RESUMEN
We tested the most widely held theory about the mode of action of petroleum spray oils (PSOs) on insects (i.e. anoxia). An nC24 petroleum oil was applied topically to cotton aphids (Aphis gossypii) and cluster caterpillars (Spodoptera litura), which then showed signs of mortality that are inconsistent with anoxia. The insects died soon after treatment, with most of the mortality occurring within the first 10min. Toxicity symptoms included loss of locomotory ability, unusual abdominal contractions associated with spiracular fluttering, and ultimately dehydration and necrosis within 24h. We therefore investigated the main mechanism(s) by which the nC24 petroleum oil interacts with the insects' cells and organs, and ultimately kills the insects. The results suggest a mode of action that relates to the liphophylic properties of the oil. This includes rapid penetration through the insect cuticle followed by accumulation in the lipid-containing tissues, mainly those of the CNS, and finally penetration into the nerve cells themselves. In vitro tests with isolated insect cells further revealed that the oil penetrates the cytoplasm and induces 100% mortality of these cells within 2min of application. No signs of oil accumulation within the tracheae were observed, so it is unlikely that anoxia is taking place at any stage of the intoxication process. Electrophysiological studies confirm that oil accumulation in the nerve ganglia has the direct effect of suppressing synaptic transmission in insect ganglia as well as in the neuromuscular junctions of vertebrates (toads and rats). These results demonstrate conclusively that at least some modern PSOs do not kill insects by anoxia, but by a range of cellular disruptions that lead to rapid insect death. The implications of our findings for the development of oil-based integrated pest management strategies are discussed.
Asunto(s)
Áfidos/fisiología , Control de Insectos/métodos , Petróleo/toxicidad , Spodoptera/fisiología , Animales , Bufo marinus , Permeabilidad de la Membrana Celular/efectos de los fármacos , Drosophila melanogaster , Ganglios de Invertebrados/efectos de los fármacos , Larva/efectos de los fármacos , Microscopía Confocal , Sistema Nervioso/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.
Asunto(s)
Venenos Elapídicos/toxicidad , Neurotoxinas/toxicidad , Receptores Nicotínicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Australia , Secuencia de Bases , Bufo marinus , Cartilla de ADN/genética , ADN Complementario/genética , Venenos Elapídicos/genética , Venenos Elapídicos/aislamiento & purificación , Elapidae/genética , Electrofisiología , Técnicas In Vitro , Datos de Secuencia Molecular , Neurotoxinas/genética , Neurotoxinas/aislamiento & purificación , Antagonistas Nicotínicos/aislamiento & purificación , Antagonistas Nicotínicos/toxicidad , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Homología de Secuencia de AminoácidoRESUMEN
N-ethylmaleimide (NEM) has been used extensively in biochemical assays as an inhibitor of the NEM sensitive fusion protein (NSF). However, examination of the effect of NEM on transmitter release in more physiologically relevant preparations has proved inconclusive. In the present study, we have examined the effect of low concentrations of NEM on synaptic transmission in intact nerve-muscle preparations from toads (Bufo marinus). Under conditions of low transmitter release probability (0.3 mM calcium, 1 mM magnesium), treatment with NEM (10 microM) caused a significant increase in the amplitude of stimulus-evoked endplate potentials (EPPs) and a significant increase in the frequency of spontaneously occurring miniature EPPS (MEPPS) without affecting the amplitude of MEPPs. When the calcium concentration in the bath was raised to 4 mM, 10 microM NEM had no effect on EPP amplitude. Under these conditions, NEM treatment reduced paired pulse facilitation and increased depression during stimulus trains. Treatment with NEM also resulted in a significant decrease in the synaptic delay. The effects of NEM on transmitter release in the present study were not due to inactivation of G-proteins. The results of the present study show a calcium-dependent facilitation of stimulus-evoked transmitter release by NEM. These results are discussed in terms of the possible sites of NEM action leading to the observed changes in transmitter release.
Asunto(s)
Etilmaleimida/farmacología , Músculo Esquelético/inervación , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Bufo marinus , Calcio/deficiencia , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Estimulación Eléctrica , Músculo Esquelético/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Receptores Purinérgicos P2/metabolismo , Sistema Nervioso Simpático/fisiología , Transmisión Sináptica/fisiología , Conducto Deferente/fisiología , Adenosina Trifosfato/farmacología , Animales , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Músculo Liso/fisiología , Norepinefrina/farmacología , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2X , Transmisión Sináptica/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Conducto Deferente/inervaciónRESUMEN
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.
