Amplitude Of Excitatory Junction Potentials Research Articles

Periplocoside P affects synaptic transmission at the neuromuscular junction and reduces synaptic excitability in Drosophila melanogaster by inhibiting V-ATPase.

Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V-Type adenosine triphosphatase (V-ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked. The bioassay revealed that Drosophila adults withdifferentgeneticbackgrounds showed varying levels of susceptibility to PSP in the order: parats1 > parats1 ;DSC1-/- ≈ w1118 > DSC1-/- . Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration-dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. Our findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V-ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides. © 2023 Society of Chemical Industry.

Sympathetic Neurotransmission in Resistance Mesenteric Arteries in Obesity‐Related Hypertension

Hyperactivity of sympathetic nerves has an important role in the pathogenesis of obesity‐related hypertension. The mechanisms linking obesity to hypertension are unclear. Small mesenteric arteries (MA, <300 mm dia.) are resistance blood vessels that are densely innervated by sympathetic nerves. Nerve varicosities release norepinephrine (NE) and ATP which cause arterial constriction. Impaired control of NE and/or ATP release and clearance would cause hypertension. Alterations in sympathetic nerves supplying MAs contribute to hypertension in several animal models. This study examined whether alterations of sympathetic neurotransmission in the resistance MAs contribute to high blood pressure in high fat‐fed (HF) Dahl salt‐sensitive rats. Sympathetic nerves supplying MAs maintained in vitro were stimulated focally. Electrically‐evoked NE and ATP release were detected by using amperometry with carbon fiber electrodes, and intracellular recording of excitatory junction potentials (EJPs) from arterial smooth muscle cells, respectively. Data were obtained from male and female rats at 10, 17, and 24 weeks of control and HF diet. Peak amplitudes of NE oxidation current and EJPs were assessed. NE oxidation current from all groups were blocked by a Na+ channel blocker, tetrodotoxin (TTX, 300 nM), suggesting that NE release in neurogenic. EJPs from all groups were abolished by TTX, and PPADS (10 μM), a P2X receptor antagonist, indicating that EJPs were neurogenic and purinergic. Frequency (1–30 Hz, 50 pulses) response curves using amperometric measurement of NE release showed similar responses in MAs from control and HF fed male and female rats. EJP frequency (0.5–10Hz, 5 pulses) response curves were also similar in MA from control and HF fed male and female rats. We investigated prejunctional α2‐adrenergic receptor function using the α2 receptor agonist (UK14,304) and antagonist (yohimbine). UK14,304 (0.0001–10 μM) inhibited NE and ATP release equally well and in a concentration‐dependent manner in MAs from control and HF rats. In the presence of 1 μM yohimbine, NE and ATP release was increased equally in MA from all rats. We conclude that adrenergic and purinergic neurotransmission by periarterial sympathetic nerves are not affected in HF Dahl salt sensitive rats. Elevated blood pressure in this animal model is not dependent on altered sympathetic neurotransmission to the mesenteric vasculature.Support or Funding InformationNIH 2P01HL070687 (For JJG), and Post‐doctoral training scholarship, Faculty of Medicine, Thammasat University, Thailand (For SS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P344: Vascular Purinergic Neurotransmission is Not Altered in High Fat-Fed Dahl S Hypertension

