Edited by: James J. Galligan, Michigan State University, USA
Reviewed by: Sumei Liu, Ohio State University, USA; Steve Vanner, Queens University, Canada
*Correspondence: Jaime Pei Pei Foong, Department of Physiology, University of Melbourne, Parkville, Victoria 3010, Australia. e-mail:
This article was submitted to Frontiers in Autonomic Neuroscience, a specialty of Frontiers in Neuroscience.
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Vasoactive intestinal peptide (VIP) immunoreactive secretomotor neurons in the submucous plexus are involved in mediating bacterial toxin-induced hypersecretion leading to diarrhoea. VIP neurons become hyperexcitable after the mucosa is exposed to cholera toxin, which suggests that the manipulation of the excitability of these neurons may be therapeutic. This study used standard intracellular recording methods to systematically characterize slow excitatory postsynaptic potentials (EPSPs) evoked in submucosal VIP neurons by different stimulus regimes (1, 3 and 15 pulse 30 Hz stimulation), together with their associated input resistances and pharmacology. All slow EPSPs were associated with a significant increase in input resistance compared to baseline values. Slow EPSPs evoked by a single stimulus were confirmed to be purinergic, however, slow EPSPs evoked by 15 pulse trains were non-purinergic and those evoked by 3 pulse trains were mixed. NK1 or NK3 receptor antagonists did not affect slow EPSPs. The group I mGluR receptor antagonist, PHCCC reduced the amplitude of purinergic and non-purinergic slow EPSPs. Blocking mGluR1 receptors depressed the overall response to 3 and 15 pulse trains, but this effect was inconsistent, while blockade of mGluR5 receptors had no effect on the non-purinergic slow EPSPs. Thus, although other receptors are almost certainly involved, our data indicate that there are at least two pharmacologically distinct types of slow EPSPs in the VIP secretomotor neurons: one mediated by P2Y receptors and the other in part by mGluR1 receptors.
Vasoactive intestinal peptide (VIP) immunoreactive neurons in the submucous plexus are the key secretomotor neurons in the enteric nervous system (Bornstein and Furness,
Several studies have found that slow EPSPs can be evoked in VIP neurons by a single stimulus pulse applied to an internodal strand (Bornstein et al.,
There is a similar lack of clarity, when one considers another candidate transmitter, glutamate. Group I metabotropic 1α and 5 glutamate receptors (mGluR1α and mGluR5) have been found via immunohistochemistry in submucosal neurons of guinea-pig ileum (Hu et al.,
One explanation for these discrepancies is that stimulus regimes differed between studies: trains of stimuli evoke slow EPSPs in submucosal neurons that lack responses to single stimulus pulses. However, the relationship between stimulus regime, input resistance and pharmacology of slow EPSPs is unknown. The present study characterized the input resistance changes and pharmacology of slow EPSPs evoked by different stimulus regimes in VIP submucosal neurons. The roles of P2, NK1 and NK3 tachykinins, and group I mGluR receptors were specifically examined.
Male or female guinea-pigs (200–400 g) were killed by applying a sharp blow to the back of the head and immediately severing the carotid arteries and spinal cord, in accordance with the guidelines of the University of Melbourne Animal Experimentation Ethics Committee (AEEC). A 3–5 cm segment of ileum, 15–30 cm oral to the ileo-caecal junction was removed from the abdominal cavity and placed in physiological saline solution (composition in mM: NaCl 118, NaHCO3 25, D-glucose 11, KCl 4.8, CaCl2 2.5, MgSO4 1.2, NaH2PO4 1.0), bubbled with 95% O2, 5% CO2. The tissue was flushed clean with physiological saline and a 1 × 1.5 cm preparation of submucous plexus was dissected as described elsewhere (Monro et al.,
Intracellular recordings were made from submucosal neurons (Monro et al.,
Antagonists were added to the superfusate. These were N-phenyl 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), BAY 36-7620, MPEP (all Tocris Bioscience, East Brisbane, QLD, Australia), idazoxan hydrochloride, pyridoxal phosphate 6 azo(benzene-2,4-disulfonic acid) (PPADS) (both Sigma Aldrich, Castle Hill, NSW, Australia), SR142801 and SR140333 (gifts from Dr Emonds-Alt, Sanofi Recherche, Montpellier, France). All were prepared as stock solutions dissolved in distilled water and/or dimethyl sulfoxide (DMSO), kept in aliquots at −20°C and dissolved to their final working concentrations in physiological saline when used in experiments.
