Non-cholinergic Slow Excitatory Postsynaptic Potential Research Articles

Pursuit of Neurotransmitter Functions: Being Attracted with Fascination of the Synapse.

In the beginning of the 1970s, only two chemical substances, acetylcholine and γ-aminobutyric acid (GABA), had been definitely established as neurotransmitters. Under such circumstances, I started my scientific career in Professor Masanori Otsuka's lab searching for the transmitter of primary sensory neurons. Until 1976, lines of evidence had accumulated indicating that the undecapeptide substance P could be released as a transmitter from primary afferent fibers into spinal synapses, although the substance P-mediated synaptic response had yet to be identified. Peripheral synapses could serve as a good model and thus, it was demonstrated in the prevertebral sympathetic ganglia by1985 that substance P released from axon collaterals of primary sensory neurons acts as the transmitter mediating non-cholinergic slow excitatory postsynaptic potential (EPSP). At that time, we also found that autonomic synapses were useful to uncover the transmitter role of the opioid peptide enkephalins, whose functions had been unknown since their discovery in 1975. Accordingly, enkephalins were found to serve a transmitter role in mediating presynaptic inhibition of cholinergic fast and non-cholinergic slow transmission in the prevertebral sympathetic ganglia. In 1990s, we attempted to devise a combined technique of brain slices and patch-clamp recordings. We applied it to study the regulatory mechanisms that operate around cerebellar GABAergic inhibitory synapses, because most of the studies then had centered on excitatory synapses and because inhibitory synapses are crucially involved in brain functions and disorders. Consequently, we discovered novel forms of heterosynaptic interactions, dual actions of a single transmitter, and receptor crosstalk, the details of which are described in this review.

Neurokinin-1 and -3 receptor blockade inhibits slow excitatory synaptic transmission in myenteric neurons and reveals slow inhibitory input

Recent studies have shown that tachykinins mediate slow synaptic transmission to myenteric AH (afterhyperpolarising) neurons via neurokinin-3 receptors (NK 3R). This study investigated a similar role for neurokinin-1 receptors (NK 1R) and compared the effect of selective receptor antagonists on non-cholinergic slow excitatory post-synaptic potentials (EPSPs) recorded in myenteric AH neurons of the guinea-pig ileum. Slow EPSPs evoked by electrical stimulation of circumferentially oriented presynaptic nerves were mimicked by application of senktide, an NK 3R agonist. [Sar 9,Met(O 2) 11]-substance P, an NK 1R agonist, depolarised a smaller number of neurons. SR142801, a selective NK 3R antagonist (100 nM), inhibited slow EPSPs and responses to senktide, but had no effect on depolarisations evoked by forskolin, an activator of adenylate cyclase. SR140333, a selective NK 1R antagonist, inhibited slow EPSPs in a subset of neurons and blocked responses to [Sar 9,Met(O 2) 11]-substance P, but not to senktide or forskolin. Slow EPSPs that were predominantly mediated by NK 1R had significantly shorter latencies than those due to activation of NK 3R. After blockade of slow EPSPs, slow hyperpolarizing responses to presynaptic nerve stimulation were revealed in one-third of neurons. These events, which were associated with a decrease in input resistance and blocked by tetrodotoxin, were equated with slow inhibitory postsynaptic potentials. They were abolished by the 5-hydroxytryptamine 1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190), but unaffected by phentolamine, an α-adrenoceptor antagonist. In conclusion, these results provide the first direct evidence that NK 1R mediate some slow excitatory synaptic input to myenteric AH neurons, and suggest that NK 1R and NK 3R activate distinct signal transduction pathways. These results also demonstrate that slow inhibitory synaptic transmission, which may be mediated by 5-hydroxytryptamine, is more prevalent in the myenteric plexus than previously indicated.

Effects of 5-HT 1A and 5-HT 4 receptor agonists on slow synaptic potentials in enteric neurons

