Fast Excitatory Postsynaptic Potentials Research Articles

Computational simulations and Ca2+ imaging reveal that slow synaptic depolarizations (slow EPSPs) inhibit fast EPSP evoked action potentials for most of their time course in enteric neurons.

Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20-50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABAA or GABAC receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABAA or GABAC ligand-gated Cl- channels to explore these interactions. Model predictions were tested by imaging Ca2+ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca2+ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABAA receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABAC receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7±2.4% of all submucosal neurons (n=6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200µM) reduced nerve-evoked chloride secretion by 34.3±18.6% (n=14 patients), whereas D-APV had no effect. Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

The inhibition of evoked excitatory postsynaptic potentials produced by ammonium chloride in rat hippocampal CA1 neurons

The depression of evoked fast excitatory postsynaptic potentials (EPSPs) following superfusion with various concentrations (3 μM-5 mM) of ammonium chloride (NH4Cl) were investigated in rat hippocampal CA1 neurons. The amplitude of the evoked fast EPSPs decreased by NH4Cl in a concentration-dependent manner. The half-maximal inhibitory concentration for the inhibition of evoked fast EPSPs was 198 ± 125 μM (n = 8). The facilitation of a pair of field EPSPs elicited by paired-pulse stimulation (40-ms interval) (paired-pulse facilitation, PPF) was recorded following superfusion with NH4Cl (200 μM and 3 mM). The PPF ratio increased to 180 ± 23% (n = 9) in the presence of 200 μM NH4Cl compared with that in the absence of NH4Cl (142 ± 24%, n = 9). In the presence of 3 mM NH4Cl, the PPF ratio increased to 172 ± 30% (n = 7) compared with that in the absence of NH4Cl (126 ± 13%, n = 7). This implies that NH4Cl suppressed the presynaptic release of glutamate. Exogenous glutamate- or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced depolarization elicited by using pressure application did not reduce following superfusion with 200 μM or 5 mM NH4Cl in the presence of 0.3 μM tetrodotoxin, suggesting that NH4Cl did not affect the postsynaptic glutamate response. Action potentials elicited by rectangular outward current injection from CA3 neurons projecting to CA1 neurons were persistent at 200 μM NH4Cl but disappeared at 5 mM NH4Cl. The abolishment of action potentials in the presence of 5 mM NH4Cl was released by increasing the amplitude of the injection current. These results suggest that NH4Cl depresses evoked fast EPSPs mainly via a presynaptic mechanism at low NH4Cl concentrations, and the failure of action potential propagation through the excitatory nerve may also contribute to the depression of evoked fast EPSPs at high NH4Cl concentrations.

Resveratrol derivative excited postsynaptic potentiation specifically via PKCβ-NMDA receptor mediation

Several decades have passed since resveratrol (RSV) was first identified in red wine. Researchers have reported the pleiotropic anti-oxidant, anti-inflammatory, anti-cancer, anti-aging, and neuronal protective effects of resveratrol and its glycosylated derivative. However, few studies have distinguished the minute differences in the properties between resveratrol and its glycosylated derivative in terms of synaptic plasticity. As an abundant natural product of glycosylated resveratrol, the derivative 2,3,4',5-tetrahydroxystilbene-2-O-β-d-glucoside (TSG) has been determined to be a better option for long-term potentiation (LTP) in the hippocampus under physiological and pathological conditions than resveratrol. TSG, as well as its parent molecule RSV, could elicit early-LTP and recover fast excitatory postsynaptic potentials (EPSPs) in the hippocampus. Using various modalities, including pre- and post-whole-cell patch clamping techniques in the calyx of Held, pharmacological inhibition of the N-methyl-d-aspartic acid receptor (NMDAr) and the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAr) as well as protein kinase C (PKC) activation, we demonstrated that TSG, unlike RSV, could merely promote NMDA-mediated EPSC via PKCβ cascade. Our results provide new knowledge that glycosylation of resveratrol could significantly improve its specificity in promoting sole NMDAr mediation of EPSPs, in addition to improving solubility and resistance against oxidation in vivo. These observations could contribute to further exploration of pharmaceutical evaluation of glycosylated stilbene in the future.

