Copine-6 is a Ca2+ sensor for activity-induced AMPA receptor exocytosis.

The recruitment of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by N-methyl-D-aspartate (NMDA) receptor-dependent postsynaptic Ca2+ influx. Synaptotagmin (Syt)-1 and -7 have been proposed as Ca2+ sensors for AMPA receptor exocytosis but are functionally redundant. Here, we identify a cytosolic C2 domain-containing Ca2+-binding protein, Copine-6, that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary neurons, which is rescued by wild-type Copine-6 but not Ca2+-binding mutants. In contrast, Copine-6 loss of function does not affect steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/Syt-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca2+-binding mutants, restores activity-dependent exocytosis of AMPA receptors in Syt-1/Syt-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca2+ sensor that mediates AMPA receptor exocytosis during synaptic potentiation.

Sites and mechanisms of action of colokinetics at dopamine, ghrelin and serotonin receptors in the rodent lumbosacral defecation centre.

Agonists of dopamine D2 receptors (D2R), 5-hydroxytryptamine (5-HT, serotonin) receptors (5-HTR) and ghrelin receptors (GHSR) activate neurons in the lumbosacral defecation centre, and act as 'colokinetics', leading to increased propulsive colonic motility, in vivo. In the present study, we investigated which neurons in the lumbosacral defecation centre express the receptors and whether dopamine, serotonin and ghrelin receptor agonists act on the same lumbosacral preganglionic neurons (PGNs). We used whole cell electrophysiology to record responses from neurons in the lumbosacral defecation centre, following colokinetic application, and investigated their expression profiles and the chemistries of their neural inputs. Fluorescence in situ hybridisation revealed Drd2, Ghsr and Htr2C transcripts were colocalised in lumbosacral PGNs of mice, and immunohistochemistry showed that these neurons have closely associated tyrosine hydroxylase and 5-HT boutons. Previous studies showed that they do not receive ghrelin inputs. Whole cell electrophysiology in adult mice spinal cord revealed that dopamine, serotonin, α-methylserotonin and capromorelin each caused inward, excitatory currents in overlapping populations of lumbosacral PGNs. Furthermore, dopamine caused increased frequency of both IPSCs and EPSCs in a cohort of D2R neurons. Tetrodotoxin blocked the IPSCs and EPSCs, revealing a post-synaptic excitatory action of dopamine. In lumbosacral PGNs of postnatal day 7-14 rats, only dopamine's postsynaptic effects were observed. Furthermore, inward, excitatory currents evoked by dopamine were reduced by the GHSR antagonist, YIL781. We conclude that lumbosacral PGNs are the site where the action of endogenous ligands of D2R and 5-HT2R converge, and that GHSR act as a cis-modulator of D2R expressed by the same neurons. KEY POINTS: Dopamine, 5-hydroxytryptamine (5-HT, serotonin) and ghrelin (GHSR) receptor agonists increase colorectal motility and have been postulated to act at receptors on parasympathetic preganglionic neurons (PGNs) in the lumbosacral spinal cord. We aimed to determine which neurons in the lumbosacral spinal cord express dopamine, serotonin and GHSR receptors, their neural inputs, and whether agonists at these receptors excite them. We show that dopamine, serotonin and ghrelin receptor transcripts are contained in the same PGNs and that these neurons have closely associated tyrosine hydroxylase and serotonin boutons. Whole cell electrophysiology revealed that dopamine, serotonin and GHSR receptor agonists induce an inward excitatory current in overlapping populations of lumbosacral PGNs. Dopamine-induced excitation was reversed by GHSR antagonism. The present study demonstrates that lumbosacral PGNs are the site at which actions of endogenous ligands of dopamine D2 receptors and 5-HT type 2 receptors converge. Ghrelin receptors are functional, but their role appears to be as modulators of dopamine effects at D2 receptors.

The association of enteric neuropathy with gut phenotypes in acute and progressive models of Parkinson's disease.

Parkinson's disease (PD) is associated with neuronal damage in the brain and gut. This work compares changes in the enteric nervous system (ENS) of commonly used mouse models of PD that exhibit central neuropathy and a gut phenotype. Enteric neuropathy was assessed in five mouse models: peripheral injection of MPTP; intracerebral injection of 6-OHDA; oral rotenone; and mice transgenic for A53T variant human α-synuclein with and without rotenone. Changes in the ENS of the colon were quantified using pan-neuronal marker, Hu, and neuronal nitric oxide synthase (nNOS) and were correlated with GI function. MPTP had no effect on the number of Hu+ neurons but was associated with an increase in Hu+ nuclear translocation (P < 0.04). 6-OHDA lesioned mice had significantly fewer Hu+ neurons/ganglion (P < 0.02) and a reduced proportion of nNOS+ neurons in colon (P < 0.001). A53T mice had significantly fewer Hu+ neurons/area (P < 0.001) and exhibited larger soma size (P < 0.03). Treatment with rotenone reduced the number of Hu+ cells/mm2 in WT mice (P < 0.006) and increased the proportion of Hu+ translocated cells in both WT (P < 0.02) and A53T mice (P < 0.04). All PD models exhibited a degree of enteric neuropathy, the extent and type of damage to the ENS, however, was dependent on the model.

Analysis of Bioavailability and Induction of Glutathione Peroxidase by Dietary Nanoelemental, Organic and Inorganic Selenium.

