Dynamics of fast synaptic excitation during trains of stimulation in myenteric neurons of guinea-pig ileum

Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A1, opioid, alpha2-adrenergic and 5-HT1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K+ (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter.     

Synaptic ultrastructure of functionally and morphologically characterized neurons of the superficial spinal dorsal horn of cat

Recordings of neuronal unitary discharges evoked by primary afferent input were made in the superficial part of the spinal cord's dorsal horn, the marginal zone and substantia gelatinosa (also known as laminae I and II), using fine micropipette electrodes filled with HRP. After physiological characterization with respect to primary afferent input, HRP was injected intracellularly iontophoretically into the recorded neuron. Following histochemical processing, the neurons so delineated were studied at the light and electron microscopic levels. No clear relationship between function and either general cellular configuration or synaptic ultrastructure appeared in these analyses, although the concentration of dendritic distribution could be related to the nature of primary afferent excitation. Nocireceptive cells had dendrites mostly branching and ending in lamina I and IIo, while the dendrites of innocuous mechanoreceptive cells arborized principally in lamina II and III. Glomerular synaptic complexes (large, complex arrays of axonic and dendritic profiles with synaptic interconnections) were found to contact a few neurons of both the nocireceptive and mechanoreceptive classes. All neurons received large numbers of simple axonic contacts (small axonic boutons with only 1 or 2 synaptic contacts with a single postsynaptic profile). A degree of specificity in the presynaptic articulations appeared to be reflected by the observations that (1) nocireceptive neurons were never found to receive synaptic contacts from boutons which resembled the known ultrastructure of peripheral innocuous mechanoreceptors, and (2) mechanoreceptive neurons were never seen to receive synaptic contacts from boutons which resembled the known ultrastructure of primary afferent nocireceptors. The axons of the labeled neurons of both nocireceptive and mechanoreceptive classes terminated in simple axonic synapses. All classes of neurons participated in dendrodendritic contacts; however, only some mechanoreceptive neurons had dendrites containing vesicles that were presynaptic to other profiles. No nocireceptive neurons, regardless of gross configuration, were found to have vesicles in their dendrites, but 3 nocireceptive neurons received synapses from presynaptic dendritic profiles.     

Synaptic processes in neurons of the cat pericruciate cortex evoked by pyramidal tract stimulation]

The influences of pyramidal tract stimulation on the activity of neurons in the pericruciate cortex were investigated on 423 neurons (81 neurons were studied intracellularly and 342--extracellularly), 78 of them having background activity. Pyramidal stimulation is shown to evoke not only antidromic spikes (0.5-16.0 ms latency) in the pyramidal cells, but also lateral and recurrent PSPs in the pyramidal and unidentified units of all cortical layers. IPSPs were observed in 46.7% of the investigated neurons, EPSPs--in 21.0%, mixed responces--in 26.0%. The latency of IPSPs was 1.5-14.0 ms, their amplitude ranged from 1.3 to 17.0 mV, the duration of the rising phase varied from 4 to 18 ms and the whole duration was 18-120 ms reaching sometimes 250-500 ms. In 30% of cases it was possible to divide the IPSPs into two phases: a fast one with a duration of 10-20 ms and a slow one. The latency of IPSPs was 2.6-19.0 ms, their amplitude--1.0--7.8 mV and duration--from 10.0 to 50.0 ms. The antidromic discharge in the pyramidal tract inhibited the background activity for 200-400 ms in 51.2% of spontaneously active units; acceleration was observed in 19.5% and mixed effect in 7.4% of units. The participation of pyramidal axonal collaterals and cortical interneurons in generation of the described processes is discussed.     

Convergence of reflex pathways excited by distension and mechanical stimulation of the mucosa onto the same myenteric neurons of the guinea pig small intestine.