Cholinergic Submucosal Neurons Display Increased Excitability Following in Vivo Cholera Toxin Exposure in Mouse Ileum.

Cholera-induced hypersecretion causes dehydration and death if untreated. Cholera toxin (CT) partly acts via the enteric nervous system (ENS) and induces long-lasting changes to enteric neuronal excitability following initial exposure, but the specific circuitry involved remains unclear. We examined this by first incubating CT or saline (control) in mouse ileal loops in vivo for 3.5 h and then assessed neuronal excitability in vitro using Ca2+ imaging and immunolabeling for the activity-dependent markers cFos and pCREB. Mice from a C57BL6 background, including Wnt1-Cre;R26R-GCaMP3 mice which express the fluorescent Ca2+ indicator GCaMP3 in its ENS, were used. Ca2+-imaging using this mouse model is a robust, high-throughput method which allowed us to examine the activity of numerous enteric neurons simultaneously and post-hoc immunohistochemistry enabled the neurochemical identification of the active neurons. Together, this provided novel insight into the CT-affected circuitry that was previously impossible to attain at such an accelerated pace. Ussing chamber measurements of electrogenic ion secretion showed that CT-treated preparations had higher basal secretion than controls. Recordings of Ca2+ activity from the submucous plexus showed that increased numbers of neurons were spontaneously active in CT-incubated tissue (control: 4/149; CT: 32/160; Fisher's exact test, P < 0.0001) and that cholinergic neurons were more responsive to electrical (single pulse and train of 20 pulses) or nicotinic (1,1-dimethyl-4-phenylpiperazinium (DMPP; 10 μM) stimulation. Expression of the neuronal activity marker, pCREB, was also increased in the CT-treated submucous plexus neurons. c-Fos expression and spontaneous fast excitatory postsynaptic potentials (EPSPs), recorded by intracellular electrodes, were increased by CT exposure in a small subset of myenteric neurons. However, the effect of CT on the myenteric plexus is less clear as spontaneous Ca2+ activity and electrical- or nicotinic-evoked Ca2+ responses were reduced. Thus, in a model where CT exposure evokes hypersecretion, we observed sustained activation of cholinergic secretomotor neuron activity in the submucous plexus, pointing to involvement of these neurons in the overall response to CT.

Hyperexcitability in synaptic and firing activities of spinal motoneurons in an adult mouse model of amyotrophic lateral sclerosis

Hyperexcitability is hypothesized to contribute to the degeneration of spinal motoneurons (MNs) in amyotrophic lateral sclerosis (ALS). Studies, thus far, have not linked hyperexcitability to the intrinsic properties of MNs in the adult ALS mouse model with the G93A-mutated SOD1 protein (mSOD1G93A). In this study, we obtained two types of measurements: ventral root recordings to assess motor output and intracellular recordings to assess synaptic properties of individual MNs. All studies were carried out in an in vitro preparation of the sacral spinal cords of mSOD1G93A mice and their non-transgenic (NT) littermates, both in the age range of 50–90days. Ventral root recordings revealed that maximum compound action potentials (coAPs) evoked by a short-train stimulation of corresponding dorsal roots were similar between the two types of mice. Although the progressive depression of coAPs was present during the train stimulation in all recordings, the coAP depression in mSOD1G93A mice was to a lesser extent, which suggests an increased firing tendency in mSOD1G93A MNs. Intracellular recordings showed no changes in fast excitatory postsynaptic potentials (EPSPs) in mSOD1G93A MNs. However, recording did show that oscillating EPSPs (oEPSPs) were induced by poly-EPSPs at a higher frequency and by less-intense electrical stimulation in mSOD1G93A MNs. These oEPSPs were dependent upon the activities of spinal network and N-methyl-d-aspartate receptors (NMDARs), and were subjected to riluzole modulation. Taken together, these findings revealed abnormal electrophysiology in mSOD1G93A MNs that could underlie ALS excitotoxicity.

