Slow excitatory post-synaptic potentials in myenteric AH neurons of the guinea-pig ileum are reduced by the 5-hydroxytryptamine7 receptor antagonist SB 269970

Serotonin (5-HT) is a key modulator of neuronal excitability in the central and peripheral nervous system. In the enteric nervous system, 5-HT causes a slow depolarization in the intrinsic sensory neurons, but the receptor responsible for this has not been correlated with known gene products. The aim of this study was to determine whether the newly characterized 5-HT7 receptor may participate in the 5-HT-mediated depolarization of, and synaptic transmission to, the intrinsic sensory neurons of the guinea-pig ileum. Intracellular electrophysiological recordings were made from intrinsic sensory neurons identified as myenteric AH neurons from guinea-pig ileum. 5-HT (5 microM) applied to the cell body evoked both a fast depolarization (5-HT3 mediated) and/or a slow depolarization (5-HT1P-like). The 5-HT1/5/7 receptor agonist 5-carboxamidotryptamine (5-CT) (5 microM) evoked only a slow depolarization. When the fast depolarization evoked by 5-HT was blocked with granisetron (1 microM, 5-HT3 receptor antagonist), only a slow depolarization remained; this was abolished by the 5-HT7 receptor antagonist SB 269970 (1 microM, control: 14+/-2 mV, granisetron+SB 269970: -1+/-2 mV). The slow depolarization evoked by 5-CT was also significantly reduced by SB 269970 (control: 14+/-1 mV, SB 269970: 5+/-2 mV) suggesting a 5-HT7 receptor was activated by exogenous application of 5-CT and 5-HT. Slow excitatory postsynaptic potentials evoked by stimulating descending neural pathways (containing serotonergic fibers) were reduced by SB 269970 (control: 8+/-3 mV, SB 269970: 3+/-1 mV). However, SB 269970 had no effect on slow excitatory postsynaptic potentials evoked by stimulation of circumferential (tachykinergic) pathways (control: 7+/-1 mV, SB 269970: 6+/-1 mV). These data are consistent with the presence on enteric AH neurons of functional 5-HT7 receptors that participate in slow synaptic transmission.     

Slow synaptic potentials in AH-type myenteric plexus neurons

Two types of slow depolarization were recorded in AH-type guinea pig myenteric plexus neurons when the myenteric plexus-longitudinal muscle preparation was stimulated transmurally with external electrodes. One depolarization was associated with a fall and the other with a rise in membrane resistance, the latter type (slow EPSP) being encountered about six times more commonly than the former. In some instances both types of potential were recorded in the same AH neuron. When this occurred the amplitude and duration of the slow EPSP was attenuated if it was timed to occur at about the same time as the other slow synaptic potential.     

Slow hyperpolarizing action of tryptamine on myenteric neurons of the isolated guinea-pig ileum

In the present study, tryptamine produced a slow hyperpolarization in a few neurons other than a slow depolarization in myenteric neurons of the isolated guinea-pig ileum. Neither the adrenergic neuron blocker, guanethidine nor the 5-hydroxytryptamine uptake inhibitor, zimelidine, which can inhibit the release of 5-hydroxytryptamine from enteric neurites induced by tryptamine (M. Takaki et al. (1985) Neuroscience 16, 223-240), affected this slow hyperpolarization. Therefore, it was concluded that the slow hyperpolarization induced by tryptamine in myenteric neurons was not mediated via the release of 5-hydroxytryptamine or noradrenaline. It might be possible that the hyperpolarization was induced by a direct action of tryptamine on myenteric neurons per se.     

