Activity in varicosities within the myenteric plexus between and during the colonic migrating motor complex in the isolated murine large intestine

Abstract

Background: Neuronal communication within the myenteric plexus occurs when action potentials along nerve fibers produce Ca(2+) transients in varicosities leading to exocytosis of vesicles and neurotransmitters release. We used Ca(2+) transients in varicosities to monitor action potential activity in myenteric nerve pathways both between and during the colonic migrating motor complex (CMMC) in the isolated murine colon.

Methods: Strips of longitudinal muscle were removed to reveal the myenteric ganglia, which were then loaded with Fluo-4.

Key results: Many varicosities, including synaptotagmin 1 labeled varicosities, exhibited ongoing Ca(2+) transients (duration of unitary Ca(2+) transient 3.9 s). Between CMMCs, varicosities fired at a frequency of 0.6 Hz, which correlated with spontaneous inhibitory junction potentials in the circular muscle, suggesting they were mainly in inhibitory nerve pathways. During a CMMC other previously quiescent varicosities fired at 1.3 Hz (max. 2.0 Hz) for the duration (24 s) of the CMMC, suggesting they were on excitatory nerve pathways. Activity in varicosities was correlated with Ca(2+) transient responses in a number of neurons. Some varicosities appeared to release an inhibitory neurotransmitter that reduced activity in nNOS-positive neurons. Varicosities along the same nerve fiber exhibited identical patterns of activity that allowed nerve fibers to be traced throughout the myenteric plexus and internodal strands. Activity in varicosities was reduced by hexamethonium (100 μmol L(-1) ), and blocked by ω-conotoxin GVIA (200 nM) and tetrodotoxin (1 μmol L(-1) ; TTX).

Conclusions & inferences: Ca(2+) imaging of varicosities allows for a determination of activity in neural pathways within the enteric nervous system.

Figure 1. Tracing varicose axons in internodal strands

A. Left hand panel (LHP): A single frame, inverted to show an active varicose nerve fiber. AI) Ca²⁺ transients taken from a single varicosity along this nerve fiber (see ** arrow, LHP) expressed as a line trace, a differentiated trace and a Spatio-temporal map (ST-Map). Bottom panel shows an inverted imaging of the summed Ca²⁺ activity over 1000 frames (Fluo-4 max, inverted image). AII) Ca²⁺ transients taken from the whole rectangular region of interest (red ROI in LHP), and expressed as an ST-Map below. Green region in ST-Map includes the varicose nerve fiber in I. AIII) Ca²⁺ transients taken from the blue rectangle. B. LHP: Summed activity in the same internodal strands shown in A. AI Ca²⁺ transients taken from 8 selected varicosities (orange traces) throughout the internodal strands that exhibited identical patterns of activity, suggesting they were on the same nerve fiber (see orange arrows in LHP). The Ca²⁺ transients in each varicosity (BI 1–8) were summed together in BI i. BI ii-vi Using the same method Ca²⁺ transients in 8 other varicosities from 6 other nerve fibers were traced within the internodal strands and summed together (BI ii–vi colored traces were sampled at the colored arrows (LHP and BI ii–vi). BII The entire summed activities of fibers i–vi are overlayed, each Ca²⁺ transient peak is represented as a colored * below.

Figure 2. Ca²⁺ transients in multiple varicosities

A. Inverted LHP shows the summed Ca²⁺activity in the ganglia, revealing numerous active varicosities (dark spots). RHP shows Ca²⁺ transients from 10 of these varicosities recorded throughout the ganglia and around an ICC-MY in the extraganglionic space. Colored vertical lines below shows the peak of every Ca²⁺ transient. B. Ca²⁺ transients from 8 varicosities lying over neuron N, which only fired once throughout the recording, show different activities. Hexamethonium (100 μM) blocked the activity in most of these particular varicosities. Several local mucosal stimuli led to firing in two varicosities and responses in neurons (N1 and N2). D. Following local mucosal stimulation, varicosities V1 and V2 eventually responded with a prolonged burst of high frequency Ca²⁺ transients. At the same time neurons N1 and N2 also responded. B. Hexamthonium (100 μM; Hex) blocked the responses in these varicosities, but only inhibited the responses in N1 and N2.

Figure 3

A plot of the frequency of varicosities both between and following CMMCs.