Differential effects of the histamine H(3) receptor agonist methimepip on dentate granule cell excitability, paired-pulse plasticity and long-term potentiation in prenatal alcohol-exposed rats.

We previously reported that prenatal alcohol-induced deficits in dentate gyrus (DG) long-term potentiation (LTP) are ameliorated by the histamine H3 receptor inverse agonist ABT-239. ABT-239 did not enhance LTP in control rats, suggesting that the possibility of a heightened H3 receptor-mediated inhibition of LTP in prenatal alcohol-exposed (PAE) offspring. To further investigate this mechanism, we examined the effect of methimepip, a selective histamine H3 receptor agonist, on DG granule cell responses and LTP in saccharin control and PAE rats. Long-Evans rat dams voluntarily consumed either a 0 or 5% ethanol solution 4 hours each day throughout gestation. Adult male offspring from these dams were anesthetized with urethane and electrodes implanted into the entorhinal cortical perforant path and the DG. In control offspring, methimepip reduced the coupling of fast excitatory postsynaptic field potentials to population spikes (E-S coupling), the probability of glutamate release, as measured by paired-pulse ratio (PPR) and diminished DG LTP. Similar reductions in E-S coupling and LTP were observed in saline-treated PAE offspring. In contrast to the control group, methimepip did not exacerbate PAE-induced reductions in E-S coupling or LTP. While the effects of methimepip in control offspring were consistent with speculation of a PAE-induced enhancement of H3 receptor-mediated inhibition of E-S coupling and LTP, the absence of an added effect of methimepip in PAE offspring could indicate either an inability to further inhibit these responses with methimepip in PAE rats or the presence of more complex regulatory neural interactions with in vivo recordings in PAE rats. Follow-up studies of H3 receptor-mediated responses in DG slice preparations are under way to provide clearer insights into the role of the H3 receptor regulation of excitatory transmission in PAE rats.

Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of guinea-pig colon

Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with 1,1′-didodecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (DiI) from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of the guinea-pig distal colon (39 impalements, n=14). Thirty-eight viscerofugal neurons were uni-axonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single- or multiple action potentials in viscerofugal neurons (range 1–25 spikes, 500ms, 100–900pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast excitatory postsynaptic potentials (EPSPs) in 19/24 cells. Focal pressure-ejection of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10μm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23±10mV, latency 350±230ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300±316ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1±4.3mV vs. 10.1±4.8mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were nitric oxide synthase (NOS) immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.

Mathematical modelling of enteric neural motor patterns

1. The enteric nervous system modulates intestinal behaviours, such as motor patterns and secretion. Although much is known about different types of neurons and simple reflexes in the intestine, it remains unclear how complex behaviours are generated. 2. Mathematical modelling is an important tool for assisting the understanding of how the neurons and reflexes can be pieced together to generate intestinal behaviours. 3. Models have identified a functional role for slow excitatory post-synaptic potentials (EPSPs) by distinguishing between fast and slow EPSPs in the ascending excitation reflex. These models also discovered coordinated firing of similarly located neurons as emergent properties of feed-forward networks of interneurons in the intestine. A model of the recurrent network of intrinsic sensory neurons identified important control mechanisms to prevent uncontrolled firing due to positive feedback and that the interaction between these control mechanisms and slow EPSPs is necessary for the networks to encode ongoing sensory stimuli. This model also showed that such networks may mediate migrating motor complexes. 4. A network model of vasoactive intestinal peptide neurons in the submucosal plexus found this relatively sparse recurrent network could produce uncontrolled firing under conditions that appear to be related to cholera toxin-induced hypersecretion. 5. Abstract modelling of the intestinal fed-state motor patterns has identified how stationary contractions can arise from a polarized network. 6. These models have also helped predict and/or explained pharmacological evidence for two rhythm generators and the requirement of feedback from contractions in the circular muscle.