Facilitation of monosynaptic excitatory synaptic potentials in spinal motoneurones evoked by internuncial impulses

1. Descending tracts and primary afferent fibres were chronically degenerated in the lumbosacral cord of the cat, and attempts were made to evoke monosynaptic e.p.s.p.s in motoneurones by stimulation of interneurones with a pair of fine electrodes inserted into the cord.2. The reversal potential of monosynaptic e.p.s.p.s so produced was more negative than that measured for monosynaptic e.p.s.p.s produced by afferent impulses.3. Monosynaptic e.p.s.p.s evoked in motoneurones by internuncial impulses showed a significantly greater facilitation than those produced by afferent impulses.4. Monosynaptic e.p.s.p.s in a motoneurone produced by supramaximal intraspinal stimuli often revealed a fluctuation in amplitude. In such cases, when two successive stimuli were applied at a short interval, the mean amplitude of the second e.p.s.p.s was greater than that of the first e.p.s.p.s. This facilitation was associated with a decrease in the coefficient of variation of the e.p.s.p. amplitude fluctuation.5. The degree of facilitation of monosynaptic e.p.s.p.s evoked by internuncial impulses was not related to the amount of transmitter released by the preceding impulses.6. It is concluded that facilitation of monosynaptic e.p.s.p.s evoked by both afferent and internuncial impulses is based on the same mechanism and that the degree of facilitation of e.p.s.p.s is entirely determined by the nature of presynaptic elements.     

Significance of slow synaptic potentials for transmission of excitation in guinea-pig myenteric plexus

Intracellular recordings were made from neurones in myenteric ganglia of the guinea-pig ileum in vitro. Synaptic potentials were evoked by electrically stimulating presynaptic fibres as they entered the ganglion, using a small focal electrode. Slow synaptic depolarizations (excitatory postsynaptic potentials) were evoked in most myenteric neurones of both types. A single stimulus was more likely to evoke a slow excitatory postsynaptic potential in cells with nicotinic synaptic input (S cells; 50%) than in cells with long-lasting after-hyperpolarizations following the soma action potential (AH cells; 20%). Two pulses often evoked a slow excitatory postsynaptic potential in AH cells when one pulse was ineffective. The optimally effective time between the pulses was about 100 ms. Ten pulses resulted in slow excitatory postsynaptic potentials even when delivered at frequencies as low as 0.5 Hz. For the same frequency of presynaptic stimulation, the duration of the slow excitatory postsynaptic potential was greater in AH cells than in S cells and the amplitude of the slow excitatory postsynaptic potential was slightly greater in S than AH cells. Spontaneous depolarizations were observed which had time-courses and amplitudes similar to the evoked slow excitatory postsynaptic potential. They were not blocked by tetrodotoxin or atropine. The calcium-dependent after-hyperpolarization which follows one or more action potentials in AH cells was reduced or even abolished during the slow excitatory postsynaptic potential. Presynaptic nerve stimulation at intensities lower than those required to cause a slow excitatory postsynaptic potential caused a reduction in the calcium dependent after-hyperpolarization. It is concluded that the slow excitatory postsynaptic potential is generated by an intracellular intermediate process which is sensitive to the intracellular calcium concentration. The results suggest that the postsynaptic action of the synaptic transmitter is to interfere with the intracellular process which couples the entry of calcium to the increase in potassium conductance.     

Antagonism of non-cholinergic excitatory junction potentials in the guinea-pig ileum by a substance P analogue antagonist

In the presence of atropine electrical transmural stimulation (using repetitive volleys, e.g. 3 pulses at 50 Hz applied every 4 s) of full thickness longitudinal strips of guinea-pig ileum produced non-cholinergic excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) in the circular muscle layer. After abolition of the IJPs with apamin, the non-cholinergic EJPs clearly showed facilitation. In the presence of apamin and the substance P analogue antagonist, [D-Arg1,D-Pro2,D-Trp7-9,Leu11]-substance P (SPA), the non-cholinergic EJPs were reduced by 60-90%; transmural stimulation now revealed an apamin-resistant IJP followed by a slow depolarization. The atropine-resistant EJPs are probably caused by the release of substance P (or a similar compound) and are likely to underlie the non-cholinergic contractions reported to occur in this tissue.     

Two descending nerve pathways activated by distension of guinea-pig small intestine.