The effects on morphologically and electrophysiologically characterized myenteric neurons of activation of intestinal reflex pathways were examined in vitro. Opened segments of guinea pig small intestine were pinned serosa down in an organ bath that had two balloons set into its base. A 5-10-mm-wide strip of myenteric plexus between the balloons was exposed from the mucosal side, and neurons were impaled with microelectrodes. Reflex pathways were stimulated by inflation of the balloons to distend the intestinal wall, and by deforming the exposed mucosal villi with a brush. Impaled neurons were classified electrophysiologically as AH-neurons or S-neurons (Hirst et al., 1974) and were injected with biocytin to determine their shapes and projections. None of the 58 AH-neurons responded to distension. In contrast, 63 of 131 S-neurons responded to distension with a burst of fast EPSPs; about one-third of the responding S-neurons received input from ascending reflex pathways, one-third received input from descending reflex pathways, and one-third received input from both ascending and descending pathways. Most neurons in this last group supplied extensive varicose branches to the tertiary plexus and were probably longitudinal muscle motor neurons. Neurons receiving input from only one pathway usually projected in the direction of that pathway; many of these were circular muscle motor neurons. Almost all neurons responding to distension were also excited by deforming the villi. Responses evoked by distension or deforming the mucosa declined when stimuli were repeated at intervals less than 10 sec. This was seen in ascending and descending pathways but was more prominent in the former. Deforming the mucosa evoked a normal response even when the response to repeated distensions had disappeared. It is concluded that distension and deforming the mucosa excite separate populations of sensory neurons to activate reflex pathways that converge onto common motor neurons and probably onto common interneurons.     

Synaptic activity of myenteric neurons in tissue culture

Intracellular recordings were made from myenteric neurons of the guinea-pig caecum grown in tissue culture. The experiments were carried out on cultures older than 15 days, in which the neurons formed ganglion-like aggregates with many interconnecting nerve fibres. Intracellular injection of horseradish peroxidase demonstrated that the neurons project processes both within and outside the aggregates. Spontaneous activity was recorded in about 50% of the cells. This activity was composed of depolarizing potentials 2-20 mV in amplitude, and in most cases 10-20 ms in duration. In some instances, the potentials occurred in short bursts. The spontaneous potentials appear to be nicotinic cholinergic excitatory postsynaptic potentials (EPSPs). Electrical stimulation of the connecting fibers elicited in the neurons nicotinic cholinergic EPSPs, since they were blocked by hexamethonium. By varying the stimulus intensity and by stimulating several fibers, it was shown that the neurons are multiple-innervated. During repetitive presynaptic stimulation at frequencies above 10 Hz, there was a progressive reduction of the EPSP amplitude. It is concluded that the cultured myenteric neurons retain many of the characteristics found in the freshly isolated preparation and may be useful for studying the synaptic properties of these cells.     

Ruthenium red antagonism of the effects of capsaicin mediated by extrinsic sensory nerves on myenteric plexus neurons of the isolated guinea-pig ileum.

The effects of Ruthenium red and its antagonism of capsaicin-induced action on the electrophysiological behavior of myenteric neurons were investigated with intracellular recording techniques in the isolated guinea-pig ileum. Ruthenium red antagonized dose-dependently (1-10 microM) a capsaicin-induced marked long-lasting slow depolarizing action associated with increased input resistance, during which the cells spiked repeatedly or displayed anodal break excitation. This action of capsaicin has been found to be mediated via a release of substance P from sensory nerve endings. The slow depolarizing response to exogenous substance P applied by pressure microejection, which mimicked the capsaicin-induced action, was not affected by Ruthenium red. Therefore, present results indicate that Ruthenium red antagonizes the specific effect of capsaicin on myenteric neurons by acting on the presynaptically located peripheral nerve terminals of sensory neurons and inhibiting the release of substance P. Electron-microscopic examination showed that the neurotoxic action of capsaicin towards extrinsic sensory nerve fibers was also dose-dependently (1-10 microM) protected by pretreatment of ruthenium red. Present results suggest that Ruthenium red inhibits a capsaicin-induced activation of cation channels at the cell membrane of sensory nerves.     