Mesenteric arteries (MAs) are supplied densely by sympathetic nerves releasing NE and ATP to increase vascular tone. Increased neuronal ATP/NE storage and release and impaired clearance cause hypertension. Our previous studies of DOCA-salt hypertensive rats showed that 1) impairment of the prejunctional autoreceptors regulating ATP and NE release contributes to elevated sympathetic neurotransmission, and 2) purinergic neurotransmission was impaired due to decreased ATP bioavailability in sympathetic nerves. As hyperactivity of sympathetic nerves occurs in obesity-related hypertension, we investigated alterations in purinergic transmission in high fat-fed (HF) Dahl S rats. MAs maintained in vitro were stimulated focally. Intracellular recording of excitatory junction potential (EJP) from smooth muscle cells was obtained from male and female, control and HF rats. EJP amplitude, facilitation and rundown were assessed. EJPs from all groups were abolished by tetrodotoxin (300 nM), a Na + channel blocker, and P2X receptor antagonist, PPADS (10 μM), indicating EJPs were neurogenic and purinergic. At 17 weeks, we found that 1) frequency (0.5-10Hz) response curves from control and HF were similar in male and female arteries. 2) When using short trains of stimulation (0.5Hz; 5 pulses), UK14304 (0.0001-1μM)-, an α2-adrenergic receptor agonist, blocked EJPs response equally well in arteries from control and HF rats. Moreover, the α-adrenergic receptor antagonist, yohimbine (1 μM), potentiated EJPs similarly in control and HF groups. 3) There were no differences in EJP rundown caused by trains of stimulation (10Hz, 50 pulses) in MA from control and HF fed rats. Differential facilitation of purinergic neurotransmission was detected in male vs female. In the presence of yohimbine, degree of EJP facilitation was higher in female arteries compared to male control arteries (2.69±0.26 vs 1.96±0.09 N=4, p<0.05) indicating higher prejunctional ATP storage in female nerves. However, HF did not affect the degree of facilitation. These data suggested that sympathetic neurovascular function is not affected in HF Dahl S rats. Elevated blood pressure in this animal model is not dependent on altered sympathetic neurovascular function.

Characterizing the physiological and behavioral roles of proctolin in Drosophila melanogaster.

The neuropeptide proctolin (RYLPT) plays important roles as both a neurohormone and a cotransmitter in arthropod neuromuscular systems. We used third-instar Drosophila larvae as a model system to differentiate synaptic effects of this peptide from its direct effects on muscle contractility and to determine whether proctolin can work in a cell-selective manner on muscle fibers. Proctolin did not appear to alter the amplitude of excitatory junctional potentials but did induce sustained muscle contractions in preparations where the CNS had been removed and no stimuli were applied to the remaining nerves. Proctolin-induced contractions were dose-dependent, were reduced by knocking down expression of the Drosophila proctolin receptor in muscle tissue, and were larger in some muscle cells than others (i.e., larger in fibers 4, 12, and 13 than in 6 and 7). Proctolin also increased the amplitude of nerve-evoked contractions in a dose-dependent manner, and the magnitude of this effect was also larger in some muscle cells than others (again, larger in fibers 4, 12, and 13 than in 6 and 7). Increasing the intraburst impulse frequency and number of impulses per burst increased the magnitude of proctolin's enhancement of nerve-evoked contractions and decreased the threshold and EC50 concentrations for proctolin to enhance nerve-evoked contractions. Reducing proctolin receptor expression decreased the velocity of larval crawling at higher temperatures, and thermal preference in these larvae. Our results suggest that proctolin acts directly on body-wall muscles to elicit slow, sustained contractions and to enhance nerve-evoked contractions, and that proctolin affects muscle fibers in a cell-selective manner.

Presynaptic active zones in invertebrates and vertebrates.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations.

Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. We previously reported that axon terminal arborization in Drosophila larval neuromuscular junctions (NMJs) is enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs) remains unaltered despite the increase in synaptic bouton numbers. Here we determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29°C). We found that synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (<15°C to >30°C). However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs), which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut) encoding adenylyl cyclase (AC) exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends our previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g., slowpoke (slo). In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Shaker (Sh) IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size.

Action of octopamine and tyramine on muscles ofDrosophila melanogasterlarvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Short-Term Synaptic Plasticity Compensates for Variability in Number of Motor Neurons at a Neuromuscular Junction

