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Selective Stimulation of Inhibitory Motorneurons in the Mouse Colon Following Light Activation of Channel Rhodopsin‐2 (ChR2) Ex Vivo
Author(s) -
PerezMedina Alberto,
Manfredsson Fredric,
Galligan James
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb572
Subject(s) - biology , electrophysiology , inhibitory postsynaptic potential , stimulation , endocrinology , medicine , nitric oxide synthase , myenteric plexus , nitric oxide , enteric nervous system , neuroscience , immunohistochemistry
Inhibitory motor neurons in the enteric nervous system (ENS) are responsible for producing relaxations of gastrointestinal (GI) smooth muscle. Neurogenic relaxations are required to produce the pressure gradients needed to propel GI content along the gut. Nitric oxide (NO) and ATP (or related purine) are the predominant neurotransmitters released by inhibitory motorneurons in the ENS. Immunohistochemical studies using antibodies against nitric oxide synthase (NOS) and the vesicular nucleotide transporter (VNUT), protein markers for nitrergic and purinergic inhibitory motorneurons respectively, showed that there are NOS+ neurons, VNUT+ neurons and NOS/VNUT+ neurons in the myenteric plexus of the mouse colon. To determine if NO and ATP were released by the same or separate neurons, we injected an adeno‐associated virus (AAV9) containing a construct with genes encoding ChR2 and yellow fluorescent protein (YFP) bordered by loxP sites into the proximal colon of NOS‐cre expressing mice. 4–6 weeks later, the colon was harvested for electrophysiological measurements of circular muscle inhibitory junction potentials (IJPs, the electrophysiological correlate of muscle relaxation) evoked by blue light (470 nm) and transmural electrical stimulation. IJPs were recorded using intracellular microelectrodes. Light stimulation (5–20 mW/mm 2 ) evoked biphasic IJPs in tissues from all mice successfully transduced by AAV9 (as measured by detection of the YFP reporter), but not in non‐transduced tissues (lacking YFP). Electrical stimulation evoked IJPs in tissues from all mice. The amplitude and duration of the light‐ and electrically‐evoked IJPs increased with the number of stimuli (1–7 stimuli). The fast, early phase of light‐ and electrically‐evoked IJPs was blocked by MRS2179 (10 μM, a P2Y1 receptor antagonist) while the slow, late phase of the IJP was blocked by nitro‐L‐arginine (100 μM, NOS inhibitor). IJPs were blocked completely by combined application of MRS2179 and nitro‐L‐arginine. These data provide direct evidence that inhibitory motorneurons in the mouse colon co‐release NO and ATP (or related purine) to cause relaxation of colon circular smooth muscle. Selective expression of ChR2 in subtypes of neurons will enable more precise identification of synaptic mechanisms and nerve pathways in the ENS. Support or Funding Information Supported by R01DK094932