Figure 4. Responses in synaptotagmin 1 labeled varicosities and calretinin positive neurons

A. I nNOS labeled neuron. II. Synaptotagmin 1 labeled varicosities. III. Superimposed nNOS with synaptotagmin 1. Synaptotagmin 1 varicosities surround nNOS neurons, but the nNOS varicosities don't appear to be colocalized with synatotagmin 1 (see arrows). III. Another ganglia showing the lack of colocalization of nNOS and synaptotagmin 1 varicosities. B. LHP shows the lack of colocalization of calretinin positive neurons with synaptotagmin 1 labeled varicosities that surrounded these neurons. C. Ca²⁺ transient responses in several synaptotagmin 1 labeled varicosities V1, V2, V3 and V4 that were closely opposed to two calretinin positive neurons (N1 and N2), respectively. The first local mucosal stimulus elicited a CMMC like response (see contraction Δy). However, despite the refractoriness associated with the CMMC these neurons responded to subsequent stimulation. There was often a close association between the responses in the synaptotagmin 1 labeled varicosities and those in the neurons (see expanded panel in B). (movement artifact shown in red dotted rectangle).

Figure 5. Effect of blocking N-type Ca²⁺ channels

A. Spontaneous activity in varicosities (V1–V4) and neurons (N1, N2). Δy = movement. B. Following ω- Conotoxin GVIA (100nM; ω-CTX GVIA) the majority of the activity was blocked, although there was an increase in spontaneous contractions (Δy). C. Anal stimulation of the mucosa evoked a robust CMMC response. Underlying this CMMC was an intense prolonged burst of Ca²⁺ transients in varicosities V1, V2 and V4, whereas V3 displayed a transient burst of activity. This increase in activity was reflected in the Ca²⁺ transients in neurons N1 and N2, the ICC-MY and the circular muscle (CM). D. Following ω- Conotoxin GVIA (100nM)all the responses to stimulation were blocked; although ICC-MY developed spontaneous activity.

Figure 6. Recruitment of varicosities and tracing nerve fibers

A. Active varicosities (red dots) are shown throughout the ganglia (inverted images) in control and following 3 successive local mucosal stimuli (puffs of N₂). B. The summed activity of all varicosities that display Ca²⁺ transients between CMMCs (see Methods; All; 10s) and 3 individual fibers selected from different parts of the ganglia thresholded in red. Traces of the % area of varicosities that were actively displaying Ca2+ transients. At any time, less than 30% of the total pool of varicosities (All) were active. Individual fibers showed different patterns of activity ranging from constant firing (Fiber 1), quiescence (Fiber 2) or clustered activity (Fiber 3). C. The summed activity of all varicosities that display Ca²⁺ transients 8 s after a brush-stroke stimulus (All). Note the much greater number of varicosities that fired during the same 10s period. The same individual fibers were selected (Fibers 1, 2 & 3). D. shows traces of the % area of varicosities that were actively displaying Ca²⁺ transients. Note the more sustained firing pattern after the brush-stroke compared to between CMMCs. The activity in the individual fibers was altered compared to between CMMCs; activity in Fiber 1 became more irregular and activity in Fibers 2 & 3 were decreased.

Figure 7. Activity in varicosities between and following the CMMC

A. By color coding varicosities with similar activity (see white insert and corresponding green arrows) 19 nerve fibers were traced throughout the ganglia. B. These varicosities were joined to show the course of the different nerve fibers. Varicosities along each nerve exhibited the same pattern of Ca²⁺ transients. C. and D. Spontaneous and evoked (local mucosal stimulation) Ca²⁺ transients in 12 varicosities selected from 12 of the 19 fibers in A & B, plus the two neurons (N1 and N2). D. Following successive local mucosal stimulation there was a pronounced increase in activity in N1 and N2, and in fibers 1, 2, 3, 4, 10, 12, 17; whereas, the activity in fiber 5 ceased firing during the CMMC response (see F).

Figure 8. Correlation between Ca²⁺ transients in varicosities and neurons

A. This particular neuron (N) exhibited spontaneous activity. 6 different active nerve fibers were traced to this neuron, whose varicose fibers are color coded are shown in the expanded panel (RHP). Despite their ongoing activity there was little correlation between their activity and that in the neuron (N). B and C. Spontaneous activity in these neurons was closely matched by the activity in a closely associated varicosity. D. The second local mucosal stimulus evoked a burst of Ca²⁺ transients in a varicosity that produced a similar burst in the neuron (N).

Figure 9. Excitatory responses in mitotracker positive neuron and inhibitory responses in neurons

A. Each local stimulation of the mucosa produced a transient response in neuron 1 and in neuron 2. Neuron 1 was mitotracker positive, whereas neuron 2 was mitotracker -ve (see lower panel). These responses were preceded by activity in their closely associated varicosities V1 and V2. B. During a spontaneous CMMC (see contraction Δy), both the nNOS positive neuron (N1) and the nNOS -ve neuron (N2) ceased their activity (see corresponding ST-Maps). However, the reduction in activity in the nNOS positive neuron was preceded by an increase in activity in varicosities V1 (see expanded panel) and V2. In contrast, the nNOS -ve neuron (N2) reduced its activity as the activity in varicosity V3 ceased.