We studied how similar postsynaptic responses are maintained in the face of interindividual variability in the number of presynaptic neurons. In the stomatogastric ganglion of the lobster, Homarus americanus, the pyloric (PY) neurons exist in variable numbers across animals. We show that each individual fiber of the stomach muscles innervated by PY neurons received synaptic input from all neurons present. We performed intracellular recordings of excitatory junction potentials (EJPs) in the muscle fibers to determine the consequences of differences in the number of motor neurons. Despite the variability in neuron number, the compound electrical response of muscle fibers to natural bursting input was similar across individuals. The similarity of total synaptic activation was not due to differences in the spiking activity of individual motor neurons across animals with different numbers of PY neurons. The amplitude of a unitary EJP in response to a single spike in a single motor neuron also did not depend on the number of PY neurons present. Consequently, the compound EJP in response to a single stimulus that activated all motor axons present was larger in individuals with more PY neurons. However, when axons were stimulated with trains of pulses mimicking bursting activity, EJPs facilitated more in individuals with fewer PY neurons. After a few stimuli, this resulted in depolarizations similar to the ones in individuals with more PY neurons. We interpret our findings as evidence that compensatory or homeostatic regulatory mechanisms can act on short-term synaptic dynamics instead of absolute synaptic strength.

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5-bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

Sympathetic nerves regulate vascular tone by releasing neurotransmitters into the vasculature. We previously demonstrated that bradykinin facilitates sympathetic neurotransmission in rat mesenteric arteries. Although little is known about the intracellular mechanism modulating this neurotransmission, recent cell line experiments have shown that the KCNQ channel, which is inhibited by the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP₂), participates in the control of neurotransmission by bradykinin. In the present study, we examined the mechanism regulating neurotransmitter release from rat perivascular sympathetic nerves. Excitatory junction potentials (EJPs) elicited by repetitive nerve stimulation (1 Hz, 11 pulses, 20 μs, 20-50 V), a measure of sympathetic purinergic neurotransmission, were recorded with a conventional microelectrode technique in rat mesenteric arteries. Bradykinin (10⁻⁷ mol l⁻¹) significantly enhanced the amplitude of EJPs (n=22, P<0.05). This enhancing effect was abolished by N-type calcium-channel inhibition with ω-conotoxin GVIA (2 × 10⁻⁹ mol ⁻¹l, n=8). The blockade of phospholipase C with U-73122 (10(-6) mol l⁻¹, n=17) also eliminated the facilitatory effect of bradykinin. In addition, the effects of bradykinin were diminished by the prevention of PIP₂ resynthesis with wortmannin (10⁻⁵ mol l⁻¹ n=7) or KCNQ channel inhibition with XE-991 (10⁻⁵ mol l⁻¹, n=7). On the other hand, depletion of intracellular calcium stores with cyclopiazonic acid (3 × 10⁻⁶ mol l⁻¹, n=6) or the inhibition of protein kinase C with bisindolylmaleimide-I (10⁻⁶ mol l⁻¹, n=9) did not alter the action of bradykinin. These data demonstrate that the hydrolysis of PIP₂ by phospholipase C, which is activated by G(q/11)-coupled receptors, and subsequent KCNQ channel inhibition enhance sympathetic purinergic neurotransmission presumably via the activation of N-type calcium channels in rat mesenteric arteries.

Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses.

The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14°C but not from those acclimatized to 21°C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.

Sympathetic overdrive in obesity involves purinergic hyperactivity in the resistance vasculature

While a close correlation exists in obese humans between sympathetic, adrenergic hyperactivity and structural and functional organ damage, a role for the co-transmitter, ATP, in vascular function remains unexplored. We therefore studied sympathetic nerve-mediated responses of pressurised small mesenteric arteries from control and obese rats. Diet-induced obesity significantly increased the amplitude of vasoconstriction to transmural nerve stimulation (1-10 Hz; P <0.05). At 1 and 5 Hz, both adrenergic and purinergic responses were significantly augmented, while only the purinergic component was increased at 10 Hz (P <0.05). Nerve stimulation at 1 Hz evoked contractions and underlying excitatory junction potentials (EJPs), which were both significantly increased in amplitude during obesity (P <0.05) and abolished by αβ-methylene ATP (1 μM; desensitises purinergic receptors). The rise time and rate of decay of these EJPs were significant decreased (P <0.05), without change in resting membrane potential. Amplitude and frequency of spontaneous EJPs and the density of perivascular sympathetic nerves were also significantly increased (P <0.05). Inhibition of sensory neurotransmitter release (capsaicin; 10 μM) significantly increased the amplitude of nerve-mediated contraction (P <0.05), with a greater effect in control than obese animals, although the density of sensory nerves was unaffected by obesity. We demonstrate that sympathetic nerve-mediated vasoconstriction is enhanced by diet-induced obesity due to upregulation of purinergic, in addition to adrenergic, neurotransmission. Changes result from increased perivascular sympathetic innervation and release of ATP. We conclude that augmented sympathetic control of vasoconstriction induced by obesity could contribute directly to hypertension and global organ damage. A decrease in sensitivity to sensory vasodilatory neurotransmitters may also affect these processes.

