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S J Brookes

Publications and source records attributed to S J Brookes.

At least 19 recordsLinked to original sources
PubMed2001

Classes of enteric nerve cells in the guinea-pig small intestine.

The guinea-pig small intestine has been very widely used to study the physiology, pharmacology and morphology of the enteric nervous system. It also provides an ideal, simple mammalian preparation for studying how nerve cells are organised into functional circuits underlying simple behaviours. Many different types of nerve cells are present in the enteric nervous system and they show characteristic combinations of morphological features, projections, biophysical properties, neurochemicals, and receptors. To identify the different functional classes is an important prerequisite for systematic analysis of how the enteric nervous system controls normal gut behaviour. Based on combinations of multiple-labelling immunohistochemistry and retrograde tracing, it has been possible to account quantitatively for all of the neurones in the guinea-pig small intestine. This article summarises that account and updates it in the light of recent data. A total of 18 classes of neurones are currently distinguishable, including primary afferent neurones, motor neurones, interneurones, secretomotor and vasomotor neurones. It is now possible to take an individual nerve cell and use a few carefully chosen criteria to assign it to a functional class. This provides a firm anatomical foundation for the systematic analysis of how the enteric nervous system normally functions and how it goes wrong in various clinically important disorders.

Animals↗
PubMed2001

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum.

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.

Anal Canal↗
PubMed2001

Amelin extracellular processing and aggregation during rat incisor amelogenesis.

Amelin (also known as ameloblastin and sheathlin) is a recently described protein that is secreted by ameloblasts during enamel formation. Here, the extracellular distribution and processing of amelin during rat incisor amelogenesis were investigated by Western blot probing using anti-recombinant rat amelin antibodies. In addition, the solubility behaviour and aggregative properties of rat amelin were investigated using a sequential extraction procedure involving (1) extraction with simulated enamel fluid to extract proteins most likely to be soluble in vivo; (2) extraction with phosphate buffer to desorb proteins bound to enamel crystal surfaces; (3) extraction with sodium dodecyl sulphate (SDS) to extract proteins present as insoluble aggregates; followed by (4) a final acid demineralization step to release any remaining proteins. Proteins immunoreactive to the anti-amelin antibodies were detectable in secretory- and transition-stage enamel. Maturation-stage enamel appeared devoid of amelin. The largest immunoreactive protein detected migrated at 68 kDa on SDS gels, corresponding to the M(r) of nascent amelin. Other immunoreactive bands at 52, 40, 37, 19, 17, 16, 15, 14 and 13 kDa were presumably amelin processing products. The sequential extraction procedure revealed that the 68-, 52-, 40-, 37- and 13-kDa amelins were completely extracted under solution conditions similar to those reported to exist in vivo. In contrast, the 19-, 17- and 16-kDa amelins were only partially extracted, whilst the 15- and 14-kDa amelins could not be extracted with simulated enamel fluid. A proportion of the remaining 17- and 16-kDa amelins was desorbed from the enamel crystals with phosphate buffer and appeared to have been mineral-bound. The 15- and 14-kDa amelins and the remainder of the 17- and 16-kDa amelins were extracted with SDS only, suggesting that these species were present in vivo as an insoluble aggregate. The results provide additional information on amelin processing and degradation, and on how such processing influences the solubility and aggregative properties of amelin-derived proteins.

Ameloblasts↗
PubMed2000

Transduction sites of vagal mechanoreceptors in the guinea pig esophagus.

