a newly synthesized VIP antagonist (10(-6) M) or alpha-chymotrypsin (1 U ml-1) considerably attenuated the amplitude of L-NAME-insensitive relaxation. 5. Single or repetitive EFS consistently evoked excitatory junction potentials (EJPs) in the central and peripheral airways. When tissues were exposed to atropine (10(-6) M) and guanethidine (10(-6) M), single or repetitive EFS did not alter the resting membrane potential. 6. These results indicate that at least two neurotransmitters, possibly NO or NO-containing compounds and VIP, are involved in i-NANC neurotransmission and the distribution profile of the two components differs in the central and peripheral airway of the cat.

Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca 2+-activated neurotransmitter release

Synaptotagmin (syt), a synaptic vesicle-specific protein known to bind Ca 2+ in the presence of phospholipids, has been proposed to mediate Ca 2+-dependent neurotransmitter release. We have addressed the role of syt in neurotransmitter release in vivo by generating mutations in synaptotagmin (syt) in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fall to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca 2+ and a 15-fold reduction in EJP amplitude in 1.0 mM Ca 2+ when compared with heterozygous controls. In contrast, we observe an increase in the frequency of spontaneous miniature EJPs in the mutants. These results provide in vivo evidence that syt plays a key role in Ca 2+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release.

Cholera toxin and G s protein modulation of synaptic transmission in guinea pig mesenteric artery

Cholera toxin (CTX) was used to test whether the presynaptic β-adrenoceptors of guinea-pig mesenteric artery are coupled via stimulatory GTP-binding proteins. The vascular smooth muscle cells were electrically quiescent unless stimulated and had a mean resting potential of −68.7 ± 2.8 mV ( n = 16) and input resistance of 12.1 ± 0.5 MΩ ( n = 4). Perivascular nerve stimulation with brief square pulses evoked excitatory junction potentials (EJPs) in the muscle cells. Isoproterenol (0.1 μM) enhanced the EJP amplitude without modifying the passive membrane properties of the muscle cells. The β-blocker, propranolol (0.5 μM), prevented the effects of isoproterenol on EJP amplitude. The permeant analogue of cyclic AMP, 8-bromocAMP, also potentiated EJP amplitude. EJP amplitude was markedly enhanced by treatment of the isolated blood vessels with CTX (10 μg/ml for 1 h). The muscle cells became hyperpolarized (−74.6 ± 2.1 mV, n = 5), and their input resistances were significantly reduced (8.2 ± 0.5 MΩ, n = 4). These effects of CTX persisted after washout. Addition of G M1 ganglioside (5 μg/ml) prevented the CTX effects. The CTX enhancement of EJP amplitude was not prevented by application of depolarizing current (ca. 0.5 nA) the muscle cells (to counter the hyperpolarization). These results suggest that CTX increases the neurotransmitter release from the nerve terminals; the hyperpolarization may be due to an increase in K + conductance. These effects of CTX may be mainly due to elevation of cAMP in the nerve terminal and in the muscle cell.