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Enteric neurons in culture

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Organotypic Culture
1.1 Demonstration of Intrinsic Enteric Neuronal Properties
1.2 Studies of Development
2 Tissue Culture
2.1 Preferred Growth Substrates
2.2 Retention of Differentiated Properties
2.3 Absent or Rare Differentiated Properties
2.4 Gaba Neurons
2.5 Fast Responses to Substance P
2.6 Distribution of Muscarinic Receptors
2.7 Properties of Enteric Glia
3 Cell Cultures
3.1 Rat Neurons
3.2 Human Neurons
4 Conclusion
4.1 Limitations of Culture Studies
4.2 Advantages of Culture Studies
Figure 1. Figure 1.

Myenteric plexus from taenia coli of a newborn guinea pig 5 h after being placed in culture. G, ganglia; arrows, interconnecting strands. Outgrowth of fibers has not started yet. Drawing (upper left), diagram of entire expiant; box, area of expiant shown in micrograph. Bar, 50 μm.

From Jessen et al.
Figure 2. Figure 2.

Neurites atop a glial sheet at edge of an expiant of guinea pig myenteric plexus cultured for 4 days. Straight arrows, neurites; curved arrows, glial cell nuclei. F, fibroblast. E, main part of expiant. Bar, 50 μm.

From Jessen et al.
Figure 3. Figure 3.

Enteric glial cells in culture. Cells can be seen to form a continuous sheet of membranous expansions and also to extend tapering processes. Cells are from a 15‐day‐old culture of the myenteric plexus that was explanted from a guinea pig taenia coli. Bar, 15 μm.

From Jessen et al.
Figure 4. Figure 4.

Preferential growth of enteric neurites on glial cells. White arrows, border between areas covered mainly by fibroblasts (left) or glia (right). Neurites above the fibroblasts are fasciculated into 3 large bundles, which divide up into small bundles and apparently single fibers to form a dense meshwork over the glial cells. FN, nucleus of fibroblast; black arrows, nuclei of 2 glial cells. These cells are part of 24‐day‐old culture of the myenteric plexus from a guinea pig taenia coli. Bar, 15 μm.

From Jessen et al.
Figure 5. Figure 5.

Nerve profiles of myenteric neurons in expiant culture. A: nerve profile containing mainly small clear vesicles (diam 40–60 nm) and a few large granular vesicles. B: nerve profile containing mostly large granular vesicles. C: nerve profile containing large opaque vesicles (diam 80–120 nm). D: nerve profile containing mixture of these vesicle types. All profiles from neurons in expiants cultured for 6–8 days. Bars, 500 nm.

From Baluk et al.
Figure 6. Figure 6.

Neurotransmitter‐like immunoreactivities in myenteric neurons in cell culture. Binding of antisera to indicated compounds was visualized by the peroxidase‐antiperoxidase procedure. A: VIP; B: substance P; C: bombesin; D: Met‐enkephalin; E: serotonin; F: somatostatin; G: cholecystokinin/gastrin; H: higher‐power photo of same field of view as in G to show more detail of positively stained small neuron. Note small process extending down larger soma adjacent to cholecystokinin‐positive neuron. Also note string of varicosities extending across upper part of photograph. Bar, 50 μm. I: higher‐power view of a culture stained with antisomatostatin. Two strongly stained somata and one lightly stained soma with punctate staining around its nucleus can be seen. The arrows point to 2 unstained somata. Bar, 50 μm. J: higher‐power photo of a culture stained with anti‐substance P. Two lightly stained somata with even distribution of reaction product within the cytoplasm are shown. The other 2 somata are unstained. Note the many strongly stained varicosities in the processes of the cells in I and J.

From Nishi and Willard
Figure 7. Figure 7.

Neurons in culture stained for intracellular AChe activity. A, B: examples of stained neurons in same field of view as unstained neurons. C: culture reacted in the presence of BW284C51, a specific AChe inhibitor. Bar, 100 μm.

Figure 8. Figure 8.

Cultured enteric neurons containing choline acetyltransferase‐like immunoreactivity. A, B: low‐power photographs (bar, 100 μm). C, D: higher‐power photographs (bar, 50 μm) show varying degrees of staining in the cell bodies. Unstained cell bodies are barely visible with bright‐field optics.

From Nishi and Willard
Figure 9. Figure 9.