Subunit-specific and homeostatic regulation of glutamate receptor localization by CaMKII in Drosophila neuromuscular junctions

For the efficient transfer of information across neural circuits, the number of synaptic components at synapses must be appropriately regulated. Here, we found that postsynaptic calcium/calmodulin dependent protein kinase II (CaMKII) modulates the localization of glutamate receptors (GluRs) at Drosophila larval neuromuscular junctions (NMJs). Expression of an inhibitory peptide of CaMKII, Ala, in muscle cells enhanced the density of GluRIIA, which is a major and calcium-permeable subunit of GluR, at synapses of third instar larval NMJs. On the other hand, postsynaptic expression of a constitutively active form of CaMKII (T287D) reduced synaptic GluRIIA. These results suggest that CaMKII regulates GluRIIA at NMJs. Moreover, postsynaptic expression of T287D abolished the accumulation of the scaffolding protein discs large (DLG) at synapses, while exerting no significant effects on the presynaptic area and the localization of cell adhesion molecule fasciclin II (FasII). The amplitude of excitatory junctional potentials (EJPs) was enhanced in Ala-expressing larvae, whereas it was unaffected in T287D-expressing larvae in spite of the prominent loss of GluRIIA. The amplitude of miniature EJPs (mEJPs) was significantly reduced and quantal content was significantly increased in T287D-expressing larvae. Notably, another class of GluR containing GluRIIB was enhanced by the postsynaptic expression of T287D. These results suggest that the homeostatic mechanism in T287D larvae works to maintain the level of synaptic responses. Thus, the Drosophila larval NMJs have several regulatory systems to ensure efficient muscle excitability which is necessary for proper larval movement.

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (Δ V) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4–5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.

Cannabinoid-1 (CB1) receptors regulate colonic propulsion by acting at motor neurons within the ascending motor pathways in mouse colon

Cannabinoid-1 (CB(1)) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate CB(1) receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the CB(1) receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB(1)(-/-) mice. Immunostaining with the cytoskeletal marker peripherin showed CB(1) immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB(1) on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for CB(1) immunostaining. Solitary spindle-shaped CB(1)-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of approximately 18 mm. The CB(1) receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB(1) activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the CB(1) antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or CB(1)(-/-) mice. Activation of the CB(1) receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This effect on electrophysiological spreading involves CB(1)-mediated effects on motor neurons and ascending interneurons and is likely to underlie the here reported in vivo reduction in colonic propulsion.

Impaired Purinergic Neurotransmission to Mesenteric Arteries in Deoxycorticosterone Acetate-Salt Hypertensive Rats