Intracellular electrophysiological methods were used to examine the effects of 5-hydroxytryptamine (5-HT), 5-carboxa-midotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), 4-amino-5-chloro-2-methoxy- N-(4-[1-azabicyclo3,3,1]nonyl]) benzamide hydrochloride (renzapride), cis-4-amino-5-chloro- N[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidiny]-2-methoxybenzamide monohydrate (cisapride) and endo- N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-3-(1-methyl)ethyl-2-oxo-1H-benzimidazole-1-carboxamidehydrochloride (BIMU 8) on noncholinergic slow excitatory postsynaptic potentials (slow EPSPs) in myenteric afterhyperpolarization (AH) neurons of guinea pig ileum. 5-HT (0.01–1 μM) and 5-CT (0.001–0.1 μM) produced a concentration-dependent inhibition of slow EPSPs. The 5-HT 1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimidobutyl]piperazine (NAN-190) produced rightward shifts in 5-HT and 5-CT concentration-response curves; facilitation of slow EPSPs was never observed. 5-MeOT caused a depolarization and inhibited spike afterhyperpolarizations in a concentration-dependent manner but this effect was not blocked by the 5- HT 3 5- HT 4 receptor antagonist, tropisetron (1 μM). Renzapride (0.01–0.3 μM), cisapride (0.01–1.0 μM) and BIMU 8 (0.01–1.0 μM) did not change the membrane potential of any neuron tested. Renzapride and BIMU 8 did not change the amplitude of slow EPSPs. In 13 of 19 neurons cisapride did not change the amplitude of slow EPSPs; in 6 neurons cisapride (1 μM) reversibly inhibited the slow EPSP. Responses to substance P which mimicked the slow EPSP were not affected by cisapride. These data indicate that excitatory 5-HT 4 receptors are not localized to cell bodies of AH neurons and are not coupled to presynaptic facilitation of noncholinergic slow EPSPs in AH neurons.

Actions of nitric oxide-generating sodium nitroprusside in myenteric plexus of guinea pig small intestine

Sodium nitroprusside (NaNP) was used as a donor of nitric oxide (NO) to investigate actions of NO on electrical and synaptic behavior of single myenteric neurons in guinea pig small intestine. NaNP (10 microM-1 mM) did not affect resting membrane properties of the neurons, except for an occasional decrease in input resistance and hyperpolarization attributable to suppression of excitatory transmitter release. NaNP did not alter fast nicotinic neurotransmission but suppressed noncholinergic slow excitatory postsynaptic potentials (slow EPSPs) in a concentration-dependent manner. Pretreatment with either methylene blue or oxyhemoglobin reduced the inhibitory action of NaNP on the slow EPSPs. Slow EPSP-like responses to microejected substance P or 5-hydroxytryptamine were unaffected by NaNP. The nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, did not affect resting membrane excitability or excitatory synaptic events in any of the myenteric neurons. The results suggest that NO may not be released extensively as a neurotransmitter at synapses within the myenteric plexus. If myenteric neurons are exposed to NO released from nonneural sources, then the principal action is expected to be presynaptic inhibition of slow synaptic excitation.

Non‐cholinergic transmission in a sympathetic ganglion of the guinea‐pig elicited by colon distension.

Sensory transmission from the colon was studied using a preparation of inferior mesenteric ganglion (i.m.g.) attached to a segment of distal colon in guinea-pigs, in vitro. Electrical responses to colon distension were recorded intracellularly from neurones of the i.m.g. Distension of the distal colon up to an intraluminal pressure of 20 cmH2O caused an increase in resting asynchronous synaptic activity and a concomitant slow depolarization. The asynchronous synaptic activity, but not the slow depolarization, was abolished by cholinergic antagonists. Distension-induced non-cholinergic depolarizations were elicited in 44% of i.m.g. neurons sampled. For distensions of 1 min at 10-20 cmH2O, depolarizations reached a mean amplitude of 3.4 +/- 0.3 mV and lasted 108 +/- 7 s. Continuous distension resulted in a tachyphylaxis of the depolarization. Tetrodotoxin (3 X 10(-7) M) superfused over the i.m.g. reversibly abolished the distension-induced non-cholinergic depolarization. Distension-induced non-cholinergic depolarizations were accompanied by an increase in input resistance of 21%. Neuronal excitability also increased, as sub-threshold potentials produced by intracellular current injection reached threshold for firing action potentials during colon distension. The amplitude of non-cholinergic depolarizations increased with colonic intraluminal pressure between 2 and 20 cmH2O, although the slope of the mean amplitude-pressure curve decreased progressively at higher pressures. The amplitude of distension-induced non-cholinergic depolarizations increased as membrane potential was manually hyperpolarized to approximately -80 mV, whereupon further hyperpolarization resulted in a decrease in response amplitude. Non-cholinergic slow excitatory post-synaptic potentials (e.p.s.p.s) evoked by repetitive presynaptic nerve stimulation were reversibly attenuated by 19 +/- 8% during depolarizations produced by distension. Systemic administration of capsaicin (50-350 mg/kg) reduced the number of i.m.g. neurones exhibiting the non-cholinergic mechanosensory response; direct superfusion of capsaicin over the i.m.g. attenuated the response in some neurones but had no effect in others. These results demonstrate the existence of a non-cholinergic mechanosensory pathway from the colon to the i.m.g., and suggest that non-cholinergic transmission in the ganglion participates in mediating gastrointestinal reflexes. One transmitter utilized by the non-cholinergic mechanosensory pathway may be substance P.