Properties of cholinergic and non‐cholinergic submucosal neurons along the mouse colon

Submucosal neurons are vital regulators of water and electrolyte secretion and local blood flow in the gut. Due to the availability of transgenic models for enteric neuropathies, the mouse has emerged as the research model of choice, but much is still unknown about the murine submucosal plexus. The progeny of choline acetyltransferase (ChAT)-Cre × ROSA26(YFP) reporter mice, ChAT-Cre;R26R-yellow fluorescent protein (YFP) mice, express YFP in every neuron that has ever expressed ChAT. With the aid of the robust YFP staining in these mice, we correlated the neurochemistry, morphology and electrophysiology of submucosal neurons in distal colon. We also examined whether there are differences in neurochemistry along the colon and in neurally mediated vectorial ion transport between the proximal and distal colon. All YFP(+) submucosal neurons also contained ChAT. Two main neurochemical but not electrophysiological groups of neurons were identified: cholinergic (containing ChAT) or non-cholinergic. The vast majority of neurons in the middle and distal colon were non-cholinergic but contained vasoactive intestinal peptide. In the distal colon, non-cholinergic neurons had one or two axons, whereas the cholinergic neurons examined had only one axon. All submucosal neurons exhibited S-type electrophysiology, shown by the lack of long after-hyperpolarizing potentials following their action potentials and fast excitatory postsynaptic potentials (EPSPs). Fast EPSPs were predominantly nicotinic, and somatic action potentials were mediated by tetrodotoxin-resistant voltage-gated channels. The size of submucosal ganglia decreased but the proportion of cholinergic neurons increased distally along the colon. The distal colon had a significantly larger nicotinic ion transport response than the proximal colon. This work shows that the properties of murine submucosal neurons and their control of epithelial ion transport differ between colonic regions. There are several key differences between the murine submucous plexus and that of other animals, including a lack of conventional intrinsic sensory neurons, which suggests there is an incomplete neuronal circuitry within the murine submucous plexus.

Rapid Effects of Oestrogen on Synaptic Plasticity: Interactions with Actin and Its Signalling Proteins

Oestrogen rapidly enhances fast excitatory postsynaptic potentials, facilitates long-term potentiation (LTP) and increases spine numbers. Each effect likely contributes to the influence of the steroid on cognition and memory. In the present review, we first describe a model for the substrates of LTP that includes an outline of the synaptic events occurring during induction, expression and consolidation. Briefly, critical signalling pathways involving the small GTPases RhoA and Rac/Cdc42 are activated by theta burst-induced calcium influx and initiate actin filament assembly via phosphorylation (inactivation) of cofilin. Reorganisation of the actin cytoskeleton changes spine and synapse morphology, resulting in increased concentrations of AMPA receptors at stimulated contacts. We then use the synaptic model to develop a specific hypothesis about how oestrogen affects both baseline transmission and plasticity. Brief infusions of 17β-oestradiol (E2 ) reversibly stimulate the RhoA, cofilin phosphorylation and actin polymerisation cascade of the LTP machinery; blocking this eliminates the effects of the steroid on transmission. We accordingly propose that E2 induces a weak form of LTP and thereby increases synaptic responses, a hypothesis that also accounts for how it markedly enhances theta burst induced potentiation. Although the effects of E2 on the cytoskeleton could be a result of the direct activation of small GTPases by oestrogen receptors on the synaptic membrane, the hormone also activates tropomyosin-related kinase B receptors for brain-derived neurotrophic factor, a neurotrophin that engages the RhoA-cofilin sequence and promotes LTP. The latter observations raise the possibility that E2 produces its effects on synaptic physiology via transactivation of neighbouring receptors that have prominent roles in the management of spine actin, synaptic physiology and plasticity.