Sympathetic nerves release norepinephrine and ATP onto mesenteric arteries. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, there is increased arterial sympathetic neurotransmission attributable, in part, to impaired prejunctional regulation of norepinephrine release. Prejunctional regulation purinergic transmission in hypertension is less well understood. We hypothesized that alpha(2)-adrenergic receptor dysfunction alters purinergic neurotransmission to arteries in DOCA-salt hypertensive rats. Mesenteric artery preparations were maintained in vitro, and intracellular electrophysiological methods were used to record excitatory junction potentials (EJPs) from smooth muscle cells. EJP amplitude was reduced in smooth muscle cells from DOCA-salt (4+/-1 mV) compared with control arteries (9+/-1 mV; P<0.05). When using short trains of stimulation (0.5 Hz; 5 pulses), the alpha(2)adrenergic receptor antagonist yohimbine (1 micromol/L) potentiated EJPs in control more than in DOCA-salt arteries (180+/-35% versus 86+/-7%; P<0.05). Norepinephrine (0.1 to 3.0 micromol/L), the alpha(2)adrenergic receptor agonist UK 14304 (0.001 to 0.100 micromol/L), the A(1) adenosine receptor agonist cyclopentyladensosine (0.3 to 100.0 micromol/L), and the N-type calcium channel blocker omega-conotoxin GVIA (0.0003 to 0.1000 micromol/L) decreased EJP amplitude equally well in control and DOCA-salt arteries. Trains of stimuli (10 Hz) depleted ATP stores more completely, and the latency to EJP recovery was longer in DOCA-salt compared with control arteries. These data indicate that there is reduced purinergic input to mesenteric arteries of DOCA-salt rats because of decreased ATP bioavailability in sympathetic nerves. These data highlight the potential importance of impaired purinergic regulation of arterial tone as a target for drug treatment of hypertension.

Moesin helps to restrain synaptic growth at the Drosophila neuromuscular junction

The precise role of actin and actin-binding proteins in synaptic development is unclear. In Drosophila, overexpression of a dominant-negative NSF2 construct perturbs filamentous actin, which is associated with overgrowth of the NMJ, while co-expression of moesin, which encodes an actin binding protein, suppresses this overgrowth phenotype. These data suggest that Moesin may play a role in synaptic development at the Drosophila NMJ. To further investigate this possibility, we examined the influence of loss-of-function moesin alleles on the NSF2-induced overgrowth phenotype. We found that flies carrying P-element insertions that reduce moesin expression enhanced the NMJ overgrowth phenotype, indicating a role for Moesin in normal NMJ morphology. In addition to the NMJ overgrowth phenotype, expression of dominant-negative NSF2 is known to reduce the frequency of miniature excitatory junctional potentials and the amplitude of excitatory junctional potentials. We found that moesin coexpression did not restore the physiology of the mutant NSF2 phenotype. Together, our results demonstrate a role for moesin in regulating synaptic growth in the Drosophila NMJ and suggest that the effect of dominant-negative NSF2 on NMJ morphology and physiology may have different underlying molecular origins.

The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens

Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species. The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques. In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes. The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.

Raised tone reveals purinergic-mediated responses to sympathetic nerve stimulation in the rat perfused mesenteric vascular bed

Noradrenaline and ATP are sympathetic co-transmitters. In rat isolated mesenteric small arteries, activation of sympathetic nerves can produce a vasoconstrictor response mediated by ATP. In contrast, the rat perfused mesenteric bed displays vasoconstrictor responses that are blocked solely by α 1-adrenoceptor antagonists. This study assessed the effect of raising tone with a vasoconstrictor on purinergic and noradrenergic responses to sympathetic nerve stimulation in the rat perfused mesentery. Rat mesenteric vascular beds were perfused with physiological salt solution and responses to nerve stimulation, or P2X-receptor agonists, were determined under basal conditions and after raising tone with endothelin-1. The contribution of noradrenaline and ATP to sympathetic nerve-mediated responses was assessed using the α 1-adrenoceptor antagonist, prazosin and the P2X-receptor desensitizing agent, α,β-methyleneATP. The effect of endothelin-1 on excitatory junction potentials generated in response to nerve stimulation in isolated mesenteric arteries was also assessed. Under baseline conditions, responses to nerve stimulation were mediated solely by activation of α 1-adrenoceptors. After raising perfusion pressure with endothelin-1 or the thromboxane mimetic 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F 2α (U44619), sympathetic nerve-mediated responses were larger than under basal conditions and the response was partly sensitive to P2X-receptor desensitization. Responses to exogenous P2X-receptor agonists were enhanced after treatment with endothelin-1, while endothelin-1 decreased the amplitude of excitatory junction potentials. These results indicate that ATP acts as an important, functional, sympathetic neurotransmitter in the perfused mesentery under raised tone conditions, where the perfusion pressure is closer to that found in vivo. This effect is due to a postjunctional enhancement of purinergic function.