Properties of choroid and ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter

Intracellular recordings were made from identified choroid and ciliary neurons in the ciliary ganglion of the embryonic chick. Choroid neurons, which were innervated by multiple preganglionic fibers, frequently displayed noncholinergic slow excitatory postsynaptic potentials (EPSPs) following repetitive stimulation of the preganglionic nerve trunk. These slow potentials were blocked by high Mg2+/low Ca2+ buffer and were closely mimicked by bath application of substance P, which is known to be present within both populations of preganglionic nerve terminals. Substance P-induced depolarizations desensitized during prolonged exposure, at which time it was no longer possible to evoke slow synaptic potentials. Following manual voltage clamp to resting membrane potential, parallel increases in input resistance were seen during the slow EPSP and the response to substance P, suggesting that the two responses share common mechanisms. Ciliary neurons, which were innervated by a single preganglionic fiber and displayed dual electrical-chemical synapses, did not exhibit slow synaptic potentials and were unaffected by bath application of substance P. The magnitude and time course of fast nicotinic EPSPs elicited in ciliary neurons by 0.5 Hz presynaptic stimulation were also unchanged in the presence of 1 to 3 microM substance P. Although the ciliary and choroid neurons share a common embryological origin in the neural crest, they are specialized for quite different physiological roles. Integration of multiple presynaptic inputs occurs at choroid synapses, mediated by the presence of both subthreshold fast nicotinic EPSPs and the slow EPSP. In contrast, synapses on ciliary neurons have specializations which preclude any integrative function, including single innervation, a high quantal content, electrical coupling potentials, and a lack of slow synaptic potentials.

An intracellular study of synaptic transmission and dendritic morphology in sympathetic neurons of the chick embryo

The characteristics of synaptic transmission in whole embryonic avian sympathetic ganglia have been examined by intracellular recording. Neurons in lumbar paravertebral ganglia of chick embryos exhibit both fast nicotinic excitatory postsynaptic potentials (EPSPs) and non-cholinergic slow EPSPs. Fast nicotinic transmission is mediated by at least 3-5 convergent preganglionic inputs and can be detected at the earliest embryonic stage examined (Stage 38; 12 days of incubation). Two types of non-cholinergic slow EPSPs have been observed and distinguished by their time course and the resulting changes in input resistance. One of these slow synaptic potentials is mimicked by exogenously applied substance P, but not by exogenous luteinizing hormone-releasing hormone (LH-RH). Muscarinic agonists also evoke slow depolarizations in the ganglia, mediated at least in part by inhibition of the M-current. Intracellular labeling with horseradish peroxidase reveals cells with 5-10 primary dendrites at Stage 42 (16 days of incubation), the earliest stage to exhibit slow EPSPs. The active and passive membrane properties of avian sympathetic neurons, including the presence of the M-current, generally resemble those of adult mammalian and amphibian sympathetic neurons. Functional activity in chick sympathetic neurons is present at a developmental stage where both biochemical and morphological indices of synapse maturation are at low levels. Since this progression has also been observed in the avian ciliary ganglion, it may be of general relevance to neuronal development.

Blockade of slow excitatory post-synaptic potential by substance P antagonists in guinea-pig sympathetic ganglia.

The effects of three substance P (SP) antagonists on the inferior mesenteric ganglion of the guinea-pig were studied using intracellular recording techniques, and the possible role of SP as a transmitter for the non-cholinergic slow excitatory post-synaptic potential (e.p.s.p.) was examined. The SP antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP, exerted a depolarizing action on the ganglion cells when applied by perfusion at a concentration of 3-16 microM or by pressure ejection from a micropipette. This depolarizing action is probably due to a release of endogenous histamine because it was abolished by treatment with a histamine antagonist, mepyramine (1-3 microM), or by a repeated application of the antagonist. When applied by pressure ejection, SP at 0.5-1 microM depolarized the ganglion cells. In the presence of mepyramine, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP suppressed the SP-induced depolarization by 41% at a concentration of 8 microM and by 75% at 16 microM. By contrast the SP antagonist did not affect the depolarizing action of angiotensin II on the ganglion cells. The non-cholinergic slow e.p.s.p. evoked in the ganglion cells by repetitive stimulation of the lumbar splanchnic nerves was suppressed by [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP at 8 or 16 microM. The degrees of suppression of both the non-cholinergic slow e.p.s.p. and the SP-induced depolarization by the SP antagonist were approximately equal. The cholinergic fast e.p.s.p. evoked by preganglionic nerve stimulation was not affected by the SP antagonist. [D-Pro2, D-Trp7,9]SP exhibited the properties of an SP antagonist similar to, but slightly weaker than [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP. [D-Pro2, D-Phe7, D-Trp9] at a concentration of 16 microM had a depolarizing action on the ganglion cells, which was not blocked by mepyramine. The peptide exerted hardly any antagonistic action against the SP-induced depolarization of the ganglion cells. Stimulation of the other preganglionic (intermesenteric) nerves and the post-ganglionic (colonic and hypogastric) nerves produced a non-cholinergic slow e.p.s.p. in the inferior mesenteric ganglion cells. The non-cholinergic slow e.p.s.p. evoked by both pre- and post-ganglionic nerve stimulation were depressed by [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP to similar extents. The present results show that [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP and [D-Pro2, D-Trp7,9]SP can serve as specific SP antagonists in the inferior mesenteric ganglion of the guinea-pig.(ABSTRACT TRUNCATED AT 400 WORDS)