Asunto(s)
Vivienda para Animales , Estrés Fisiológico/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Transmisión Sináptica , Animales , Conductividad Eléctrica , Electrofisiología , Uniones Intercelulares/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Neurotransmisores/metabolismo , Ratas , Aislamiento SocialRESUMEN
1. Previous studies have demonstrated that chronic pre-synaptic inhibition of transmitter release by morphine evokes a counter-adaptive response in the sympathetic nerve terminals that manifests itself as an increase in transmitter release during acute withdrawal. In the present study we examined the possibility that other pre-synaptically acting drugs such as clonidine also evoke a counter-adaptive response in the sympathetic nerve terminals. 2. In chronically saline treated (CST) preparations, clonidine (0.5 microM) completely abolished evoked transmitter release from sympathetic varicosities bathed in an extracellular calcium concentration ([Ca(2+)](o)) of 2 mM. The inhibitory effect of clonidine was reduced by increasing [Ca(2+)](o) from 2 to 4 mM and the stimulation frequency from 0.1 to 1 Hz. 3. The nerve terminal impulse (NTI) was not affected by concentrations of clonidine that completely abolished evoked transmitter release. 4. Sympathetic varicosities developed a tolerance to clonidine (0.5 microM) following 7-9 days of chronic exposure to clonidine. 5. Acute withdrawal of preparations following chronic clonidine treatment (CCT) resulted in a significant (P < 0.005) enhancement of neurotransmitter release (3.75 times) above control levels observed in CST preparations. 6. The present findings demonstrate an enhancement of neurotransmitter release from sympathetic varicosities following acute withdrawal from chronic clonidine treatment.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Clonidina/farmacología , Neurotransmisores/metabolismo , Conducto Deferente/efectos de los fármacos , Animales , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Fibras Simpáticas Posganglionares/efectos de los fármacos , Fibras Simpáticas Posganglionares/metabolismo , Conducto Deferente/inervación , Conducto Deferente/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Potenciales Postsinápticos Excitadores/fisiología , Músculo Liso/metabolismo , Receptores Purinérgicos P2/metabolismo , Conducto Deferente/metabolismo , Adenosina Trifosfato/farmacología , Animales , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/efectos de los fármacos , Músculo Liso/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2X , Conducto Deferente/efectos de los fármacos , Conducto Deferente/crecimiento & desarrolloRESUMEN
1. The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha(2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2. In chronically saline treated (CST) preparations, morphine (1 microM) and the alpha(2)-adrenoceptor agonist (clonidine, 1 microM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 microM) and clonidine (1 microM) decreased mean EJP amplitude by 68+/-8% (n=7) and 79+/-8% (n=7) respectively. 3. When stimulating the sympathetic axons at 0.03 Hz, the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n=7 compared with 9.9+/-0.3 mV, n=7). 4. Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5. Results from the present study indicate that the effectiveness of alpha(2)-adrenoceptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha(2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Neurotransmisores/metabolismo , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Conducto Deferente/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Animales , Axones/efectos de los fármacos , Axones/ultraestructura , Clonidina/farmacología , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Músculo Liso/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Conducto Deferente/inervaciónRESUMEN
1 The effects of stress in rats were evaluated by measuring changes in body weight and in responsiveness to noradrenaline (NA) in the isolated vas deferens and atria after the animals had been exposed to restraint or restraint and isolation. 2 Animals which were subjected to restraint alone (1 h day-1 for 3, 7 or 28 days) had a significantly reduced rate of body weight gain. This effect was not potentiated by the additional stress of isolation. 3 Restraint alone did not produce significant changes in the responsiveness of the vas deferens to NA. However, adding isolation to restraint, as an additional stress, produced a further leftward shift of the NA dose-response curve for the vas deferens, so that there was a significant increase in sensitivity compared with control. 4 There was a significant rightward shift in the NA dose-response curves or reduction in sensitivity in the atria from animals which had been restrained for 7 or 28 days. Isolation did not produce a further rightward shift in the NA dose-response curve. 5 The results from this study indicate that the stress associated with repeated restraint reduces the rate of weight gain and reduces the responsiveness of the atria to NA. The responsiveness of the vas deferens to NA was increased by stress, but the combined effect of isolation and restraint was required to produce a significant effect. The differences in the effects of stress on these tissues could be associated with differences in presynaptic receptor populations.