Obesity and plasticity: how your gut learns to deal with your diet

Today you may be eating a sugar- and fat-rich diet, but tomorrow you may become a strict vegetarian who's also anti-sugar. These dramatic dietary changes are a challenge to your body. Your intestine needs to continue to harvest nutrients to maintain health, but is using outdated methods to do so. Much of the recent research has been on the important role of gut bacteria (Lupp et al. 2012), and how they respond to diet in a way that may benefit the host. However, new evidence published in The Journal of Physiology (Reichardt et al. 2013) suggests that our gastrointestinal tracts are not as helpless as we may think, but can adapt quickly and efficiently to dietary challenges. A defining feature of the gastrointestinal tract is the enteric nervous system (ENS), a rich and complex network of nerves in its wall that is often called the ‘little brain’ or ‘second brain’. At the turn of the last century, John Langley defined the ENS as the third division of the autonomic nervous system. Since then, many studies have found the ENS to be unlike other peripheral nerves and more closely related to the CNS. It has been difficult to link enteric neuronal behaviour with whole organ behaviour. The nerve circuits in the gut are complex, and the numbers and types of neurons make traditional research methods – like intracellular electrophysiological approaches – difficult, time-consuming and prone to sampling errors. New data from Reichardt et al. (2013) have overcome this problem by using a voltage-sensitive dye to record action potentials from many neurons at once. This has allowed them to quickly gather daa about the electrophysiological properties of a wide range of ENS neuronal phenotypes. That the small intestine responds rapidly to nutrients has been known for some time. When rodent models are fed a high-sugar diet, sugar transporters (SGLT1 and GLUT2) are rapidly up-regulated – an effect which is under the control of intestinal sweet taste receptors. In people with diabetes, faulty regulation can cause a sustained up-regulation of sweet taste receptors and of glucose absorption (Young et al. 2013). Now information is coming to light that the behaviour of enteric neurons is also very sensitive to feeding state. Recently, Baudry et al. (2011) showed that a Western diet, high in fat and simple sugars, had a neuroprotective effect on nitric oxide-producing neurons in mouse gastric antrum, which was coupled with increased gastric emptying. Similarly, Roosen et al. (2012) used calcium imaging to show that enteric neurons from fasted and then fed guinea pigs were more sensitive to serotonin but less sensitive to ghrelin versus a fasted-only group. Reichardt et al. (2013) took this line of research one step further showing that a high-fat diet enriched with simple sugars can change how the ENS processes information – in effect reprogramming the ENS. Using a voltage-sensitive dye technique, they have shown that the neurons of mice fed a Western diet are more sensitive to neurotransmitters that normally mediate enteric fast excitatory postsynaptic potentials than those of mice fed a standard chow. Acetylcholine and serotonin participate in fast synaptic transmission via nicotinic and 5-HT3 receptors, respectively. The authors found a strong correlation between body weight and the numbers of neurons responding to a nicotinic and 5-HT3 receptor agonist, or to the tissue availability of the endogenous neurotransmitters. Previously, changes in neurotransmission have been linked with gut inflammation, but the neuronal changes in this paper occurred without inflammation or increased mucosal permeability. This reprogramming translated directly into faster colonic transit in mice fed the Western diet for 12 weeks but not in those on the diet for 4 weeks, suggesting that the neuronal changes take time to develop. A key follow-up study will be to examine in detail which enteric neurons became sensitised and whether this can be prevented by dietary or lifestyle changes. The gut is equipped to extract all of the nutrients the body needs from a meal. In times of food scarcity this is a real benefit, but in times of plenty this system may extract too many nutrients. Stopping the gut from increasing its absorptive capacity may be a way to reduce excess nutrient absorption (similar to the action of lipase inhibitors). In fact, the ENS is likely to be smart enough to ignore very large nutrient loads altogether, but reprogramming may prove difficult with a lifetime of experience to work against.