Extrinsic afferent neurons play an essential role in both sensation and reflex control of visceral organs, but their specialized morphological peripheral endings have never been functionally identified. Extracellular recordings were made from fine nerve trunks running between the vagus nerve and esophagus of the guinea pig. Mechanoreceptors, which responded to esophageal distension, fired spontaneously, had low thresholds to circumferential stretch, and were slowly adapting. Calibrated von Frey hairs (0.12 mN) were used to probe the serosal surface at 100-200 sites, which were mapped on a video image of the live preparation. Each stretch-sensitive unit had one to three highly localized receptive fields ("hot spots"), which were marked with Indian ink applied on the tip of the von Frey hair. Recorded nerve trunks were then filled anterogradely, using biotinamide in an artificial intracellular solution. Receptive fields were consistently associated with intraganglionic laminar endings (IGLEs) in myenteric ganglia, but not with other filled neuronal structures. The average distance of receptive fields to IGLEs was 73 +/- 14 microm (24 receptive fields, from 12 units; n = 5), compared to 374 +/- 17 microm for 240 randomly generated sites (n = 5; p < 0.001). After maintained probing on a single receptive field, spontaneous discharge of units was inhibited, as were responses to distension. During adapted discharge to maintained distension, interspike intervals were distributed in a narrow range. This indicates that multiple receptive fields interact to encode mechanical distortion in a graded manner. IGLEs are specialized transduction sites of mechanosensitive vagal afferent neurons in the guinea pig esophagus.

Animals↗
PubMed2000

The chemical composition of tooth enamel in junctional epidermolysis bullosa.

The junctionalis form of epidermolysis bullosa (EBJ) is associated with a number of clinical problems involving tooth enamel, including increased susceptibility to caries. The aim here was to carry out a chemical characterization of the enamel of teeth from EBJ patients compared with that of unaffected controls. The results showed that while protein concentration, amino acid composition and carbonate content were similar in both groups, EBJ enamel contained a significantly reduced mineral per volume content, resulting in enamel hypoplasia. In addition, Western blotting revealed the presence of serum albumin (a known inhibitor of enamel crystal growth) in EBJ enamel. This was not detected in control enamel or in enamel of teeth from patients with the dystrophic form of the disease. It is concluded that EBJ enamel is developmentally compromised and that the enamel defects are commensurate with the reported genetic lesions.

Amelogenesis↗
PubMed2000

Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies.

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.

Amelogenesis↗
PubMed2000

Evidence for charge domains on developing enamel crystal surfaces.

The control of hydroxyapatite crystal initiation and growth during enamel development is thought to be mediated via the proteins of the extracellular matrix. However, the precise nature of these matrix-mineral interactions remains obscure. The aim of the present study was to use a combination of atomic and chemical force microscopy to characterize developing enamel crystal surfaces and to determine their relationship with endogenous enamel matrix protein (amelogenin). The results show regular and discrete domains of various charges or charge densities on the surfaces of hydroxyapatite crystals derived from the maturation stage of enamel development. Binding of amelogenin to individual crystals at physiological pH was seen to be coincident with positively charged surface domains. These domains may therefore provide an instructional template for matrix-mineral interactions. Alternatively, the alternating array of charge on the crystal surfaces may reflect the original relationship with, and influence of, matrix interaction with the crystal surfaces during crystal growth.

Amelogenesis↗
PubMed2000

The chemistry of enamel caries.

The chemical changes which occur during the process of carious destruction of enamel are complex due to a number of factors. First, substituted hydroxyapatite, the main component of dental enamel, can behave in a very complex manner during dissolution. This is due not only to its ability to accept substituent ions but also to the wide range of calcium phosphate species which can form following dissolution. In addition, the composition, i.e., the extent of substitution, changes throughout enamel in the direction of carious attack, i.e., from surface to interior. Both surface and positively birefringent zones of the lesion clearly illustrate that carious destruction is not simple dissolution. Selective dissolution of soluble minerals occurs, and there is the probability of reprecipitation. The role of fluoride here is crucial in that not only does it protect enamel per se but also its presence in solution means that rather insoluble fluoridated species can form very easily, encouraging redeposition. The role of organic material clearly needs further investigation, but there is the real possibility of both inhibition of repair and facilitation of redeposition. For the future, delivering fluoride deep into the lesion would appear to offer the prospect of improved repair. This would entail a delivery vehicle which solved the problem of fluoride uptake by apatite at the tooth surface. Elucidation of the role of organic material may also reveal putative mechanisms for encouraging repair and/or protecting the enamel mineral.