Asunto(s)
Frecuencia Cardíaca/efectos de los fármacos , Estrés Fisiológico/fisiopatología , Conducto Deferente/fisiopatología , Glándulas Suprarrenales/efectos de los fármacos , Animales , Conducta Animal , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Atrios Cardíacos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Norepinefrina/farmacología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Restricción FísicaRESUMEN
ATP released by sympathetic varicosities of the mouse vas deferens binds to P2X receptors which activate fast, ligand-gated channels, resulting in depolarisation of smooth muscle cells. We examined the development of fast neuromuscular transmission at surface longitudinal smooth muscle fibres of the mouse vas deferens. Sympathetic varicosities were visualised using DiOC(2)(5)-fluorescence to aid in positioning loose patch electrodes over small sets of sympathetic varicosities to record the nerve terminal impulse (NTI) and excitatory junction currents (EJCs) evoked during nerve stimulation. At the earliest age at which EJCs could be detected, 21 days postnatal (PN), most recording sites rarely showed a detectable EJC over 100 trials, even though NTIs were recorded without failure. The extent of such intermittence in transmitter release progressively declined between 21 and 42 days PN. In addition, the mean amplitude of spontaneous EJCs (SEJCs) and EJCs increased by 2- and 2.4-fold, respectively, between 21 and 42 days PN. The rise time of EJCs varied widely at each age but declined with development (e.g., 7-14 ms at 28 days PN, 3-12 ms at 42 days PN). All EJCs were abolished by suramin (100 microM). Fast rise time EJCs were rapidly abolished by alpha,beta-methylene ATP (20 microM) while some (34%) of the slower rise time EJCs were resistant to rapid desensitisation of this kind. P2X(1) and P2X(2) mRNAs were detected by reverse transcription and polymerase chain reaction (RT-PCR) to determine whether levels of expression of the receptor subunits might explain the increased EJC amplitude. Between 10 and 42 days PN no marked change was observed in the P2X(2) receptor mRNA or beta-actin mRNA (control). In contrast, the intensity of the RT-PCR band for P2X(1) receptor showed a progressive approximately 4.3-fold developmental increase relative to the P2X(2) band. These observations suggest that both prejunctional and postjunctional mechanisms cause the maturation of fast purinergic junctional transmission at the longitudinal muscle of the mouse vas deferens between 21 and 42 days PN.
Asunto(s)
Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Transmisión Sináptica/fisiología , Conducto Deferente/inervación , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Fibras Adrenérgicas/fisiología , Animales , Antineoplásicos/farmacología , Clonación Molecular , Electrofisiología , Expresión Génica/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Músculo Liso/inervación , ARN Mensajero/análisis , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X2 , Suramina/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.
Asunto(s)
Acetilcolina/metabolismo , Canales de Calcio/fisiología , Ganglios Autónomos/metabolismo , Terminaciones Nerviosas/metabolismo , Amilorida/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo P/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Técnicas In Vitro , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/fisiología , Pelvis/inervación , Ratas , Glándula Submandibular/inervación , Transmisión Sináptica/fisiologíaRESUMEN
Activation of the sympathetic nervous system is an important component of the response to stress, but the effects of prolonged stress on sympathetic neurotransmission have not been assessed. In the present study we have examined the effect of 3 to 10 days exposure to stress induced by frequent handling and sham injections on neurotransmitter release from sympathetic varicosities of the mouse vas deferens. DiOC2(5)-fluorescence was used to visualise the sympathetic varicosities so that extracellular electrodes could be placed over known numbers of varicosities to monitor transmitter release using electrophysiological techniques. The frequency of excitatory junction currents (EJCs) increased with increasing duration of exposure to stress. The mean and maximum EJC amplitude significantly increased by 107% and 43%, respectively after 10 days of exposure to stress. The density of sympathetic varicosities innervating smooth muscle of the mouse vas deferens was not changed throughout the duration of the exposure to stress. The findings from this study demonstrate that the efficacy of transmitter release from the sympathetic varicosities is altered by repeated exposure of mice to stressful stimuli, such as handling and sham injections. Since such procedures are routine in many pharmacological experiments, it is important that investigators are aware of these changes so that due consideration is given when interpreting the data obtained from animals treated in this way.