NGX426, an Oral AMPA-Kainate Antagonist, Is Effective in Human Capsaicin-Induced Pain and Hyperalgesia

Non-N-methyl-D-aspartate receptor subtypes modulate neurotransmitter release and mediate fast excitatory postsynaptic potentials. This study evaluated the effects of an oral prodrug to tezampanel, a selective α-amino-3-hydroxy-5-methly-4-isoxazole-proprionic acid/kainate receptor antagonist, on intradermal capsaicin-induced pain and hyperalgesia. This was a randomized, double blind, crossover, placebo-controlled study. Eighteen subjects received 150 or 90 mg NGX426, or placebo, separated by a washout of 6 ± 2 days. In each treatment period, two intradermal injections of capsaicin were given in the volar region of alternate forearms at 30- and 120-minute drug/placebo administration. Spontaneous pain, elicited pain, and area of hyperalgesia were determined at certain time points after each injection. Subjects were asked to rate the painfulness of a 1-minute long 45°C heat stimulus (brief thermal stimulation [BTS]) applied to the anterior thigh at 4 hours and 30 minutes following drug administration, then every 30 minutes through 6 hours following drug administration. The 150-mg dose produced a statistically definitive reduction in spontaneous pain for all time points relative to placebo. The 90-mg dose produced a statistically significant reduction for the early time point and the entire time interval. Both doses significantly reduced elicited pain at all time points. For the BTS, the 150-mg group reached statistical significance compared with placebo at the 270-minute time point only. This study demonstrated that NGX426 reduces capsaicin-induced pain and hyperalgesia in human volunteers with low incidence of side effects that suggests that this class of drug may be effective in the treatment of clinical pain.

Activation of metabotropic glutamate receptors improves the accuracy of coincidence detection by presynaptic mechanisms in the nucleus laminaris of the chick

Interaural time difference (ITD) is a major cue for localizing a sound source and is processed in the nucleus laminaris (NL) in birds. Coincidence detection (CD) is a crucial step for processing ITD and critically depends on the size and time course of excitatory postsynaptic potentials (EPSPs). Here, we investigated a role of metabotropic glutamate receptors (mGluRs) in the regulation of EPSP amplitude and CD in the NL of chicks. A non-specific agonist of mGluRs ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) reduced the amplitude and extent of depression of excitatory postsynaptic currents (EPSCs) during a stimulus train, while the paired pulse ratio and coefficient of variation of EPSC amplitude were increased. In contrast, the amplitudes of spontaneous EPSCs were not affected, but the frequency was reduced. Thus, the effects of t-ACPD were presynaptic and reduced the release of neurotransmitter from terminals in the NL. Expression of group II mGluRs was graded along the tonotopic axis and was stronger towards the low frequency region in the NL. Both group II (DCG-IV) and group III (l-AP4) specific agonists reduced EPSC amplitude by presynaptic mechanisms, and the reduction was larger in the low frequency region; however, we could not find any effects of group I-specific agonists on EPSCs. The reduced EPSP amplitude in DCG-IV improved CD. A specific antagonist of group II mGluRs (LY341495) increased the amplitude of both EPSCs and EPSPs and enhanced the depression during a stimulus train, indicating constitutive activation of mGluRs in the NL. These observations indicate that mGluRs may work as autoreceptors and regulate EPSP size to improve CD in the NL.

Targeting Neuronal Nicotinic Receptors in Cancer: New Ligands and Potential Side-Effects

In the nervous system, the neuronal nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory postsynaptic potentials as well as slower paracrine actions of ACh. They are also widely expressed in non-nervous tissue, including the neoplastic, which is intriguing as smoking is an established risk factor for cancer. Moreover, recent evidence attributes to the gene cluster coding for the α3/α5/β4 nAChR subunits a role in both development of lung cancer and nicotine addiction. Many cellular effects of nicotine and the tobacco-derived carcinogenic N-nitrosamines are probably caused by nAChR activation, which regulates cell proliferation, migration, apoptosis and neoangiogenesis. Nonetheless, the precise nAChR roles in tumors are difficult to determine because cancer cells express a wide variety of nicotinic subunits, whose function is unclear. Patented compounds which selectively target nAChRs subtypes are increasingly available and will hopefully allow better understanding of the physiology of these channels in specific cell types, as well as suggest novel diagnostic and therapeutic approaches. At the present state, however, thorough functional studies of these compounds are still limited and whether they act as agonists, antagonists or partial agonists is often unclear. Such a blurred distinction between activators and inhibitors makes detailed studies in expression systems sorely needed for both physiological understanding and outlining the possible side-effects.