1. Intracellular recordings were made from myenteric neurones of the guinea-pig small intestine in preparations which had a synaptic input from an orally situated segment of intestine. 2. Excitatory synaptic potentials could be evoked in most neurones by distension of the attached intestinal segment. 3. It was possible to distinguish two distinct firing patterns of synaptic potentials in response to distension. A transient short latency discharge was recorded from some neurones. From the others, a persistent synaptic discharge was recorded only after a long latency (2-11 sec). 4. Distension of intestinal segments evoked short latency transient inhibitory junction potentials in the circular muscle layer followed by excitatory junction potentials in both the circular and longitudinal muscle layers. 5. It is suggested that distension may cause both descending inhibition and, after a delay, descending excitation of the guinea-pig small intestine.     

Intracellular recording from the myenteric plexus of the guinea-pig ileum

1. Ganglion cells of the myenteric plexus of the guinea-pig ileum have been studied with intracellular micro-electrodes.2. Three types of cell were distinguished. Type 1 cells had a high resistance (58 MOmega) and had properties similar to guinea-pig sympathetic ganglion cells. Type 2 cells were also excitable but had a lower resistance (21 MOmega) and showed accommodation to depolarizing current pulses. Type 3 cells were inexcitable.3. Point stimulation within 150 mum excited neurones either antidromically or orthodromically, sometimes both.4. Antidromic responses had a small all-or-nothing component which was subthreshold for the soma spike. Two or more such components sometimes occurred, and were probably due to stimulation of two or more cell processes.5. Excitatory post-synaptic potentials (e.p.s.p.s) were blocked by hexamethonium (400 muM). They progressively declined in amplitude when elicited at frequencies of 0.05 Hz or more, and this is discussed in relation to studies on acetylcholine (ACh) output.6. Many cells often showed a slow after-hyperpolarization following a direct or antidromic spike. Its mechanism and significance are discussed.7. Spontaneous e.p.s.p.s and spikes were occasionally seen.8. Intracellular injection of a fluorescent dye reveals that the neurones have one to seven processes, which usually arise from the poles of the oval soma.     

Projections and neurochemistry of interneurones in the myenteric plexus of the guinea-pig gastric corpus

Recently, motor neurones of the myenteric plexus innervating the muscle layers or the mucosa have been identified in the guinea-pig stomach. We applied the neuronal tracer DiI (1,1'-didodecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorat) onto myenteric ganglia in order to identify populations of interneurones in the myenteric plexus of the guinea-pig stomach. The tracing was combined with the immunohistochemical detection of calbindinD28k (CALB), choline acetyltransferase (ChAT), neuropeptide Y (NPY) and 5-hydroxytryptamine (serotonin) (5-HT) and the results were compared to the neurochemical coding of target specific motor neurones. Long projecting ( approximately 5.4 mm) ChAT/CALB/+/-5-HT-, nitric oxide synthase (NOS)/CALB- and short projecting ( approximately 1.1 mm) ChAT/NPY-neurones were identified as descending interneurones. CALB positive ascending interneurones contained ChAT but rarely 5-HT (code: ChAT/CALB). This study identified ascending and descending interneurones in the gastric myenteric plexus and revealed the neurochemical coding of some of the interneurone populations.     

Morphological studies of electrophysiologically-identified myenteric plexus neurons of the guinea-pig ileum

The morphology of neurons in the myenteric plexus of the guinea-pig ileum has been studied by means of the intracellular application of Procion Yellow. Sixty-six electrophysiologically-unidentified cells showed a great variety of soma shapes and number of processes, the vast majority of the longer of which were circumferentially-orientated.The electrophysiological properties of an additional 47 neurons were ascertained; 29 were neurons with a slow after-hyperpolarization (AH-neurons), 14 showed fast excitatory synaptic potentials (S-neurons) and 4 were of neither category.Twenty-two of the AH-neurons had a smooth soma outline and, on average, each had 5 processes, of which the great majority of long processes were circumferentially-orientated and intraganglionic. The projection ratios of oral:circumferential:aboral processes were 6:61:9. Branching was a prominent feature of the processes.In contrast, a large soma with many broad, short processes was a feature of 8 of 14 S-neurons studied. The average number of processes was 8.6 per cell and relatively more of them were aborally-directed, giving projection ratios of 2:21:7. There was, however, such a variation and overlap in the morphology of AH- and S-neurons that it was not possible to achieve a simple, reliable classification.It is concluded that many neuronal processes may be intraganglionic and that longitudinal ones are mainly aboral. From the varied morphological characteristics of AH-neurons, it is unlikely that these neurons subserve a single function in the plexus. For the same reasons S-neurons may fulfil different physiological roles.     