Asunto(s)
Neurotransmisores/metabolismo , Estrés Psicológico/fisiopatología , Sistema Nervioso Simpático/patología , Conducto Deferente/inervación , Conducto Deferente/fisiopatología , Animales , Estimulación Eléctrica , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Unión Neuromuscular/efectos de los fármacosRESUMEN
Excitatory postsynaptic currents (EPSCs) were recorded with loose patch electrodes placed over visualized boutons on the surface of rat pelvic ganglion cells. At 34 degrees C the time to peak of the EPSC was about 0.7 ms, and a single exponential described the declining phase with a time constant of about 4.0 ms; these times were not correlated with changes in the amplitude of the EPSC. The amplitude-frequency histogram of the EPSC at individual boutons was well described by a single Gaussian-distribution that possessed a variance similar to that of the electrical noise. Nonstationary fluctuation analysis of the EPSCs at a bouton indicated that about 120 ACh receptor channels were available beneath boutons for interaction with a quantum of ACh. The characteristics of these EPSCs were compared with the results of Monte Carlo simulations of the quantal release of 9000 acetylcholine (ACh) molecules onto receptor patches of density 1400 microns-2 and 0.41 micron diameter, using a kinetic scheme of interaction between ACh and the receptors similar to that observed at the neuromuscular junction. The simulated EPSC generated in this way had temporal characteristics similar to those of the experimental EPSC when either the diffusion of the ACh is slowed or allowance is made for a finite period of transmitter release from the bouton. The amplitude of the simulated EPSC then exhibited stochastic fluctuations similar to those of the experimental EPSC.
Asunto(s)
Acetilcolina/metabolismo , Ganglios Simpáticos/metabolismo , Terminales Presinápticos/metabolismo , Receptores Colinérgicos/metabolismo , Transmisión Sináptica , Animales , Cinética , Microelectrodos , Método de Montecarlo , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas , Procesos EstocásticosRESUMEN
1. The effect of opiates on neurotransmission between visualized hypogastric nerve boutons and postganglionic cell bodies has been examined using extracellular recording of nerve bouton impulses (NBIs) and excitatory postsynaptic currents (e.p.s.cs). 2. Morphine (10 to 40 microM) did not affect neurotransmission in the ganglia. Dynorphin-A (4 microM) and U50488H (1 microM) decreased quantal transmitter release and naloxone (10 microM) reversed these effects. 3. Morphine (10 microM), dynorphin-A (4 microM) and U50488H (1 microM) did not affect either the time course or consistency with which the NBI was recorded. 4. Dynorphin-A (1 to 4 microM) and U50488H (1 microM) decreased the average amplitude of e.p.s.cs by increasing the number of failures to release quanta from single or small groups of 2 to 4 boutons during continuous nerve stimulation at 0.1 Hz. 5. The decrease in quantal release induced by dynorphin-A and U50488H in 0.2 to 0.5 mM [Ca2+]zero was readily reversed by increasing the extracellular calcium ion concentration to 1 mM. 6. It was concluded that kappa-opioid receptors are located on the boutons of the hypogastric nerve and when activated by kappa-opioid receptor agonists reduce quantal release without affecting the NBI.
Asunto(s)
Plexo Hipogástrico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Narcóticos/farmacología , Neurotransmisores/metabolismo , Animales , Dinorfinas/farmacología , Morfina/farmacología , Naloxona/farmacología , RatasRESUMEN
The spatial relationships between nerve varicosities and smooth muscle cells in the longitudinal muscle layer of the mouse vas deferens have been determined from serial section reconstructions of individual varicosities at the ultrastructural level. Bundles of up to five axons, together with single axons, occurred frequently at the surface of the muscle as well as at about 3-6 muscle cell diameters into the muscle. Varicosities within axon bundles at the muscle surface each became partially divested of Schwann cell processes. The smallest distance separating varicosity membrane from muscle cell membrane (apposition distance) was 100 nm (mean 170 nm) for varicosities contained in bundles. Varicosities from six single axons on the muscle surface were reconstructed and 11 of the 12 possessed a mean apposition distance of 48 nm. Varicosities in axon bundles at about 12 microns deep into the muscle came into an apposition distance of 50-90 nm (mean = 67 nm). All varicosities of single axons at this depth came into about 50 nm apposition (mean = 53 nm). These results indicate that the varicosities lie at varying distances from the muscle cells in the longitudinal muscle layer of the vas deferens.
