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Cholinergic neurones in the submucosal plexus mediate reflex vasodilation in guinea pig ileum
202
fEPSPs during distension and often continuing for some seconds after distension. On the basis of synaptic responses to transmural electrical stimulation and direct soma depolarisation, the cells were classified as S-neurones. Responses to distension were observed in only 71% (48/68) of S-neurones; the short latency 'burst-type' response occurred in 42% of these (20/48). Subsequent immunohistochemical examination revealed that 60% (21/35) of S-neurones responding to distension were enkephalin-immunoreactive (Enk-IR) in contrast to 40% (75/189; 69 neurones tested for distension and 120 in another study) of S-neurones as a whole. The proportion of Enk-IR-S-neurones responding to distension with a short latency response was twice that of non-Enk-IR neurones of this type. The high probability that an S-neurone responding to distension is Enk-IR would appear to indicate that Enk-IR neurones are involved in this
intrinsic reflex. The existence of a population of Enk-IR myenteric plexus neurones projecting anally to the circular muscle [1] and the correlation of the short latency response in neurones and UPs in smooth muscle with the onset of distension would seem to indicate that the Enk-IR neurones are likely to be inhibitory motor neurones. The financial assistance of the Medical Research Council and SmithKline (1982) Foundation is gratefully acknowledged. DJL was supported by Medical Endowments, University of Aberdeen.
References 1 Furness, J.B., Costa, M. and Miller, R.J., Neuroscience, 8 (1983) 653-664. 2 Bornstein, J.C., Costa, M., Furness, J.B. and Lees, G.M, J. Physiol., 351 (1984) 313-325. 3 Hirst, G.D.S. and McKirdy, H.C., J. Physiol,, 238 (1974) 129-143.
neurones in the saltt etml in guinea ilig iletan Steve Vanner and Annmarie
reflex vasmllla¢l
Surprenant
Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, U.S.A.
Mechanical or chemical stimulation of the intestinal mucosa is known to produce a reflex vasodilation of submucosal arterioles. Enteric neurones have been implicated in this reflex but the pathways involved in this response have not been determined. We examined this question by using an in vitro preparation from guinea pig ileum which consisted of a segment of submucosal plexus in which overlying mucosa had been stripped away adjacent to a segment of plexus xn which the mucosa was left intact. Visceral smooth muscle and myenteric plexus were removed in all preparations. The outside diameter of submucosal arterioles was monitored using Diamtrak software [Nield.
T.O., Blood Vessels. 26 (1989) 45-52]. The mueosa was lightly stroked with a feather or fine paint brush attached to a mieromanipulator. Mechanical stimulation of the mucosa caused arteriolar vasodilation in vessels which had been preconstricted with vasopressin or phenylephrine; the amplitude of the vasoditation increased with the number of strokes applied to the mueosa. Tetrodotoxin (0.5 # M ) abolished the vasodilation produced by mucosal stimulation (n = 7), as d i d t h e muscarinic receptor antagonist, 4 - D A M P (200 riM, n = 9); hexamethonium (200 ~ M ) did not alter the response. Mucosal stimulation had no effect on arteriolar diameter in vessels which had not been preconstricted. Vasodilation in response to
203
mucosal stimulation was observed in preparations in which the sympathetic nerve supply had been surgically removed five days prior to the experiment. We suggest the following reflex pathway is present in guinea pig ileum:
ACh
submuco~sal
/
~--J
mucos~
Regeneration of severed nerve plexuses in the guinea pig small intestine at lesions produced by argon laser irradiation S. Kobayashi 1, T. Uchida 1, S. Torihashi, H. Kuramoto 1, M. Suzuki 2 and T. Nishisaka 3 I Department of Anatomy, Yamanashi Medical College, Yamanashi 409-38, z Yamanashi-Gakuin Junior College, Kofu 400, and ~ Medical Physics Laboratory, Health Science Center, Tokyo University of Agriculture and Technology, Tokyo 184, Japan
