Abstract
Glutamate is a classic excitatory neurotransmitter in the central nervous system (CNS), but despite several studies reporting the expression of glutamate together with its various receptors and transporters within the enteric nervous system (ENS), its role in the gut remains elusive. In this study, we characterized the expression of the vesicular glutamate transporter, vGluT2, and examined the function of glutamate in the myenteric plexus of the distal colon by employing calcium (Ca2+)-imaging on Wnt1-Cre; R26R-GCaMP3 mice which express a genetically encoded fluorescent Ca2+ indicator in all enteric neurons and glia. Most vGluT2 labeled varicosities contained the synaptic vesicle release protein, synaptophysin, but not vesicular acetylcholine transporter, vAChT, which labels vesicles containing acetylcholine, the primary excitatory neurotransmitter in the ENS. The somata of all calbindin (calb) immunoreactive neurons examined received close contacts from vGluT2 varicosities, which were more numerous than those contacting nitrergic neurons. Exogenous application of L-glutamic acid (L-Glu) and N-methyl-D-aspartate (NMDA) transiently increased the intracellular Ca2+ concentration [Ca2+]i in about 25% of myenteric neurons. Most L-Glu responsive neurons were calb immunoreactive. Blockade of NMDA receptors with APV significantly reduced the number of neurons responsive to L-Glu and NMDA, thus showing functional expression of NMDA receptors on enteric neurons. However, APV resistant responses to L-Glu and NMDA suggest that other glutamate receptors were present. APV did not affect [Ca2+]i transients evoked by electrical stimulation of interganglionic nerve fiber tracts, which suggests that NMDA receptors are not involved in synaptic transmission. The group I metabotropic glutamate receptor (mGluR) antagonist, PHCCC, significantly reduced the amplitude of [Ca2+]i transients evoked by a 20 pulse (20 Hz) train of electrical stimuli in L-Glu responsive neurons. This stimulus is known to induce slow synaptic depolarizations. Further, some neurons that had PHCCC sensitive [Ca2+]i transients were calb immunoreactive and received vGluT2 varicosities. Overall, we conclude that electrically evoked release of endogenous glutamate mediates slow synaptic transmission via activation of group I mGluRs expressed by myenteric neurons, particularly those immunoreactive for calb.
Highlights
Glutamate is the primary excitatory transmitter in the central nervous system (CNS)
We found vGluT2 immunoreactivity in varicosities and terminals, but not in neuronal cell bodies, in the myenteric plexus of the mouse distal colon (Figures 1A,B)
To establish the nature of vGluT2 varicosities and terminals in the myenteric plexus, we quantified the co-localization of vGluT2 with two key markers of enteric varicosities
Summary
Glutamate is the primary excitatory transmitter in the central nervous system (CNS). But its role the enteric nervous system (ENS) of the gut has remained elusive despite several reports of possible function (Kirchgessner, 2001; Filpa et al, 2016; Seifi and Swinny, 2016).Several ultrastructural and immunofluorescence studies in rodent models provide evidence for the expression of glutamate, its release and re-uptake transporters, and its various receptor subtypes within the ENS (Liu et al, 1997; Tong et al, 2001; Giaroni et al, 2003; Tsai, 2005; Brumovsky et al, 2011; Seifi and Swinny, 2016). EAAT is expressed within the ENS, by enteric glia (Liu et al, 1997; Seifi and Swinny, 2016). The ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), as well as subtypes of metabotropic glutamate receptors (mGluRs) are found on enteric neurons (Broussard et al, 1994; Burns et al, 1994; Liu et al, 1997; Liu and Kirchgessner, 2000; McRoberts et al, 2001; Chen and Kirchgessner, 2002; Tong and Kirchgessner, 2003; Del Valle-Pinero et al, 2007; Foong and Bornstein, 2009; Seifi and Swinny, 2016)
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