Fast nicotinic excitatory postsynaptic potential (EPSP). Two cultured myenteric neurons were impaled with intracellular microelectrodes. Injection of depolarizing current into one of them caused an action potential in that cell (middle trace), which in turn caused a fast EPSP in the other neuron (bottom trace). Addition of 20 μM hexamethonium blocked the EPSP reversibly. Top trace, output of the current monitor.

From Willard and Nishi
Figure 10. Figure 10.

Slow noncholinergic synaptic potential. Two cultured myenteric neurons were impaled with intracellular microelectrodes. When one of them was stimulated at 10 Hz, a slow depolarization was recorded in the other. Trace 1, chart recording from the presynaptic neuron during stimulation. Traces 2–6, chart recordings from the postsynaptic neuron. Trace 2, slow depolarization evoked in control perfusion fluid. Trace 3, recorded while sufficient hyperpolarizing current was being injected into the postsynaptic neuron to maintain its membrane potential at its resting level during the slow synaptic event (the so‐called manual clamp). Downward deflections are the voltage responses to constant amplitude hyperpolarizing pulses of current injected through the recording electrode. Note that apparent resistance of postsynaptic neuron increases during time when slow depolarization would have occurred. Trace 4, recording of the slow depolarization evoked in 1 mM hexamethonium and 10 μM atropine. Trace 5, slow depolarization was blocked by perfusion fluid containing low (0.36 mM) CaCl2 and elevated (10 mM) MgCl2. Trace 6, slow depolarization returns when control perfusion fluid is washed back into the culture.

From Willard and Nishi
Figure 11. Figure 11.

High‐frequency stimulation of cholinergic neuron causes slow depolarization. Upper and lower traces, chart recordings from the post‐ and presynaptic neurons, respectively. When presynaptic neuron is stimulated at 1 Hz (left), fast excitatory postsynaptic potentials (EPSP) are evoked in the postsynaptic neuron. When the stimulation frequency is increased to 10 Hz, the fast EPSP are depressed and slow depolarization is elicited in postsynaptic neuron.

From Willard and Nishi
Figure 12. Figure 12.

Noncholinergic depolarization evoked by a cholinergic neuron. In each panel upper and lower traces, chart recordings from the post‐ and presynaptic neurons, respectively. A: stimulation of presynaptic neuron at 10 Hz evokes fast excitatory postsynaptic potential (EPSP) and slow depolarization. B: fast EPSP, but not the slow depolarization, are blocked by hexamethonium (1 mM) plus atropine (10 μM). C: fast EPSP return when the cholinergic antagonists are washed out.

From Willard and Nishi


Figure 1.

Myenteric plexus from taenia coli of a newborn guinea pig 5 h after being placed in culture. G, ganglia; arrows, interconnecting strands. Outgrowth of fibers has not started yet. Drawing (upper left), diagram of entire expiant; box, area of expiant shown in micrograph. Bar, 50 μm.

From Jessen et al.


Figure 2.

Neurites atop a glial sheet at edge of an expiant of guinea pig myenteric plexus cultured for 4 days. Straight arrows, neurites; curved arrows, glial cell nuclei. F, fibroblast. E, main part of expiant. Bar, 50 μm.

From Jessen et al.


Figure 3.

Enteric glial cells in culture. Cells can be seen to form a continuous sheet of membranous expansions and also to extend tapering processes. Cells are from a 15‐day‐old culture of the myenteric plexus that was explanted from a guinea pig taenia coli. Bar, 15 μm.

From Jessen et al.


Figure 4.

Preferential growth of enteric neurites on glial cells. White arrows, border between areas covered mainly by fibroblasts (left) or glia (right). Neurites above the fibroblasts are fasciculated into 3 large bundles, which divide up into small bundles and apparently single fibers to form a dense meshwork over the glial cells. FN, nucleus of fibroblast; black arrows, nuclei of 2 glial cells. These cells are part of 24‐day‐old culture of the myenteric plexus from a guinea pig taenia coli. Bar, 15 μm.

From Jessen et al.


Figure 5.

Nerve profiles of myenteric neurons in expiant culture. A: nerve profile containing mainly small clear vesicles (diam 40–60 nm) and a few large granular vesicles. B: nerve profile containing mostly large granular vesicles. C: nerve profile containing large opaque vesicles (diam 80–120 nm). D: nerve profile containing mixture of these vesicle types. All profiles from neurons in expiants cultured for 6–8 days. Bars, 500 nm.

From Baluk et al.


Figure 6.