A slow EPSP in mammalian inferior mesenteric ganglion persists after in vivo capsaicin

The non-cholinergic slow excitatory postsynaptic potential (EPSP) in the guinea pig inferior mesenteric ganglion (IMG) is believed to be mediated by substance P (SP). Yet, in ganglia of guinea pigs treated with capsaicin (50, 100, or 350 mg/kg, s.c.), a compound that depletes SP from sensory neurons, slow EPSPs were only 50% smaller in amplitude than those in vehicle-treated controls; this was true at all dosages. When ganglia from capsaicin-treated animals were desensitized to SP, slow EPSP amplitude was decreased by 50% from predesensitization values. These data indicate that capsaicin does not eliminate the slow EPSP in the IIMG and suggest that both SP and another transmitter are involved in co-mediating this response.

Substance P as an excitatory transmitter of primary afferent neurons in guinea-pig sympathetic ganglia

Electrophysiological and neurochemical experiments were carried out to examine a possible transmitter role of substance P in the prevertebral ganglia of the guinea-pig. When potentials were recorded intracellularly from neurons of the isolated ganglia, stimulation of the pre- or postganglionic nerves elicited a non-cholinergic slow excitatory postsynaptic potential (EPSP). This synaptic potential was compared with the effects of substance P. Brief application of substance P caused a depolarization of the ganglion cells with a similar time course to that of the non-cholinergic slow EPSP. Changes in membrane resistance during the substance P-induced depolarization resembled those associated with the non-cholinergic slow EPSP. During the substance P-induced depolarization the non-cholinergic slow EPSP was markedly depressed. Attempts were made to determine the origin of the fibers eliciting the non-cholinergic slow EPSP. In the inferior mesenteric ganglia isolated together with preganglionic nerves that retained intact connections with spinal nerve roots, dorsal root stimulation evoked a non-cholinergic slow EPSP but not a cholinergic fast EPSP in the ganglion cells, whereas ventral root stimulation caused only cholinergic fast EPSPs. Following the prolonged treatment with capsaicin, the non-cholinergic slow EPSP was greatly depressed or abolished. Radioimmunoassay revealed that after ligation or section of pre- or postganglionic nerves an accumulation of substance P occurred in the proximal stumps of the interrupted nerves. Stimulation with high potassium medium evoked a release of immunoreactive substance P from the prevertebral ganglia and the release was calcium-dependent. The present findings suggest that axon collaterals of certain visceral primary afferents form synapses with principal cells in the prevertebral ganglia and release substance P as a transmitter for the noncholinergic slow EPSP.

Role of substance P as a sensory transmitter in spinal cord and sympathetic ganglia.

The hypothesis that substance P (SP) might be a transmitter of primary sensory neurons was first proposed by Lembeck in 1953. A large amount of evidence supporting this hypothesis has recently accumulated, particularly since the elucidation of the chemical structure of SP by Leeman and her colleagues in 1971, which made a number of new approaches possible (e.g. radioimmunoassay for SP, immunohistochemistry and electrophysiological tests of SP action on central and peripheral neurons). SP is concentrated in certain primary afferent terminals in the spinal cord, is released therefrom when the dorsal roots are electrically stimulated, and exerts a powerful excitant action on spinal neurons. It is therefore likely that SP produces excitatory postsynaptic potentials (EPSPs) in spinal neurons, although the characteristics of SP-mediated EPSPs, i.e. their time course, ionic mechanisms, etc., remain to be revealed. Recent electrophysiological and neurochemical studies on the prevertebral ganglia of the guinea-pig strongly suggest that SP is released from axon collaterals of visceral primary afferent neurons in the ganglia and serves as a transmitter that generates non-cholinergic slow EPSPs in principal cells. There is evidence that this SP-mediated synaptic transmission in the sympathetic ganglia is under the influence of enkephalinergic presynaptic inhibition. Some preliminary experiments on the interaction between SP and enkephalins in the spinal cord are described.