Our recent histological and immunohistological studies have shown that in the guinea pig small intestine the autonomic end apparatus consists of a glial cell framework and bundles of axonal terminals. The glial cells containing S-100b protein are arranged in a three dimensional lattice [1]. The axonal terminals of different neurones converge into anastomosing bundles which are ensheathed and supported by the glial cell framework. Although the concept of such an autonomic ground plexus has been known for many years, it is unknown whether the nerve plexus and the accompanying glial cells can regenerate after injury. The purpose of our experiments was to determine whether the enteric nerve plexuses can regenerate after injury, how rapidly the regeneration occurs and whether complete recovery takes place. Our strategy was to make 0.5 m m circular lesions in the nerve plexus using Argon laser irradiation of the serosal surface of the guinea pig small intestine. From 3 hours to 120 days thereafter the nerve plexus in and around the lesions was investigated by immunocytochemistry using antisera specific for S-100b (a glial specific protein) and for the neuropeptides methionine-enkephalin-Arg6-GlyV-LeuS(Enk-8), substance P and somatostatin. In addition, tissue blocks stained by the Champy-Maillet (zinc iodide-osmium) method were examined by light microscopy and transmis-
sion electron microscopy and the three-dimensional structure of the severed and regenerating nerve plexuses was examined by scanning electron microscopy. We found that 3 hours after the laser irradiation, a conspicuous accumulation of neuropeptide immunoreactivity occurred in the severed nerve stumps at the periphery of the laser wound. Somatostatin immunoreactivity was present exclusively on the oral side of the lesion, but substance P and Enk-8 immunoreactivities were present on both oral and aboral sides. The neuropeptide immunoreactivity of the axotomized nerve cell bodies in the area surrounding the laser lesion was much stronger 3-10 days after laser irradiation. After 15-70 days, regenerated nerve plexuses had covered the scar tissue in the region of the lesion. In the damaged ganglia, glial cell bodies, but n o t nerve cell bodies, underwent mitosis. The regenerated nerve plexuses consisted of a framework of glial cells immunoreactive for S-100b protein and nerve fibre bundles containing the various neuropeptides. These results suggest that, in the regenerated enteric plexuses, the proliferating glial cells support and guide the nerve fibre bundles which grow from the intact neuronal cell bodies in ganglia around the lesion. Such laser-induced lesions may provide a simple approach for studying the remodelling of the autonomic ground plexus in the enteric nervous system.
fEPSPs during distension and often continuing for some seconds after distension. On the basis of synaptic responses to transmural electrical stimulation and direct soma depolarisation, the cells were classified as S-neurones. Responses to distension were observed in only 71% (48/68) of S-neurones; the short latency 'burst-type' response occurred in 42% of these (20/48). Subsequent immunohistochemical examination revealed that 60% (21/35) of S-neurones responding to distension were enkephalin-immunoreactive (Enk-IR) in contrast to 40% (75/189; 69 neurones tested for distension and 120 in another study) of S-neurones as a whole. The proportion of Enk-IR-S-neurones responding to distension with a short latency response was twice that of non-Enk-IR neurones of this type. The high probability that an S-neurone responding to distension is Enk-IR would appear to indicate that Enk-IR neurones are involved in this
intrinsic reflex. The existence of a population of Enk-IR myenteric plexus neurones projecting anally to the circular muscle [1] and the correlation of the short latency response in neurones and UPs in smooth muscle with the onset of distension would seem to indicate that the Enk-IR neurones are likely to be inhibitory motor neurones. The financial assistance of the Medical Research Council and SmithKline (1982) Foundation is gratefully acknowledged. DJL was supported by Medical Endowments, University of Aberdeen.
References 1 Furness, J.B., Costa, M. and Miller, R.J., Neuroscience, 8 (1983) 653-664. 2 Bornstein, J.C., Costa, M., Furness, J.B. and Lees, G.M, J. Physiol., 351 (1984) 313-325. 3 Hirst, G.D.S. and McKirdy, H.C., J. Physiol,, 238 (1974) 129-143.
neurones in the saltt etml in guinea ilig iletan Steve Vanner and Annmarie
reflex vasmllla¢l
Surprenant
Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, U.S.A.
