Abstract
γ-Aminobutyric Acid (GABA) and its receptors, GABAA,B,C, are expressed in several locations along the gastrointestinal tract. Nevertheless, a role for GABA in enteric synaptic transmission remains elusive. In this study, we characterized the expression and function of GABA in the myenteric plexus of the mouse ileum. About 8% of all myenteric neurons were found to be GABA-immunoreactive (GABA+) including some Calretinin+ and some neuronal nitric oxide synthase (nNOS+) neurons. We used Wnt1-Cre;R26R-GCaMP3 mice, which express a genetically encoded fluorescent calcium indicator in all enteric neurons and glia. Exogenous GABA increased the intracellular calcium concentration, [Ca2+]i of some myenteric neurons including many that did not express GABA or nNOS (the majority), some GABA+, Calretinin+ or Neurofilament-M (NFM)+ but rarely nNOS+ neurons. GABA+ terminals contacted a significantly larger proportion of the cell body surface area of Calretinin+ neurons than of nNOS+ neurons. Numbers of neurons with GABA-induced [Ca2+]i transients were reduced by GABAA,B,C and nicotinic receptor blockade. Electrical stimulation of interganglionic fiber tracts was used to examine possible effects of endogenous GABA release. [Ca2+]i transients evoked by single pulses were unaffected by specific antagonists for each of the 3 GABA receptor subtypes. [Ca2+]i transients evoked by 20 pulse trains were significantly amplified by GABAC receptor blockade. These data suggest that GABAA and GABAB receptors are not involved in synaptic transmission, but suggest a novel role for GABAC receptors in modulating slow synaptic transmission, as indicated by changes in [Ca2+]i transients, within the ENS.
Highlights
GABAA receptor subunits and GABAB receptors are expressed in the large intestine (Casanova et al, 2009; Seifi et al, 2014); the presence of the 3 receptor subtypes in the small intestine has been deduced from functional contractility studies (Sanger et al, 2002; Zizzo et al, 2007) GABAC receptor expression and function has been elusive in comparison to its counterparts (Auteri et al, 2015)
GABA+ neurons are relatively uncommon in the myenteric plexus of the mouse ileum and very little is known about their functional classification in this gut region (Sang and Young, 1996; Li et al, 2011). nNOS+ neurons have been described mainly as inhibitory motor neurons, while Calretinin+ neurons are intrinsic sensory neurons, interneurons and excitatory neurons to the muscle layers (Sang and Young, 1996; Li et al, 2011)
GABA+ terminals and varicosities were present within the myenteric ganglia and muscle layers (Figure 1A). nNOS immunoreactivity was observed in 24.3 ± 2%, while Calretinin immunoreactivity was found in 33 ± 1.4% of Hu+ neurons
Summary
Γ-Aminobutyric Acid (GABA), a prominent neurotransmitter in the central nervous system (CNS) is expressed by neurons of the enteric nervous system (ENS), enteroendocrine cells in the mucosa and nerve terminals in the smooth muscle layers of the gastrointestinal tract (Jessen et al, 1986; Krantis et al, 1994; Sang and Young, 1996; Krantis, 2000). GABA may have a role in synaptic transmission in this region as there are many GABA-immunoreactive myenteric terminals in the mouse small intestine (Jessen et al, 1986; Hills et al, 1987; Sang and Young, 1996), but these have not been examined in depth. GABA can act through GABAA and GABAC receptor subtypes, which are both pentameric chloride channels, to excite enteric neurons (Auteri et al, 2015). While several studies have reported modulatory roles for GABA and its receptors on contractile activity in the gut (Tonini et al, 1989a,b; Sanger et al, 2002; Zizzo et al, 2007; Auteri et al, 2014), a role for GABA in synaptic transmission has not been identified
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