The mean amplitudes of slow EPSPs and IPSPs were analyzed using Axoscope 9.2.0.12 (Axon Instruments, Inc., Union City, CA, USA) in control, drug and, where possible, after washout (at least 15–30 min). All data are presented as mean ± SEM. A two-tailed or one-tailed Student's paired
A subset of electrophysiology preparations was fixed in Zamboni's fixative (2% formaldehyde and 0.2% picric acid in 0.1-M phosphate buffer, pH 7.0) at 4°C overnight. They were then cleared of fixative and permeabilized using 3 × 10 min washes with DMSO followed by 3 × 10 min washes with phosphate buffered saline (PBS) and processed to reveal the presence of VIP and/or biocytin by incubating in rabbit anti-VIP antibody (1:500; Euro-Diagnostica, East Brisbane, QLD, Australia) for 48 h at room temperature, washing with PBS, and incubating with donkey anti-rabbit fluorescein isothiocyanate (FITC) antibody (1:40; Amersham Biosciences Pty Ltd, Castle Hill, NSW, Australia) and Streptavidin-Texas Red (1:200; Amersham Biosciences) for 2.5 h. After removing excess antibody by washing with PBS, preparations were mounted on slides in buffered glycerol (pH 8.7), and viewed under a fluorescence microscope.
A total of 213 neurons were impaled in 67 preparations and 72 neurons were studied in detail as they had stable impalements and displayed slow EPSPs. Pharmacological experiments were conducted on 34 of the 72 neurons characterized.
IPSPs were evoked by trains (3 or 15 pulse) of focal stimuli in 69 out of the 72 neurons. Idazoxan (2 μM), which depresses IPSPs, was present for two of the other three neurons. IPSPs identify VIP neurons, as they are confined to this neuronal subtype (Bornstein et al.,
All 34 neurons that were used for pharmacology displayed fast EPSPs, followed by intermediate EPSPs or IPSPs and then slow EPSPs in response to a 1 (22 of 34), 3 (31 of 34) and/or 15 pulse (30 of 34) stimulus (Figures
Hyperpolarizing pulses (0.1 nA, 60–80 ms, 1 Hz) were used to measure the change in input resistance associated with slow EPSPs. The amplitude of the hyperpolarization evoked by a current pulse at the baseline membrane potential and that of one from the peak of the slow EPSP were measured for each stimulus protocol. Input resistances were calculated from these values and are shown in Figure
PPADS (30 μM) reversibly abolished 1 pulse slow EPSPs (
Possible roles for NK1 tachykinin receptors in slow EPSPs evoked in VIP neurons were tested using the specific NK1 receptor antagonist SR140333 at a concentration (200 nM) that depresses slow EPSPs in myenteric neurons (Johnson and Bornstein,
Possible roles for NK3 tachykinin receptors in the slow EPSPs of VIP neurons were tested using the specific NK3 receptor antagonist SR142801 at a concentration that blocks slow EPSPs in myenteric neurons (Johnson and Bornstein,
Roles for group I metabotropic glutamate receptors were initially tested using the group I mGluR antagonist PHCCC, at a concentration that we have found to depress colonic propulsion. PHCCC (30 μM) reversibly reduced the amplitude of purinergic (
To determine if mGluR1 receptors play a role in the slow EPSPs of VIP neurons, the specific mGluR1 antagonist BAY 36-7620 (10 μM) was tested against responses to 1, 3 and 15 pulse trains. A one-tailed test was used to minimize the number of neurons analyzed, as the PHCCC results indicated that a decrease in the slow EPSP amplitude was to be expected. BAY 36-7620 did not affect the amplitude of purinergic slow EPSPs (
When individual neurons were examined, BAY 36-7620 was found to reduce the amplitude of three pulse slow EPSPs in six neurons, but caused a slight increase (two neurons) or had no effect (three neurons) in others. Similarly, its effects on 15 pulse non-purinergic slow EPSPs were variable: six neurons showed a marked reduction in slow EPSP amplitude, but others showing no effect (three neurons) or a slight increase (two neurons). Three neurons whose slow EPSPs were depressed by BAY 36-7620 were held long enough to wash out the drug and in each case the slow EPSPs recovered. Effects on purinergic one pulse slow EPSPs were examined in four of the neurons whose 15 pulse slow EPSPs were depressed and the one pulse responses were increased in two neurons, reduced in one neuron and unchanged in the last.