Endogenous GABA mediates presynaptic inhibition of spontaneous and evoked excitatory synaptic potentials in the rat neostriatum

The effect of the blockade of the gamma-aminobutyric acid (GABA) uptake system on the amplitude of glutamatergic synaptic potentials was studied by using a corticostriatal slice preparation. Nipecotic acid (0.1-1 mM), a GABA uptake blocker, produced a dose-dependent decrease of the amplitude of kynurenate-sensitive excitatory synaptic potentials recorded in the neostriatum following cortical stimulation. Nipecotic acid did not affect the postsynaptic responses to exogenously applied glutamate. The presynaptic effect of endogenous GABA was bicuculline-resistant and was mimicked by baclofen (0.3-3 microM). This effect was not blocked by phaclofen (0.5-1 mM). These findings show that phaclofen-insensitive GABAB receptors, activated by endogenous GABA, mediate presynaptic inhibition of cortical glutamatergic inputs in the neostriatum.     

Motilin inhibits ganglionic transmission in the myenteric plexus of the guinea-pig ileum

Motilin is a key factor in triggering interdigestive migrating contractions. Our preceding study demonstrated that motilin caused membrane depolarizations in a minority of S and AH neurons in the myenteric plexus of the guinea-pig ileum after 18 h-fasting period; motilin depolarizations were small and seldom triggered action potentials. Then, the present study was undertaken to examine possible electrophysiological actions of motilin on the ganglionic transmission in the myenteric plexus. Intracellular recordings with sharp glass microelectrodes were made from myenteric S neurons having fast excitatory postsynaptic potentials (EPSPs), evoked by focal electrical stimulation. Motilin inhibited the fast EPSPs in amplitude, associated either with or without membrane depolarizations. Results obtained with the paired stimulus method suggested that the site for motilin-induced inhibition of fast EPSPs might be presynaptic. Furthermore, motilin did not decrease postsynaptic sensitivity to ACh, a main neurotransmitter mediating the fast EPSPs. Therefore, it is concluded that motilin might inhibit presynaptically ganglionic transmission in the myenteric plexus of the guinea-pig ileum.     

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.     

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y₁ receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y₁ purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y₁ receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca²⁺ from intracellular stores.     

Naloxone-induced depolarization and synaptic activation of myenteric neurons in morphine-dependent guinea pig ileum

To investigate the cellular basis of opiate dependence, intracellular microelectrodes were used to record from both electrophysiologically defined classes of neurons (S and AH) in myenteric plexus longitudinal muscle preparations from morphine pretreated guinea pigs. These preparations responded to naloxone with the characteristic contraction of the longitudinal smooth muscle, indicative of morphine dependence. Depolarization in response to naloxone was observed in 42% of S neurons, but there were no consistent changes in input resistance. In some cells the depolarization was reduced or abolished after blockade of synaptic transmission, suggesting that it was due in part to the release of an excitatory transmitter producing a slow depolarization in the impaled neuron. Synaptic activation of S neurons during withdrawal was further indicated by the observation that fast postsynaptic potentials appeared after abrupt displacement of morphine from its receptors by naloxone. Morphine withdrawal, therefore, involves both the final motor neurons and interneurons. During naloxone-induced withdrawal, 25% of S neurons discharged action potentials. In contrast, no action potentials were discharged in AH neurons. Furthermore, naloxone did not alter the resting membrane potential, input resistance, soma action potential configuration, or slow hyperpolarization following a soma spike in AH neurons. The specificity of the withdrawal response for S neurons and the relatively small proportion of neurons involved suggests that morphine withdrawal occurs in quite specific neuronal circuits in the myenteric plexus.     

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1).Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.