Animals↗
PubMed1999

Neuronal control of the gastric sling muscle of the guinea pig.

The gastric sling (oblique) muscle (GSM), located close to the lower esophageal sphincter (LES), is involved in gastric motor function and may cooperate with the LES in controlling propulsion between the esophagus and stomach. Neuronal pathways and transmission to the GSM were investigated in isolated esophagus-stomach preparations by using intracellular recording with the focal electrical stimulation and neuroanatomical tracing method. Focal stimulation on the GSM evoked inhibitory junction potentials (IJPs) that were reduced to 45% by 100 microM N-nitro-L-arginine and subsequently blocked by 0.5 microM apamin, thereby unmasking excitatory junction potentials (EJPs), which were abolished by 1 microM hyoscine. Vagal and esophageal stimulation evoked IJPs that were blocked by 100 microM hexamethonium. Vagal stimulation also evoked EJPs after blockade of IJPs. Application of 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate to the GSM labeled muscle motor neurons located in the stomach mainly close to the GSM, with a few neurons (2%) in the esophagus. The majority (79%) of labeled neurons were immunoreactive for choline acetyltransferase and, hence, excitatory motor neurons. Inhibitory motor neurons (nitric oxide synthase immunoreactive; 15%) were clustered in the midline near the gastroesophageal region. These results demonstrate that the GSM is innervated primarily by gastric excitatory and inhibitory motor neurons and some esophageal neurons. Both excitatory (acetylcholine) and inhibitory (nitric oxide and apamin-sensitive component) transmission can be activated via vagal-enteric pathways.

Animals↗
PubMed1999

Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.

1. Peristalsis was evoked in guinea-pig small intestine by slow fluid infusion and recorded onto video and digitized. Spatio-temporal maps of diameter and longitudinal movement were constructed and parameters of motion were calculated. 2. During the filling of the isolated segments of intestine, rhythmic local longitudinal movements were observed at several points along the preparation. These phasic longitudinal muscle contractions were associated with small but significant local increases in diameter and probably reflect a passive mechanical coupling by connective tissue in the gut wall. In addition, occasional synchronized longitudinal muscle contractions caused net shortening of the preparation and always preceded the onset of peristaltic emptying. 3. Peristaltic emptying was characterized by a contraction of the circular muscle which usually started at the oral end of the preparation, that propagated aborally, propelling the contents. However, in 19 % of trials, the first circular muscle contraction occurred in the aboral half of the preparation. 4. The propagation of peristalsis consisted of separate sequential circular muscle contractions several centimetres long, particularly in the oral half of the preparation, giving a 'step-like' appearance to the spatio-temporal map. The gut was transiently distended aboral to the propagating circular muscle contraction due to the propulsion of contents. 5. At each point in the preparation, the longitudinal muscle remained contracted during the propulsive part of the circular muscle contraction. Only when the circular muscle contraction became lumen occlusive did lengthening of the longitudinal muscle take place. 6. Spatio-temporal maps are a powerful tool to visualize and analyse the complexity of gastrointestinal motility patterns.

Animals↗
PubMed1999

Initiation of peristalsis by circumferential stretch of flat sheets of guinea-pig ileum.

1. Segments of isolated guinea-pig intestine, 12 mm long, were distended slowly by intraluminal fluid infusion or by mechanical stretch as either a tube or flat sheet. In all cases, at a constant threshold length, a sudden, large amplitude contraction of the circular muscle occurred orally, corresponding to the initiation of peristalsis. 2. Circumferential stretch of flat sheet preparations evoked graded contractions of the longitudinal muscle (the 'preparatory phase'), which were maintained during circular muscle contraction. This suggests that the lengthening reported during the emptying phase of peristalsis is due to mechanical interactions. 3. The threshold for peristalsis was lower with more rapid stretches and was also lower in long preparations (25 mm) compared with short preparations (5-10 mm), indicating that ascending excitatory pathways play a significant role in triggering peristalsis. 4. Stretching a preparation beyond the threshold for peristalsis evoked contractions of increasing amplitude; thus peristalsis is graded above its threshold. However, during suprathreshold stretch maintained at a constant length, contractions of the circular muscle quickly declined in amplitude and frequency. 5. Circular muscle cells had a resting membrane potential approximately 6 mV more negative than the threshold for action potentials. During slow circumferential stretch, subthreshold graded excitatory motor input to the circular muscle occurred, prior to the initiation of peristalsis. However, peristalsis was initiated by a discrete large excitatory junction potential (12 +/- 2 mV) which evoked bursts of smooth muscle action potentials and which probably arose from synchronized firing of ascending excitatory neuronal pathways.

Animals↗
PubMed1999

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro.

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.

Animals↗
PubMed1999

Projections of nitric oxide synthase and vasoactive intestinal polypeptide-reactive submucosal neurons in the human colon.

BACKGROUND: The submucosal plexus is important in the control of secretomotor and motor function of the intestine. Our aim was to describe the projections of submucosal neurons to the mucosa within the submucosal plexus and to the circular muscle of human colon and to determine whether submucosal neurons that projected to different layers were located at different levels of the submucosa. METHODS: A retrogradely transported fluorescent dye was applied to the mucosa, submucosa or circular muscle layer of human colon which was then maintained in organotypic culture for 5 days. The submucosa was then dissected into two preparations, one containing the inner layer of the submucosal plexus and the other containing both the intermediate and outer layers. The dissected preparations were labelled with antibodies to nitric oxide synthase (NOS) or vasoactive intestinal peptide (VIP). RESULTS: Submucosal neurons projected to the mucosa, submucosa and circular muscle layers for mean distances of 3.7, 3.0 and 4.3 mm, respectively. Ninety-seven per cent of submucosal neurons labelled from the circular muscle were located in the outer or the intermediate layers, while 51% of those projecting to the mucosa were in inner layer and 49% in the intermediate/outer layers of the submucosal plexus. Eleven per cent of submucosal neurons projecting to the circular muscle were immunoreactive for NOS and 12% were immunoreactive for VIP. Forty-five per cent of those projecting within the submucosa were immunoreactive for VIP and 38% of those projecting to the mucosa were immunoreactive for VIP. CONCLUSIONS: Submucosal neurons in the human colon innervate the mucosa, circular muscle and submucosa and different functional classes of neurons are located in different layers of the submucosal plexus.

Adult↗
PubMed1998

Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine.

The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of a single myenteric ganglion, from 2 to 15 DiI-labelled nerve cell bodies were labelled in the submucous plexus up to 7.9 mm circumferentially, 4.5 mm orally, and 3.4 mm aborally to the DiI application site. No cells were labelled in preparations in which connections between myenteric and submucous plexuses had been severed prior to DiI application. Cells that were immunoreactive for vasoactive intestinal polypeptide (VIP) or for substance P (SP) accounted for about 75% and 11% of DiI-labelled cells, respectively. Neither neuropeptide Y- nor calretinin-immunoreactive submucous neurons were labelled by DiI, indicating that these classes of neurons do not project to the myenteric plexus. Retrograde tracing from the myenteric plexus with Neurobiotin revealed that labelled VIP-immunoreactive neurons had several short, filamentous processes and a single long axon that could be followed through the circular muscle to myenteric ganglia without branches to the mucosa. The previously described projection of submucous, SP-immunoreactive putative sensory neurons to the myenteric plexus was confirmed. However, this study has identified a considerably larger population of presumed interneurons that are immunoreactive for VIP that likely transmit information from the submucous plexus to the myenteric plexus and presumably coordinate activity between the two ganglionated plexuses.

Animals↗
PubMed1998

Identification of motor neurons to the circular muscle of the guinea pig gastric corpus.

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.

Animals↗
PubMed1998

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig.

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.

Animals↗