Asunto(s)
Músculo Liso/inervación , Terminales Presinápticos/ultraestructura , Fibras Simpáticas Posganglionares/ultraestructura , Conducto Deferente/inervación , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica/métodos , Conducto Deferente/ultraestructuraRESUMEN
Synaptic transmission from single preganglionic hypogastric nerves innervating monopolar pelvic ganglion neurones has been studied with intracellular electrodes to record transmission from all the boutons and with extracellular electrodes placed over boutons visualized with DiOC2(5) in order to record transmission from selected boutons. Intracellular electrodes revealed spontaneous excitatory postsynaptic potentials (EPSPs) with amplitude histograms that show increasing numbers of large EPSPs as the external calcium ([Ca2+]o) was increased from 0.15 to 1.0 mM. These histograms were in general well fitted by a Poisson mixture of gamma distributions. Extracellular electrodes placed over visualized boutons revealed evoked excitatory postsynaptic potentials (extracellular EPSPs) with amplitude histograms that were best described by single gamma distributions in most cases in low [Ca2+]o (less than 0.5 mM). The standard deviation of these gammas was not much larger than that of the electrical noise. In a minority of extracellular recordings the amplitude histogram of evoked extracellular EPSPs was best described by a gamma distribution in which the standard deviation was much greater than that of the noise. Confocal microscopy of boutons orthogradely labelled with dextran-rhodamine showed that about 30% of these formed closely apposing pairs on the surface of the neurones. These observations are discussed in terms of the hypothesis that multiquantal release at boutons occurs as a consequence of the coupled secretion from closely apposed boutons.
Asunto(s)
Ganglios Autónomos/metabolismo , Ganglios Autónomos/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Neurotransmisores/metabolismo , Transmisión Sináptica/fisiología , Animales , Carbocianinas , Electrofisiología , Espacio Extracelular/metabolismo , Colorantes Fluorescentes , Ganglios Autónomos/citología , Técnicas In Vitro , Microscopía Confocal , Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/fisiología , Ratas , Ratas EndogámicasRESUMEN
1 The effect of morphine on both the propagation of the nerve terminal impulse along the sympathetic varicose axons as well as the evoked and spontaneous transmitter release has been evaluated. 2 Morphine (1 microM) did not significantly change the shape or the regularity by which the nerve terminal impulse was recorded while evoked transmitter release was greatly reduced. 3 Morphine induced a uniform decrease in evoked transmitter release irrespective of the release probability of individual varicosities of their position along terminal branches. 4 Procedures which are thought to increase intracellular calcium concentration such as increasing the extracellular calcium concentration, stimulation of the nerve with trains of impulses and increasing the duration of the action potential with 4-aminopyridine reduced the ability of morphine to decrease evoked transmitter release. 5 Morphine had to act directly on the varicosities to induce a decrease in evoked transmitter release. 6 The decrease in evoked quantal release does not involve an affect on the nerve terminal impulse or the vesicle release process and morphine may affect the dependence of the secretory process on calcium.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Músculo Liso/inervación , Terminaciones Nerviosas/efectos de los fármacos , Neurotransmisores/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Axones/fisiología , Potenciales Evocados/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/metabolismo , Terminaciones Nerviosas/metabolismo , Conducto Deferente/inervaciónRESUMEN
1 Transmitter release from sympathetic varicosities of mouse vasa deferentia removed from animals which were chronically treated with morphine for 7 to 9 days has been evaluated. 2 In control preparations increasing the extracellular calcium concentration ([Ca2+]o) from 1 to 2 mM increased transmitter release by 3 fold while increasing [Ca2+]o from 6 to 8 mM increased transmitter release by about 0.9 fold. Introduction of morphine (1.0 microM) produced a uniform decrease in transmitter release, shifting the relationship between transmitter release and [Ca2+]o to the right. 3 Only sympathetic varicosities with probabilities of transmitter release greater than 0.01 were chosen for this study. In these varicosities the decrease in transmitter release induced by morphine in control preparations (bathed in [Ca2+]o 2.0 mM) was not observed following 7 to 9 days of morphine treatment. When the morphine was acutely withdrawn from these preparations transmitter release was more than 6 times the average level of transmitter release from control preparations. 4 The morphine induced increase in facilitation of transmitter release while stimulating with short trains of nerve impulses was not observed when the preparations were removed from animals which had been exposed to morphine for 7 to 9 days. When these preparations were acutely withdrawn from morphine there was a further decrease in the level of facilitation and a significant increase in depression of transmitter release when compared to control. 5 The morphine induced decrease in probability of transmitter release when naive sympathetic varicosities in vitro were bathed with morphine (1 microM) was not observed following chronic morphine treatment of the animals for 7 to 9 days. When the morphine was acutely withdrawn from chronically morphine treated preparations the underlying increase in probabilities of transmitter release of sympathetic varicosities was unmasked.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Músculo Liso/inervación , Terminaciones Nerviosas/efectos de los fármacos , Neurotransmisores/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Calcio/metabolismo , Potenciales Evocados/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Microelectrodos , Músculo Liso/metabolismo , Conducto Deferente/inervaciónRESUMEN