Targeting Neuronal Nicotinic Receptors in Cancer: New Ligands and Potential Side-Effects

In the nervous system, the neuronal nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory postsynaptic potentials as well as slower paracrine actions of ACh. They are also widely expressed in non-nervous tissue, including the neoplastic, which is intriguing as smoking is an established risk factor for cancer. Moreover, recent evidence attributes to the gene cluster coding for the 3/5/4 nAChR subunits a role in both development of lung cancer and nicotine addiction. Many cellular effects of nicotine and the tobacco-derived carcinogenic N-nitrosamines are probably caused by nAChR activation, which regulates cell proliferation, migration, apoptosis and neoangiogenesis. Nonetheless, the precise nAChR roles in tumors are difficult to determine because cancer cells express a wide variety of nicotinic subunits, whose function is unclear. Patented compounds which selectively target nAChRs subtypes are increasingly available and will hopefully allow better understanding of the physiology of these channels in specific cell types, as well as suggest novel diagnostic and therapeutic approaches. At the present state, however, thorough functional studies of these compounds are still limited and whether they act as agonists, antagonists or partial agonists is often unclear. Such a blurred distinction between activators and inhibitors makes detailed studies in expression systems sorely needed for both physiological understanding and outlining the possible side-effects.

The play is still being written on opening day: postnatal maturation of enteric neurons may provide an opening for early life mischief

The play is still being written on opening day: postnatal maturation of enteric neurons may provide an opening for early life mischief

489 Endogenous H2S Produced in Prevertebral Sympathetic Ganglia Predominantly by Cse in Neurons and Glia Cells Modulates Central Cholinergic Synaptic Input

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

490 Studies of the Psychophysiological Markers and the Brain Processing of Nausea in Healthy Humans Using a Novel Virtual Reality Video

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

Myenteric neurons of the mouse small intestine undergo significant electrophysiological and morphological changes during postnatal development

Organized motility patterns in the gut depend on circuitry within the enteric nervous system (ENS), but little is known about the development of electrophysiological properties and synapses within the ENS. We examined the electrophysiology and morphology of myenteric neurons in the mouse duodenum at three developmental stages: postnatal day (P)0, P10–11, and adult. Like adults, two main classes of neurons could be identified at P0 and P10–11 based on morphology: neurons with multiple long processes that projected circumferentially (Dogiel type II morphology) and neurons with a single long process. However, postnatal Dogiel type II neurons differed in several electrophysiological properties from adult Dogiel type II neurons. P0 and P10–11 Dogiel type II neurons exhibited very prominent Ca(2+)-mediated after depolarizing potentials (ADPs) following action potentials compared to adult neurons. Adult Dogiel type II neurons are characterized by the presence of a prolonged after hyperpolarizing potential (AHP), but AHPs were very rarely observed at P0. The projection lengths of the long processes of Dogiel type II neurons were mature by P10–11. Uniaxonal neurons in adults typically have fast excitatory postsynaptic potentials (fEPSPs, ‘S-type' electrophysiology) mainly mediated by nicotinic receptors. Nicotinic-fEPSPs were also recorded from neurons with a single long process at P0 and P10–11. However, these neurons underwent major developmental changes in morphology, from predominantly filamentous neurites at birth to lamellar dendrites in mature mice. Unlike Dogiel type II neurons, the projection lengths of neurons with a single long process matured after P10–11. Slow EPSPs were rarely observed in P0/P10–11 neurons. This work shows that, although functional synapses are present and two classes of neurons can be distinguished electrophysiologically and morphologically at P0, major changes in electrophysiological properties and morphology occur during the postnatal development of the ENS.