Dietary organic selenium (Se) is commonly utilized to increase formation of selenoproteins, including the major antioxidant protein, glutathione peroxidase (GPx). Inorganic Se salts, such as sodium selenite, are also incorporated into selenoproteins, and there is evidence that nanoelemental Se added to the diet may also be effective. We conducted two trials, the first investigated inorganic Se (selenite), organic Se (L-selenomethionine) and nanoelemental Se, in conventional mice. Their bioavailability and effectiveness to increase GPx activity were examined. The second trial focused on determining the mechanism by which dietary Se is incorporated into tissue, utilising both conventional and germ-free (GF) mice. Mice were fed a diet with minimal Se, 0.018 parts per million (ppm), and diets with Se supplementation, to achieve 0.07, 0.15, 0.3 and 1.7 ppm Se, for 5 weeks (first trial). Mass spectrometry, Western blotting and enzymatic assays were used to investigate bioavailability, protein levels and GPx activity in fresh frozen tissue (liver, ileum, plasma, muscle and feces) from the Se fed animals. Inorganic, organic and nanoelemental Se were all effectively incorporated into tissues. The high Se diet (1.7 ppm) resulted in the highest Se levels in all tissues and plasma, independent of the Se source. Interestingly, despite being ~11 to ~25 times less concentrated than the high Se, the lower Se diets (0.07; 0.15) resulted in comparably high Se levels in liver, ileum and plasma for all Se sources. GPx protein levels and enzyme activity were significantly increased by each diet, relative to control. We hypothesised that bacteria may be a vector for the conversion of nanoelemental Se, perhaps in exchange for S in sulphate metabolising bacteria. We therefore investigated Se incorporation from low sulphate diets and in GF mice. All forms of selenium were bioavailable and similarly significantly increased the antioxidant capability of GPx in the intestine and liver of GF mice and mice with sulphate free diets. Se from nanoelemental Se resulted in similar tissue levels to inorganic and organic sources in germ free mice. Thus, endogenous mechanisms, not dependent on bacteria, reduce nanoelemental Se to the metabolite selenide that is then converted to selenophosphate, synthesised to selenocysteine, and incorporated into selenoproteins. In particular, the similar efficacy of nanoelemental Se in comparison to organic Se in both trials is important in the view of the currently limited cheap sources of Se.

Dopamine and ghrelin receptor co-expression and interaction in the spinal defecation centers.

BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.

Chronic isolation stress is associated with increased colonic and motor symptoms in the A53T mouse model of Parkinson's disease.

BACKGROUND: Chronic stress exacerbates motor deficits and increases dopaminergic cell loss in several rodent models of Parkinson's disease (PD). However, little is known about effects of stress on gastrointestinal (GI) dysfunction, a common non-motor symptom of PD. We aimed to determine whether chronic stress exacerbates GI dysfunction in the A53T mouse model of PD and whether this relates to changes in α-synuclein distribution. METHODS: Chronic isolation stress was induced by single-housing WT and homozygote A53T mice between 5 and 15 months of age. GI and motor function were compared with mice that had been group-housed. KEY RESULTS: Chronic isolation stress increased plasma corticosterone and exacerbated deficits in colonic propulsion and whole-gut transit in A53T mice and also increased motor deficits. However, our results indicated that the novel environment-induced defecation response, a common method used to evaluate colorectal function, was not a useful test to measure exacerbation of GI dysfunction, most likely because of the reported reduced level of anxiety in A53T mice. A53T mice had lower corticosterone levels than WT mice under both housing conditions, but single-housing increased levels for both genotypes. Enteric neuropathy was observed in aging A53T mice and A53T mice had a greater accumulation of alpha-synuclein (αsyn) in myenteric ganglia under both housing conditions. CONCLUSIONS & INFERENCES: Chronic isolation stress exacerbates PD-associated GI dysfunction, in addition to increasing motor deficits. However, these changes in GI symptoms are not directly related to corticosterone levels, worsened enteric neuropathy, or enteric αsyn accumulation.

Cellular and sub-cellular localisation of oxyntomodulin-like immunoreactivity in enteroendocrine cells of human, mouse, pig and rat.

We use a monoclonal antibody against the C-terminal of oxyntomodulin (OXM) to investigate enteroendocrine cells (EEC) in mouse, rat, human and pig. This antibody has cross-reactivity with the OXM precursor, glicentin (Gli) but does not recognise glucagon. The antibody stained EEC in the jejunum and colon of each species. We compared OXM/Gli immunoreactivity with that revealed by antibodies against structurally related peptides, GLP-1 and glucagon and against GIP and PYY that are predicted to be in some EEC that express OXM/Gli. We used super-resolution to locate immunoreactive vesicles. In the pancreas, OXM/Gli was in glucagon cells but was located in separate storage vesicles to glucagon. In jejunal EEC, OXM/Gli and GIP were in many of the same cells but often in separate vesicles, whereas PYY and OXM/Gli were commonly colocalised in the same storage vesicles of colonic EEC. When binding of anti-GLP-1 to the structurally related GIP was removed by absorption with GIP peptide, GLP-1 and OXM/Gli immunoreactivities were contained in the same population of EEC in the intestine. We conclude that anti-OXM/Gli is a more reliable marker than anti-GLP-1 for EEC expressing preproglucagon products. Storage vesicles that were immunoreactive for OXM/Gli were almost always immunoreactive for GLP-1. OXM concentrations, measured by ELISA, were highest in the distal ileum and colon. Lesser concentrations were found in more proximal parts of small intestine and pancreas. Very little was in the stomach. In EEC containing GIP and OXM/Gli, these hormones are packaged in different secretory vesicles. Separate packaging also occurred for OXM and glucagon, whereas OXM/Gli and PYY and OXM/Gli and GLP-1 were commonly contained together in secretory vesicles.

Evidence that central pathways that mediate defecation utilize ghrelin receptors but do not require endogenous ghrelin.

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild-type rats, WAS-induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex.