Intrinsic innervation patterns of the smooth muscle in the rumen and reticulum of lambs.

The rumen and reticulum of sheep serve as a fermentation chamber. Both compartments exhibit specific motility patterns. With developmental changes, the size of the reticulorumen dramatically increases when newborn lambs mature to adult sheep. This makes it possible to investigate the intrinsic innervation of the reticuloruminal muscles in lambs by taking the entire reticulum and rumen into account. The aim of the study was to analyse the projections and neurochemistry of myenteric neurons in the rumen and reticulum, which project to the inner or outer muscle layer, respectively. Therefore, we applied retrograde tracing with the fluorescent dye 1,1'-didodecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and subsequent immunohistochemical detection of choline acetyltransferase (ChAT), substance P (SP) and vasoactive intestinal peptide (VIP). In both compartments innervation of both the inner and the outer muscle layer consisted mainly of cholinergic neurons (65-70%). The majority of them co-localized SP. The non-cholinergic neurons projecting to the muscle expressed immunoreactivity for VIP. Polarized innervation of the muscle layers was found neither in the rumen nor in the reticulum. Consequently, intrinsic innervation patterns for the smooth muscle layers in the rumen and reticulum differ from all gastrointestinal regions examined thus far.

Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep.

The mycotoxic tremorgens penitrem, paxilline and lolitrem B had profound effects on electromyographic (EMG) activity of smooth muscle of the reticulorumen in conscious sheep, with a similar time course of action to their respective characteristic effects on the induction (1 to 2, 15 to 20 and 20 to 30 minutes) and the duration (1 to 2, 1 to 2 and 8 to 12 hours) of tremoring. Responses to penitrem revealed a greater sensitivity of smooth muscle than skeletal muscle. Effects included an inhibition of the vagally-dependent cyclical A and B sequences of contraction of the reticulorumen, an increase in their amplitude and an excitation of local intrinsic activity contributing to elevated baselines and the occurrence of chaotic activity of the reticulum. The excitatory local effects were partially blocked by atropine, indicating that stimulation of muscarinic cholinoceptors was involved. Increased local activity may mediate a reflex inhibition of cyclical contractions. A non-tremorgenic isomer of lolitrem B (31-epilolitrem B) had no effect on the reticulorumen. The intensity and duration of the effects of lolitrem B, up to 12 hours, indicate that severe disruption of digestion may occur in animals grazing endophyte-infected pasture.

Antagonist of nitric oxide synthesis inhibits nerve-mediated relaxation of isolated strips of rumen and reticulum.

The present study investigated the possibility that nitric oxide is a nonadrenergic, noncholinergic neurotransmitter of nerves that are intrinsic to the forestomach. Tunica muscularis, myenteric plexus preparations of bovine reticulum and rumen were maintained in vitro in a physiological solution of buffer that contained scopolamine. Trains of electric field stimulation transiently reduced (relaxed) the tone induced by BaCl2. NG-Nitro-L-arginine methyl ester, a nitric oxide synthase competitive antagonist, inhibited relaxation of the rumen and reticulum preparations that had been induced by the electrical field. The actions of NG-nitro-L-arginine methyl ester were partially reversed by L-arginine. These data suggest that nitric oxide, or a related substance, is an inhibitory neurotransmitter of nerves that are intrinsic to tunica muscularis, myenteric plexus preparations of the bovine forestomach.

Structural differences of the enteric nervous system in the cattle forestomach revealed by whole mount immunohistochemistry.

The specific motility patterns of the forestomach of ruminants, composed of three structurally distinct compartments (rumen, reticulum, omasum), require an elaborate intramural innervation. To demonstrate the complex structure of the enteric nervous system (ENS), whole mount preparations obtained from different sites of the bovine forestomach were submitted to immunohistochemical procedures in which neuronal (protein gene product 9.5, neurofilament 200) and glial (protein S-100, glial fibrillary acid protein) markers were applied. Immunohistochemistry performed on whole mounts allowed a detailed two-dimensional assessment of the architecture of the intramural nerve networks. Generally, the myenteric and submucosal plexus layers were composed of ganglia and interconnecting nerve fiber strands, whereas the mucosal plexus consisted of an aganglionated nerve network. However, the texture of the ENS showed considerable regional differences concerning the ganglionic size, shape and density and the arrangement of nerve fiber strands. The myenteric plexus of the ruminal wall, showing a low ganglionic density and wide polygonal meshes, contrasted with the nerve network within the ruminal pillar which consisted of ropeladder-like nerve fiber strands and parallel orientated ganglia. The highest ganglionic density was observed at the reticular groove, the most prominent ganglia were found within the omasal wall. Branches of the vagal nerve frequently ramified within the myenteric plexus layers. The submucosal plexus of the rumen was divided into an external and internal layer; the reticular submucosal plexus followed the cristae and cellulae reticuli, the omasal submucosal (sublaminar) plexus showed intra- and parafascicular ganglia apart from ganglia located at the junctions of the nerve network. The mucosal plexus of the rumen consisted of thin nerve fascicles ramifying between the ruminal papillae, and reticular mucosal nerve fibers passed throughout the base of the cellulae reticuli. The highly specialised nerve network of the intralaminar omasal plexus showed radial and transverse trajectories reflecting the spatial arrangement of the intralaminar musculature. The demonstrated structural complexity of the ENS reflects the functional complexity of the ruminant forestomach and indicates the relatively high degree of autonomy in coordinating the different motility patterns required for the processing of the ingesta.

Neuropeptides in the myenteric ganglia and nerve fibres of the forestomach and abomasum of grey, white and black Karakul lambs.

Previous studies indicated large, thin-walled, milk-filled rumens in lethal grey and white Karakul lambs. There was also a significant decrease in the number and size of the myenteric plexuses and the number of ganglion cells in these lambs. The purpose of this study was to determine whether the myenteric ganglia of the affected lambs are functional, by testing for the presence of vaso-active intestinal peptide, somatostatin, neurotensin, neuropeptide Y, met-enkephalin, calcitonin gene-related peptide and substance P in the myenteric ganglia and nerve fibres in the forestomach and abomasum of grey, white and black Karakul lambs. Four 1-cm2 samples were taken from analogous areas of the wall of the rumen, reticulum, omasum and abomasum of five grey, five white and five black newborn Karakul lambs. They were pinned to wax squares, fixed for 18 h in Zamboni's fixative, dehydrated and rehydrated through graded alcohols and stored in phosphate-buffered saline. The outer longitudinal muscle layer of each sample of the rumen, reticulum, omasum and abomasum was separated from the rest of the tissue layers, stained for each of the seven neuropeptides by employment of the immunofluorescence technique, and studied with a Leitz Orthoplan fluorescent microscope. All the material studied tested positive for all the neuropeptides. It is concluded that all the peptides tested for were present in all the lambs and that the myenteric ganglia are therefore functional in the lethal lambs.

Substance P-immunoreactive neurons of the bovine forestomach mucosa: their presumptive role in a sensory mechanism.

Substance P-immunoreactive nerve fibers and cell bodies were examined in the forestomach mucosa of the calf and cow pretreated with colchicine, using thick (100 microns) floating sections. Intraepithelial nerve fibers were identified, appearing only rarely in the rumen and reticulum, and completely absent from the omasum. Nerve fibers were observed in the lamina propria of all the regions of the forestomach examined. A few thin nerve fibers were seen in the core of the ruminal papillae of the calf, whereas in the cow they appeared very coarse in nature. Flocculent and complicated nerve fiber networks were seen in the connective tissue of the reticular papillae. Mucosal nerve fibers formed a peculiar glomerulus-like architecture in the omasal papillae of the calf, while in the cow, the nerve fibers were largely restricted in distribution to the vicinity of the epithelium within the connective tissue pegs. Immunoreactive nerve cell bodies were found in the ruminal atrium, the dorsal sac and the ventral sac of the rumen of the calf and in the reticulum of both the calf and cow. Some of these neurons exhibited processes that appeared to course toward the papillae. In total, substance P-immunoreactive nerve fibers and cell bodies were more abundant in the calf than in the cow. These distributions demonstrate that the neural circuitry of the bovine forestomach contains substance P immunoreactivity in the mucosa as well as in the muscle layer, pointing to its possible importance in the regulation of the forestomach function. Substance P-immunoreactive nerve fibers were numerous in the reticular papillae of the calf and cow and in the omasal papillae of the calf. The positive fibers at these localities may act as mucosal receptors.

Ultrastructure of the myenteric ganglia in the rumen, reticulum, omasum and abomasum of grey, white and black Karakul lambs.

Homozygous grey and white Karakul lambs die after they have reached weaning age. This is due to a lethal gene causing digestive disturbances. Previous studies revealed that grey and white lambs have large, milk-filled rumens; the phenomenon was attributed to a significant decrease in the number of myenteric ganglia and neurons in the rumen wall. This study was undertaken to determine whether any morphological differences exist in the ultrastructure of the myenteric ganglia in the forestomach and abomasum of grey, white and black Karakul lambs. Samples of the forestomach and abomasum of grey, white and black Karakul lambs were prepared routinely for electron microscopy and studied with a Phillips electron microscope. No morphological differences could be detected in the structure of the components of the myenteric ganglia in the forestomachs and abomasums of grey, white and black Karakul lambs. It was therefore concluded that the lethal gene in grey and white Karakul lambs results in a paucity of the myenteric ganglia, but does not affect the ultrastructure of these structures.

A comparative histological study of the number and size of the myenteric ganglia and neurones in the fore-stomach and abomasum of grey, white and black Karakul lambs.

Homozygous grey Karakul lambs are born with a lethal genetic factor responsible for death at weaning age. When put on a high roughage diet under field conditions they develop distended, thin-walled rumens and sand impacted abomasa. Homozygous white Karakul lambs have a similar factor but survive for a longer period. Black Karakul lambs are not affected. The present study was undertaken to compare by image analysis the number and size of the myenteric ganglia, and the number of myenteric neurones in the walls of the fore-stomach and abomasum of 24-h-old grey, white and black Karakul lambs. One square centimetre samples were taken from analogous areas of the rumen, reticulum, omasum and abomasum of 38 embalmed Karakul lambs. Haematoxylin and eosin stained histological sections of each sample were studied with a Vids 2 Image Analyzer. One way analysis of variance indicated a significant difference between the groups regarding the number and size of the myenteric ganglia and in the number of myenteric neurones in the reticulum, rumen and abomasum. The number and size of the ganglia and the number of neurones was greatest in the black lambs and decreased progressively in the white and grey lambs. The omasum was not affected. It is suggested that the paucity of myenteric ganglia and neurones in the regions examined is instrumental in causing the lethal condition described above.

Ruminal lactic acidosis: forestomach epithelial receptor activation by undissociated volatile fatty acids and rumen fluids collected during loss of reticuloruminal motility.

Forty-three reticuloruminal epithelial receptors, with excitatory receptive fields within the reticulum, were isolated in 19 anaesthetised sheep. The responsiveness of these receptors to acetic, propionic, butyric and DL-lactic acids were assessed as well as their ability to be activated by rumen fluids obtained from sheep with induced ruminal lactic acidosis. Eighteen (41.9 per cent) receptors were excited by all three volatile fatty acids (VFAs) and 17 (39.5 per cent) responded to two VFAs. Butyric acid was the most potent volatile fatty acid eliciting responses in 41 (95.3 per cent) receptors. Acetic acid activated 33 (76.7 per cent) receptors and propionic acid excited 22 (53.5 per cent) receptors. DL-lactic acid only activated 13 (30.2 per cent) receptors, six of which required concentrations of 200 mM to be activated. Rumen fluids obtained when forestomach motility was impaired, activated 37 (81.1 per cent) of the 43 receptors, whereas rumen fluids obtained when forestomach motility was normal did not activate any receptors. Despite the high levels of lactic acid in rumen fluids which activated epithelial receptors, this acid was not responsible for receptor excitation.

[Masticatory cycles of the rumination type in vagotomized lambs]. / Cycles masticatoires de type mérycique chez la brebis vagotomisée.

Contractions of the fluid-filled RR dependent upon the activity of the myenteric plexus were observed within 2 weeks after vagotomy in sheep maintained on a liquid diet. Spontaneous but not adrenergic evoked rumination was recorded beyond the 5th week after vagotomy. The observation of ruminating-like activity during 120 to 200 min per day in chronically-vagotomized sheep indicates that the onset of rumination does not require a gastric afferent pathway.

Distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of cattle.

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of calves and cows was studied, using immunohistochemical methods. Methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves were found in all regions of the bovine forestomach. Cell bodies were found mainly in myenteric ganglia, and nerve fibers were found principally in muscular layers. Nerve fibers and ganglia were concentrated in the lips of the reticular groove, but were least numerous in ruminal and omasal walls. The reduced number of immunoreactive nerves in cows, compared with the number of those in calves, was striking, especially in the lips of the reticular groove and the omasal leaves. Results may indicate that the importance of methionine-enkephalin-Arg6-Gly7-Leu8-containing nerves in the physiologic regulation of the forestomach of the calf is different from that of the cow.

Hexamethonium: a probe to assess autonomic nervous system involvement in upper gastrointestinal functions in conscious sheep.

Hexamethonium, which inhibits cholinergic transmission by preventing acetylcholine release, has been considered an ideal reference drug for the blockade of autonomic ganglia, Auerbach plexus and reflex gastrointestinal secretions. The degree of inhibition of ruminant gastrointestinal functions with this reference drug were as follows: cyclical contractions of the reticulo-rumen and abomasal motility greater than gastric acid secretion and duodenal migrating myoelectrical complexes. Although reduced at high dosages, the initiation of migrating myoelectric complexes was enhanced at clinically used dosages. The duration of the inhibition of reticular contractions was dose-related varying from 0.5 to 5 h for 1.25 to 20 mg/kg subcutaneously. Abomasal motility and acid secretion were similarly reduced but exhibited strong and long-lasting rebound effects. Inhibition of the reticulum by the blockade of muscarinic receptors by atropine was also dose-related lasting from 0.5 to 3 h for 0.5 to 2 mg/kg, whereas inhibition of the abomasal motor and secretory functions lasted from 1 to 6 h. These results suggest a higher degree of impingement of the parasympathetic pathways on abomasal acid secretion and motility than on the cyclical activity of the reticulum and only a modulatory role of the extrinsic neural activity on the cyclical motor events of the duodenum.

Assessment of alpha-2 adrenergic antagonists on the central nervous system using reticular contraction in sheep as a model.

In contrast to others species, the activity of ruminant forestomach (reticulum) is under the control of the central nervous system. Xylazine, a potent alpha-2 agonist, was shown to inhibit reticular activity. The potential usefulness of this physiological response to evaluate drugs acting on central adrenoreceptors was assessed. In conscious sheep, reticular activity was recorded either by electromyographic or manometric techniques. It was demonstrated that xylazine inhibited reticular contraction in a dose-dependent manner. This inhibition, which displayed limited intra- and intersubject variation, was related to a central action of xylazine because vagal stimulation was able to elicit reticular contraction. The so-called specific alpha-2 antagonists (tolazoline and yohimbine) or nonspecific (phentolamine) antagonists reduced xylazine inhibition. In contrast, the so-called alpha-1 antagonists (chlorpromazine, chlorprothixene, nicergoline, prazosin and thymoxamine) were without effect. Schild plots for the antagonists tolazoline and yohimbine in competition with xylazine were linear and had slopes that were not significantly different from unity, indicating competitive antagonism. These results suggest that reticular contractions, a discrete and periodic phenomenon of central origin easily recorded in conscious sheep, could be a useful physiological model to assess the effect of drugs on the adrenoreceptors of the central nervous system.

Central projections of gastric afferent vagal inputs.

1. The medulla oblongata of thirteen halothane-anaesthetized, decerebellate sheep was explored systematically with recording micro-electrodes.2. The nervous activity recorded was designated either ;cyclical', if it was related to the periodic vagal outflow responsible for primary cycle movements of the reticulo-rumen, or ;afferent-like', if it was directly related to mechanical stimulation of the reticulum or the abomasum.3. Forms of mechanical stimulation were used which were known (from earlier ;single afferent fibre' studies) to evoke characteristic and distinguishable responses from four receptor types, i.e. reticular tension receptors, reticular epithelial receptors, abomasal tension receptors and abomasal mucosal receptors.4. Micro-electrode penetrations were made within a zone 4 mm lateral to the mid line, 6 mm rostral to and 2.5 mm caudal to the obex. In certain regions, ;afferent-like activity' was recorded, which corresponded to the discharge properties of one or more of the above receptor types.5. Units were sometimes excited or inhibited by more than one kind of receptor excitation, thus demonstrating the possibilities of a convergence of afferent projections and either a direct or a reciprocal relationship between receptor activity and central nervous ;afferent-like activity'.6. It is concluded that most, if not all, of the ;afferent-like activity' was recorded from the vicinity of interneurones.7. There was a partially overlapping viscerotopic organization of ;afferent-like activity' derived from abomasal and from reticular receptors.8. Regions showing ;afferent-like activity' were located dorso-laterally to regions showing ;cyclical activity' although there was some overlap between the two regions.9. ;Afferent-like activity' was recorded from regions which included the dorso-lateral part of the dorsal vagal motor nucleus, the adjacent reticular formation and the nucleus of the solitary tract between transverse planes 3 mm rostral to the obex and 1 mm caudal to the obex.10. Some of the gastric afferent vagal inputs were projected to contralateral locations via unidentified commissural connexions.

The effects of peripheral and central nervous influences on gastric centre neuronal activity in sheep.

1. Responses of identified vagal reticulo-ruminal motoneurones and gastric centre interneurones to changes in vagal afferent activity were examined in anaesthetized, decerebellate sheep.2. Procedures which reflexly modified the form of forestomach movements caused corresponding changes in the activities of motoneurones and Type A interneurones, whereas the activity patterns of Type B, and many Type C, interneurones were not affected.3. Distension of the pyloric region of the abomasum reduced the number of spikes in the periodic discharges of gastric centre neurones (motoneurones and Type A interneurones) with reticular activity, although the frequency of periods of activity was often increased. The afferent pathway for both effects was probably vagal.4. Unilateral vagotomy usually had little effect on the frequency and amplitude of forestomach movements, and did not influence the temporal relation between ipsilateral gastric centre discharges and the movements.5. Median division of the medulla oblongata only in the region between the gastric centres caused a loss of synchronization in the activities of the two centres, indicating the existence of commissural connexions at this level.6. Bilateral vagotomy abolished forestomach movements and motoneuronal activity, but rhythmic activity in gastric centre interneurones continued with a periodicity of approximately 1 min. This persisting periodic activity was unaffected by spinal section, but was not present after transection of the brain stem rostral to the medulla.7. Cyclical gastric centre activity could be elicited by reticular distension in preparations in which the medulla oblongata was isolated from higher regions of the brain, but, in contrast to many sheep in which the brain stem was intact, the existence of the activity was totally dependent upon peripheral afferent activity.8. The evidence indicates that medullary neurones responsible for periodic activation of vagal preganglionic reticulo-ruminal motoneurones may be excited by either or by both vagal afferent fibres from the fore-stomach or by descending, as yet unidentified, influences from the central nervous system.9. Possible roles for gastric centre interneurones in neural networks which control the periodic activation of motoneurones and which control the form of individual activity cycles are discussed.

Effects of stimulation of efferent fibres of the vagus on the reticulo-omasal orifice of the sheep.

1. The reticulo-omasal orifice has been observed and its activity recorded in decerebrate and spinal preparations of lambs and in lambs and sheep anaesthetized with chloralose.2. Spontaneous closure movements at about 6/min were seen, or recorded from balloons placed in the orifice in all types of preparation when both vagus nerves had been cut and after atropine, hexamethonium, phentolamine and propranolol had been given I.V.3. The effects on the reticulo-omasal orifice of stimulation in the neck of efferent fibres of the vagus nerves on either side differed according to the form of the stimulus applied.4. Threshold stimulation of the vagus nerve caused closing of the orifice which was blocked by atropine.5. Opening of the orifice and disappearance of spontaneous closing movements followed more intense stimulation of the vagus nerves.6. Vagal opening effects were obtained after atropine was given and after adrenergic opening and closure effects had been blocked with phentolamine and propranolol.