The probability of detecting first, second, and later quanta secreted at release sites of a motor-nerve terminal during the early release period following a nerve impulse has been addressed. The possibility that early quantal release autoinhibits later quantal release during this period has also been ascertained. In this investigation, a model for the secretion of a quantum at a release site is developed in which, following the influx and diffusion of calcium ions to a release site protein associated with synaptic vesicles, kappa steps of association of the ions with the protein then occur at rate alpha. The release site protein then undergoes a conformational change which may not go on to completion if calcium ions dissociate from the protein at rate gamma. If this process does reach completion then a fusion-pore between the vesicle and the presynaptic membrane is created; this happens at rate delta. Key assumptions of this fusion-pore model are that the quantal secretions from each site are independent of each other, and that there is a large number of vesicles, each with a small probability of secretion, so that the number of secretions is Poisson in nature. These assumptions allow analytical expressions to be obtained for predicting the times at which first, second and later quanta are secreted during the early release period following an impulse. To test the model, experiments were performed in which the times of first, second and later quantal releases were determined at discrete regions along the length of visualized motor-terminal branches in toad (Bufo marinus) muscles. Estimates of model rate constants and of kappa from the times for first quantal secretions failed to give satisfactory predictions of the observed times of later secretions. Therefore, either the model fails, or the procedure used for detecting later quantal events as a consequence of their being masked by earlier quantal events is inadequate. To solve this detection problem, a two-dimensional analysis of the spread of charge following the secretion of a quantum at a random site on the motor-terminal branch has been done. This allows determination of the probability that later quanta will be detected following secretion of earlier quanta. The detection model was then incorporated into the fusion-pore model to predict the times at which second and later quanta occur during the early release period, based on the estimates of the model parameters derived from the analysis of first quantal releases. Good estimates were now obtained for the observed times of second and later quantal releases, indicating that appropriate procedures must be adopted for adequate detection of quantal secretions. Furthermore, the experiments provide support for the fusion-pore model. It has been suggested that the binomial nature of quantal release from the entire motor-nerve terminal may be explained if early quantal release inhibits later quantal release during the early quantal release phase (M. R. Bennett & J. Robinson 1990, Proc. R. Soc. Lond. B 239, 329-358). Although the fusion-pore detection error model gave good predictions of the observed times of first, second and later quantal releases, these may be improved if a model for autoinhibition is included. In this model the first quantum was taken as giving rise to an inhibition of secretion that propagates to surrounding release sites with a constant velocity, v. A combined model incorporating the fusion-pore detection error model and that for autoinhibition was then used to predict second and later quantal latencies, by using the first quantal latencies to determine the estimates for the parameters in the combined model. When this analysis was done on the times for quantal secretion at sites on thirteen different motor-nerve terminals, the value of v was estimated as zero in each case, so that no autoinhibitory effect was observed.
Asunto(s)
Modelos Neurológicos , Neuronas Motoras/metabolismo , Porinas/metabolismo , Terminales Presinápticos/metabolismo , Animales , Bufo marinusRESUMEN
A method is described for recording the electrical signs of transmission at single boutons. Monopolar rat pelvic ganglion cells are electrically compact and receive an innervation from either single hypogastric or pelvic nerves that consists of a set of boutons which innervate the soma of the cells; each bouton possesses an active zone and can be observed with the confocal microscope after dextran-rhodamine orthograde labelling of the hypogastric nerves. Extracellular electrodes of about 8 microns diameter can be positioned in the loose-patch configuration over visualized boutons following their fluorescence with the vital dye, 3,3-diethyloxardicarbocyanine iodide (DiOC2(5)). Excitatory post-synaptic currents (EPSCs), due to stimulation of the hypogastric nerve, can be recorded from boutons in this way, provided that [Ca2+]o is lowered sufficiently to ensure failure of the initiation of the soma action potential by the EPSCs.