Mechanical or chemical stimulation of the intestinal mucosa is known to produce a reflex vasodilation of submucosal arterioles. Enteric neurones have been implicated in this reflex but the pathways involved in this response have not been determined. We examined this question by using an in vitro preparation from guinea pig ileum which consisted of a segment of submucosal plexus in which overlying mucosa had been stripped away adjacent to a segment of plexus xn which the mucosa was left intact. Visceral smooth muscle and myenteric plexus were removed in all preparations. The outside diameter of submucosal arterioles was monitored using Diamtrak software [Nield.
T.O., Blood Vessels. 26 (1989) 45-52]. The mueosa was lightly stroked with a feather or fine paint brush attached to a mieromanipulator. Mechanical stimulation of the mucosa caused arteriolar vasodilation in vessels which had been preconstricted with vasopressin or phenylephrine; the amplitude of the vasoditation increased with the number of strokes applied to the mueosa. Tetrodotoxin (0.5 # M ) abolished the vasodilation produced by mucosal stimulation (n = 7), as d i d t h e muscarinic receptor antagonist, 4 - D A M P (200 riM, n = 9); hexamethonium (200 ~ M ) did not alter the response. Mucosal stimulation had no effect on arteriolar diameter in vessels which had not been preconstricted. Vasodilation in response to
203
mucosal stimulation was observed in preparations in which the sympathetic nerve supply had been surgically removed five days prior to the experiment. We suggest the following reflex pathway is present in guinea pig ileum:
ACh
submuco~sal
/
~--J
mucos~
Regeneration of severed nerve plexuses in the guinea pig small intestine at lesions produced by argon laser irradiation S. Kobayashi 1, T. Uchida 1, S. Torihashi, H. Kuramoto 1, M. Suzuki 2 and T. Nishisaka 3 I Department of Anatomy, Yamanashi Medical College, Yamanashi 409-38, z Yamanashi-Gakuin Junior College, Kofu 400, and ~ Medical Physics Laboratory, Health Science Center, Tokyo University of Agriculture and Technology, Tokyo 184, Japan
Our recent histological and immunohistological studies have shown that in the guinea pig small intestine the autonomic end apparatus consists of a glial cell framework and bundles of axonal terminals. The glial cells containing S-100b protein are arranged in a three dimensional lattice [1]. The axonal terminals of different neurones converge into anastomosing bundles which are ensheathed and supported by the glial cell framework. Although the concept of such an autonomic ground plexus has been known for many years, it is unknown whether the nerve plexus and the accompanying glial cells can regenerate after injury. The purpose of our experiments was to determine whether the enteric nerve plexuses can regenerate after injury, how rapidly the regeneration occurs and whether complete recovery takes place. Our strategy was to make 0.5 m m circular lesions in the nerve plexus using Argon laser irradiation of the serosal surface of the guinea pig small intestine. From 3 hours to 120 days thereafter the nerve plexus in and around the lesions was investigated by immunocytochemistry using antisera specific for S-100b (a glial specific protein) and for the neuropeptides methionine-enkephalin-Arg6-GlyV-LeuS(Enk-8), substance P and somatostatin. In addition, tissue blocks stained by the Champy-Maillet (zinc iodide-osmium) method were examined by light microscopy and transmis-
sion electron microscopy and the three-dimensional structure of the severed and regenerating nerve plexuses was examined by scanning electron microscopy. We found that 3 hours after the laser irradiation, a conspicuous accumulation of neuropeptide immunoreactivity occurred in the severed nerve stumps at the periphery of the laser wound. Somatostatin immunoreactivity was present exclusively on the oral side of the lesion, but substance P and Enk-8 immunoreactivities were present on both oral and aboral sides. The neuropeptide immunoreactivity of the axotomized nerve cell bodies in the area surrounding the laser lesion was much stronger 3-10 days after laser irradiation. After 15-70 days, regenerated nerve plexuses had covered the scar tissue in the region of the lesion. In the damaged ganglia, glial cell bodies, but n o t nerve cell bodies, underwent mitosis. The regenerated nerve plexuses consisted of a framework of glial cells immunoreactive for S-100b protein and nerve fibre bundles containing the various neuropeptides. These results suggest that, in the regenerated enteric plexuses, the proliferating glial cells support and guide the nerve fibre bundles which grow from the intact neuronal cell bodies in ganglia around the lesion. Such laser-induced lesions may provide a simple approach for studying the remodelling of the autonomic ground plexus in the enteric nervous system.