Synaptic processes in the rubrospinal neurons evoked by stimulation of the cerebellar nucleus interpositus in the cat

Activity of red nucleus rubrospinal neurons to electrical stimulation of cerebellar nucleus interpositus was studied in nembutalized cats. Facilitation of cerebello-rubral transmission was shown to take place in the majority of neurons whose focal potentials and EPSPs to paired and frequency stimulation of cerebellar nucleus interpositus were analyzed. The mentioned facilitation was found not to be caused by changes in presynaptic volleys. Discussed modification of the transmission efficiency was supposed to be determined by peculiarities of functioning of cerebellar synapses on red nucleus neurons.     

Effects of mesenteric nerve stimulation on the electrical activity of myenteric neurons in the guinea pig ileum

Effects of mesenteric nerve stimulation on the electrical activity of 28 myenteric neurons were investigated in the myenteric flaps innervated with mesenteric nerves. Mesenteric nerve stimulation evoked slow excitatory postsynaptic potentials (EPSPs), whose amplitude and duration were 24.5 +/- 5.5 mV and 374.6 +/- 58.9 s in 7 AH/Type 2 neurons, respectively. Such slow EPSPs mimic the slow depolarizing action induced by exogenous substance P. It is, therefore, likely that slow EPSPs might be in part mediated by the release of substance P.     

Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum

Abstract Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron‐doped diamond microelectrode held at 1 V vs a Ag|AgCl reference electrode. This potential was sufficient to oxidize NO. Longitudinal muscle‐myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 μmol L^?1 nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N‐nitro‐l ‐arginine (l ‐NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation, stimulated release produced a maximum current that corresponded nominally to 46 nmol L^?1 of NO. The oxidation currents decreased to 10 and 2 nmol L^?1, respectively, when the tissue was perfused with tetrodotoxin and l ‐NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were threefold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real‐time electroanalytical techniques.     

Measurement of the intracellular calcium concentration in guinea-pig myenteric neurons by using fura-2

Intracellular free calcium concentration [( Ca2+]i) was measured in guinea-pig myenteric neurons by using the fluorescent calcium indicator, fura-2; at the same time, intracellular recordings were made from the myenteric neurons. The [Ca2+]i of the myenteric neurons was about 100 nM at the resting state. The slow after-hyperpolarization that followed an action potential was associated with an increase in [Ca2+]i [Ca2+]i decreased during superfusion with calcium-free/high (6 mM) magnesium solution, and increased during superfusion with high (20 mM) potassium solution. However, [Ca2+]i did not change when the depolarizations caused in the high potassium solution were prevented by passing inward current through the recording electrode. The present experiments provide direct evidence that depolarization of the myenteric neurons allows calcium to enter the cell, and that rises in intracellular calcium concentration hyperpolarize the neuron.     

Synaptic inhibition of accessory motoneurons evoked by stimulation of the trigeminal nerve in the cat.

Stimulation of the trigeminal nerve produced polysynaptic inhibitory postsynaptic potentials (IPSPs) in accessory motoneurons of the cat. This contrasts with the observation that dorsal cervical motoneurons responded with EPSPs to trigeminal stimulus. Stimulation of the rostral part of spinal trigeminal nucleus elicited di- or polysynaptic IPSPs in accessory motoneurons. Transection of the anterior funiculus at the upper cervical cord selectively abolished the IPSPs. The IPSPs were antagonized by systematically administrated strychnine but not bicuculline.     

Intracellular responses of raphe magnus neurons during the jaw-opening reflex evoked by tooth pulp stimulation