SUPPRESSION OF AP4-ENHANCED EXCITATORY JUNCTIONAL POTENTIALS BY THE D3 STEREOISOMER OF A CARBOXY DERIVATIVE OF BUCKMINSTERFULLERENE.

Purpose In spinal cord injury (SCI), glutamate excitotoxicity can cause secondary injury. NMDA glutamate receptor blockade is a potential protective strategy following SCI. A crayfish neuromuscular junction (NMJ) model is useful for studying NMDA receptor activity. The D3 stereoisomer of the malonic acid (carboxy) derivative of buckminsterfullerene suppresses excitatory junctional potentials (EJPs) of NMJs of the opener muscle of crayfish (Procambarus clarkii and simulans) walking limbs. DL-2-amino-4-phosphonobutyric acid (AP4, a specific NMDA receptor agonist) potentiates EJPs in this preparation. This study examines whether D3 can suppress AP4-enhanced EJPs. Materials/Methods A first or second walking limb was removed, and the meropodite and propodite were dissected to expose the excitatory axon and the opener muscle, respectively. The preparation was bathed in Van Harreveld9s Solution (VH, pH 7.2 ± 0.1) for collection of pretreatment data. Following a pretreatment control, a solution of 3.75 μm AP4 in VH was applied. EJPs were recorded immediately following application and at 1 and 2 minutes, using standard intracellular electrophysiologic techniques. Short-term facilitation (10-second stimulus at 35 Hz) averaging eight tracings per second for 10 seconds was used to record EJPs. AP4/VH was then replaced with 3.75 μm AP4 and 25 μm D3 in VH. EJPs were recorded immediately and at 1, 2, 3, and 4 minutes. Post-treatment control data collection followed three complete exchanges of the bath with VH. Results Data are reported as percent change from control amplitude; 3.75 μm AP4 enhanced EJP amplitude 30%, with a subsequent depression of 35% in 3.75 μm AP4 and 25 μm D3. The final washout returned to within 0.27% of precontrol. Qualitatively similar results were found in three additional preparations. Discussion 25 μm D3 can suppress 3.75 μm of AP4-induced EJP potentiation. This further suggests that D3 may be a useful agent in suppressing glutamate excitotoxicity induced by NMDA receptor stimulation. Supported by the Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund, the IU School of Medicine Brain and Spinal Cord Injury Research Program, and the Hillblom Foundation (L.D.).

The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity

In the moth, Manduca sexta, anterior foregut motility is modulated during the larval-larval molts in order to control the timing of molting fluid (MF) ingestion. MF is the enzymatic mixture that destroys the outer cuticle so that it can be shed at the end of the molt. The onset of the larval-larval molt is characterized by a dramatic decline in the amplitude of the anterior foregut contractions so that MF is not prematurely ingested. As the end of the molt approaches, the robust contractions of the anterior foregut return and the MF is ingested, enabling the larva to free itself from its old cuticle. In the present study we examine possible mechanisms involved in modulating anterior foregut motility during a larval-larval molt. Our results reveal that the release of a blood-borne factor plays a role in the decline in anterior foregut peristaltic activity during the molt. This blood-borne factor reduces the efficacy of the presynaptic endings of the motorneurons, resulting in a reduction in the amplitude of the excitatory junctional potential (EJP) recorded from the anterior foregut musculature. We also present evidence that crustacean cardioactive peptide (CCAP) targets the motorneuron terminals and its actions are sufficient to trigger the dramatic increase in EJP amplitude and anterior foregut contractions. Finally, the surgical ablation of the subesophageal ganglion, which has been previously described to be a source of CCAP neurons and the CCAP projections to the anterior foregut region, blocks both the increase in anterior foregut motility and the ingestion of MF that normally occur at the end of a larval-larval molt.