Neurotransmitter‐like immunoreactivities in myenteric neurons in cell culture. Binding of antisera to indicated compounds was visualized by the peroxidase‐antiperoxidase procedure. A: VIP; B: substance P; C: bombesin; D: Met‐enkephalin; E: serotonin; F: somatostatin; G: cholecystokinin/gastrin; H: higher‐power photo of same field of view as in G to show more detail of positively stained small neuron. Note small process extending down larger soma adjacent to cholecystokinin‐positive neuron. Also note string of varicosities extending across upper part of photograph. Bar, 50 μm. I: higher‐power view of a culture stained with antisomatostatin. Two strongly stained somata and one lightly stained soma with punctate staining around its nucleus can be seen. The arrows point to 2 unstained somata. Bar, 50 μm. J: higher‐power photo of a culture stained with anti‐substance P. Two lightly stained somata with even distribution of reaction product within the cytoplasm are shown. The other 2 somata are unstained. Note the many strongly stained varicosities in the processes of the cells in I and J.

From Nishi and Willard


Figure 7.

Neurons in culture stained for intracellular AChe activity. A, B: examples of stained neurons in same field of view as unstained neurons. C: culture reacted in the presence of BW284C51, a specific AChe inhibitor. Bar, 100 μm.



Figure 8.

Cultured enteric neurons containing choline acetyltransferase‐like immunoreactivity. A, B: low‐power photographs (bar, 100 μm). C, D: higher‐power photographs (bar, 50 μm) show varying degrees of staining in the cell bodies. Unstained cell bodies are barely visible with bright‐field optics.

From Nishi and Willard


Figure 9.

Fast nicotinic excitatory postsynaptic potential (EPSP). Two cultured myenteric neurons were impaled with intracellular microelectrodes. Injection of depolarizing current into one of them caused an action potential in that cell (middle trace), which in turn caused a fast EPSP in the other neuron (bottom trace). Addition of 20 μM hexamethonium blocked the EPSP reversibly. Top trace, output of the current monitor.

From Willard and Nishi


Figure 10.

Slow noncholinergic synaptic potential. Two cultured myenteric neurons were impaled with intracellular microelectrodes. When one of them was stimulated at 10 Hz, a slow depolarization was recorded in the other. Trace 1, chart recording from the presynaptic neuron during stimulation. Traces 2–6, chart recordings from the postsynaptic neuron. Trace 2, slow depolarization evoked in control perfusion fluid. Trace 3, recorded while sufficient hyperpolarizing current was being injected into the postsynaptic neuron to maintain its membrane potential at its resting level during the slow synaptic event (the so‐called manual clamp). Downward deflections are the voltage responses to constant amplitude hyperpolarizing pulses of current injected through the recording electrode. Note that apparent resistance of postsynaptic neuron increases during time when slow depolarization would have occurred. Trace 4, recording of the slow depolarization evoked in 1 mM hexamethonium and 10 μM atropine. Trace 5, slow depolarization was blocked by perfusion fluid containing low (0.36 mM) CaCl2 and elevated (10 mM) MgCl2. Trace 6, slow depolarization returns when control perfusion fluid is washed back into the culture.

From Willard and Nishi


Figure 11.

High‐frequency stimulation of cholinergic neuron causes slow depolarization. Upper and lower traces, chart recordings from the post‐ and presynaptic neurons, respectively. When presynaptic neuron is stimulated at 1 Hz (left), fast excitatory postsynaptic potentials (EPSP) are evoked in the postsynaptic neuron. When the stimulation frequency is increased to 10 Hz, the fast EPSP are depressed and slow depolarization is elicited in postsynaptic neuron.

From Willard and Nishi


Figure 12.

Noncholinergic depolarization evoked by a cholinergic neuron. In each panel upper and lower traces, chart recordings from the post‐ and presynaptic neurons, respectively. A: stimulation of presynaptic neuron at 10 Hz evokes fast excitatory postsynaptic potential (EPSP) and slow depolarization. B: fast EPSP, but not the slow depolarization, are blocked by hexamethonium (1 mM) plus atropine (10 μM). C: fast EPSP return when the cholinergic antagonists are washed out.

From Willard and Nishi
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Alan L. Willard, Rae Nishi. Enteric neurons in culture. Compr Physiol 2011, Supplement 16: Handbook of Physiology, The Gastrointestinal System, Motility and Circulation: 331-347. First published in print 1989. doi: 10.1002/cphy.cp060109