In contrast, the specific mGluR5 receptor antagonist MPEP (10 μM, previously reported to be effective on submucosal slow EPSPs (Liu and Kirchgessner,
This is the first time the input resistance, pharmacology and stimuli regimes have been studied together to characterize slow EPSPs in VIP secretomotor neurons of the submucosal plexus. The results show that sensitivity to blockade of P2 receptors is stimulus regime dependent with single pulses evoking slow EPSPs that appear to be entirely due to P2 receptor activation and 15 pulse trains evoking responses that are insensitive to blockade of these receptors. Both the purinergic slow EPSPs and the non-purinergic slow EPSPs were associated with an increase in input resistance at the peak of the response suggesting they are due to a decrease in K+ conductance. The non-purinergic EPSPs are not mediated by tachykinins acting at either NK1 or NK3 receptors, but a subset are probably mediated by mGluR1 receptors with others mediated by neurotransmitter/receptor combinations that remain to be identified.
The finding that slow EPSPs evoked by single pulses applied to an internodal strand are blocked by the P2 receptor antagonist PPADS is consistent with previous reports that such slow EPSPs are mediated by P2Y1 receptors (Hu et al.,
Previous studies did not test whether altering the stimulus changes the character of the slow EPSPs. While Bornstein et al. (
The present results support earlier findings that all slow EPSPs in VIP neurons involve increased input resistance, due to inactivation of K+ conductance (Bornstein et al.,
Substance P (SP) acting on NK1 receptors has often been considered a candidate for a neurotransmitter mediating slow EPSPs in submucosal neurons. Bath application (Surprenant,
The conclusion that tachykinins do not play a role in synaptic transmission to VIP neurons does not exclude roles for them in transmission to other submucosal neurons. Immunohistochemical studies indicate that both NK1 (Moore et al.,
The other transmitter candidate whose role we investigated was glutamate acting on group I metabotropic glutamate receptors. The data indicate that mGluR1 receptors mediate a component of the non-purinergic slow EPSPs in some VIP neurons. This is consistent with the finding that submucosal neurons express mGluR1 receptors (Hu et al.,
Another possible explanation for some of the discrepancies comes from the report that glutamate suppresses slow EPSPs and potentiates slow IPSPs in S-type submucosal neurons via group I mGluRs (Ren et al.,
The conclusion that mGluR1 receptors play a significant role depends on the specificity of BAY 36-7620. This compound has been shown to be highly selective for mGluR1 receptors over other metabotropic glutamate receptors (Carroll et al.,
Group I mGluRs are positively coupled to phospholipase C (PLC), while group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6–8) are negatively coupled to adenylate cyclase (Coutinho and Knopfel,
While the results implicate mGluR1 receptors in some slow EPSPs of VIP neurons, they also clearly indicate that other neurotransmitter receptor combinations are involved. Several potential candidates have been identified in the literature. Pan and Gershon (
This study shows that the VIP neurons of the submucosal plexus exhibit two qualitatively different slow EPSPs that can be distinguished by their sensitivity to P2 receptor antagonists and the stimulus regimes that evoke them. While there is evidence that P2 mediated slow EPSPs play a role in the regulation of secretion in the colon, whether they play a role in the small intestine is unknown. However, overactivity of VIP neurons plays a critical role in the hypersecretion produced by cholera toxin and some other toxins that produce secretory diarrhoea (Banks et al.,
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The study was supported by a grant from the National Health and Medical Research Council Australia (NHMRC Grant No. 40053) and an Australian Postgraduate Award (JPPF).