Neurons in the nucleus raphe magnus (RM) may play an important role in the modulation of nociception. To determine how RM neurons are activated during a nociceptive reflex, the intracellular responses of raphe neurons were studied during the jaw-opening reflex (JOR) elicited by tooth pulp shock in lightly anesthetized cats. Tooth pulp stimulation produces reflex EMG activation of the digastric muscle at a latency of 7-11 ms, resulting in jaw opening. Tooth pulp shock that elicits the JOR also produces an EPSP in a subset of raphe neurons. This EPSP consists of an early small depolarization that occurs at a latency of 10-15 ms followed by a larger depolarization at a latency of 20-60 ms. In all cases the latency to EPSP is longer than the latency to digastric EMG onset. Electrical stimulation of the 4 paws elicits oligosynaptic EPSPs in the same cells at a latency of 16-20 ms. Electrical train stimulation of the midbrain periaqueductal gray region (PAG) suppresses the JOR. Single shock stimulation at the same PAG sites that suppress the JOR evokes monosynaptic EPSPs in the large majority of raphe neurons recorded. In all cases, the threshold for EPSP is below the threshold for suppression of the JOR. The EPSP amplitude is a direct function of PAG stimulus intensity and there is temporal summation of EPSPs evoked by paired PAG shocks. At condition-test intervals of 40-90 ms, train stimulation of PAG suppresses the tooth pulp-evoked EPSP in raphe neurons. The threshold for EPSP suppression occurs at a PAG stimulation intensity below that required for suppression of the JOR. The present findings provide evidence that RM neurons may play an important role in the modulation of the tooth pulp-evoked JOR, but only after the initial withdrawal reflex has occurred.     

Synaptic responses of cortical pyramidal neurons to light stimulation in the isolated turtle visual system

The resistance of the turtle brain to hypoxic injury permits a unique in vitro preparation in which the organization and function of visual cortex can be explored. Intracellular recordings from cortical pyramidal neurons revealed biphasic responses to flashes of light, consisting of an early phase (50-100 msec) of concurrent inhibitory and excitatory activation, followed by a longer, inhibitory phase (250-600 msec) composed of summated Cl- -dependent postsynaptic potentials mediated by GABA. This response sequence results from the coactivation of pyramidal and GABAergic non-pyramidal cells, followed by feed-forward and possibly feed-back pyramidal cell inhibition, and is partly dependent on differences in the membrane properties of pyramidal and non-pyramidal neurons.     

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea‐pig ileum

Abstract Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea‐pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post‐synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.     

Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea-pig ileum

Flat sheet preparations of guinea-pig ileum were stretched circumferentially and the propagation of circular muscle contractions along the preparation was investigated. Slow stretch, at 100 microm s-1, of a 50-mm long flat sheet of intestine, evoked circular muscle contraction orally, which propagated, without decrement, for up to 30 mm. This occurred despite circular muscle shortening being prevented, and in the absence of propulsion of contents. Thus, propagation in this flat sheet preparation could not explained on the basis of neuro-mechanical interactions, as previously proposed. Irrespective of the length of preparations, contraction amplitude decreased significantly in the most aboral 10-15 mm of intestine. This was not due to descending inhibitory pathways, but was associated with interruption of ascending excitatory pathways near the aboral end. Slow waves were not detected in circular muscle cells in any preparation (n=8). Smooth muscle action potentials evoked in circular muscle cells, in the presence of tetrodotoxin (TTX, 0.6 micromol L-1), did not propagate for more than 1 mm in the longitudinal axis. Propagation of circular muscle activity, evoked by slow stretch of flat sheet preparations, reveals the presence of a mechanism other than myogenic spread or the neuro-mechanical interactions previously proposed to account for propagation; the nature of this mechanism remains to be determined.     

Excitatory actions of motilin on myenteric neurons of the guinea-pig small intestine

Motilin is considered as a key factor in controlling interdigestive migrating contractions. The present electrophysiological experiments were performed in vitro to examine actions of motilin on myenteric neurons of guinea-pigs after 18-h fasting period. Superfusion of motilin depolarized both S and AH neurons; the lowest effective concentration was 10 nM, and motilin depolarization was observed in 9 of 23 S neurons and in 5 of 25 AH neurons. The motilin depolarizations were associated with an increase in neuronal input resistance. The motilin responses were preserved in Ca2+ free/high Mg2+ solution in which no Ca2+ dependent synaptic transmission occurred. The reversal potential of the motilin responses was estimated about -95 mV, close to the equilibrium potential for K+. Furthermore, muscarinic depolarizations were blocked during the motilin responses. All of these indicated that motilin directly excited myenteric neurons mainly by inactivating K+ channels. It is concluded that motilin might modulate neuronal excitability of the myenteric plexus, leading to the control of interdigestive migrating contractions.