Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris.

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg(-1) chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (< or = 1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field.

Neuronal location of 5-hydroxytryptamine3 receptor-like immunoreactivity in the rat colon.

The presence of 5-hydroxytryptamine(3) receptors on enteric neurons is known from pharmacological data that date back more than 40 years. However, an adequate account of which neurons bear these receptors has not been made because suitable antisera have not been available. We have found that the majority of antisera that have been raised against sequences from the 5-hydroxytryptamine(3) receptor also recognize pre-prosomatostatin. We report that this source of false labeling can be eliminated by pre-incubating the antisera with a peptide designed for this purpose. We have used the pre-absorbed antiserum to localize 5-hydroxytryptamine(3) receptors in the rat colon. Immunoreactive nerve cell bodies occurred in the myenteric and submucosal ganglia. The majority had smooth cell bodies and long, smooth processes, that is, Dogiel type II morphology. The initial segments of the long processes of the Dogiel type II neurons were strongly immunoreactive. About 12% of immunoreactive myenteric nerve cells were of the same or smaller size, and had multiple short filamentous processes. Some of the immunoreactive Dogiel type II neurons were also immunoreactive for calretinin in both plexuses, and the majority were immunoreactive for calbindin in submucosal ganglia. Specific immunoreactivity occurred in non-varicose, but not in varicose, fibers in the myenteric and submucosal ganglia, and in fiber bundles that traversed the longitudinal and circular muscle layers. Immunoreactive varicose fibers were observed only in the mucosa. It is concluded that 5-hydroxytryptamine(3) receptors occur on intrinsic sensory neurons in the rat colon, and on extrinsic sensory nerve fibers that innervate the colon.

Ultrastructural study of relationships between c-kit immunoreactive interstitial cells and other cellular elements in the human colon.

C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".

Ultrastructural analysis of spinal primary afferent fibers within the circular muscle of the cat lower esophageal sphincter.

We have analyzed the ultrastructural characteristics and environment of spinal primary afferent fibers that run within the circular muscle of the cat lower esophageal sphincter. These were selectively labeled by anterogradely transported cholera toxin B subunit conjugated with horseradish peroxidase. Most of the labeled fibers were perpendicular to the muscle cells but some ran sinuously or parallel to the muscle cells. All the labeled fibers were unmyelinated and exhibited relatively rare varicosities. Most of the fibers were in large nerve fiber bundles surrounded by perineurium and probably project to the mucosa. Only some fibers that were in small nerve fiber bundles with no perineurium ran parallel to the musculature and established close relationships with smooth muscle cells. They might be a small subpopulation of the spinal tension receptors, most of the other spinal tension receptors being located in the myenteric plexus area, between the circular and longitudinal muscle.

Ultrastructural relationships of spinal primary afferent fibres with neuronal and non-neuronal cells in the myenteric plexus of the cat oesophago-gastric junction.

Spinal primary afferent fibres innervating the myenteric area in the oesophago-gastric junction of the cat were selectively labelled by anterogradely transported cholera toxin B subunit-horseradish peroxidase conjugate injected into thoracic dorsal root ganglia. The ultrastructure of these labelled primary afferent fibres was studied in order to determine whether they display close relationships with specific cell types in the myenteric plexus. Horseradish peroxidase was revealed with tetramethylbenzidine stabilized with ammonium heptamolybdate or with the tetramethylbenzidine/tungstate reaction in order to visualize the cytoplasmic organelles and the axolemma, respectively. The labelled primary afferent fibres were unmyelinated. Two kinds of profiles of labelled fibres containing vesicles and mitochondrial accumulations were found: (i) fibres running in myenteric connectives in isolated nerve bundles, and (ii) fibres within the myenteric ganglia. The first kind had small areas of axolemma with no glial cell covering, whereas the second kind had little or no glial cell covering (termed naked primary afferent fibres). In addition, labelled fibres containing few vesicles and mitochondria and running in nerve bundles surrounded by perineurium were numerous. Within the myenteric ganglia, naked primary afferent fibres contacted myenteric neurons. The contacts were mainly axosomatic. No synaptic specializations were distinguished. In the interganglionic area, some labelled fibres terminated close to blood vessels. The intraganglionic naked primary afferent fibres are suggested to be mechanoreceptors. Their exposed axolemma might allow both mechanotransduction and release of neurotransmitters which could act on myenteric neurons. Because they are protected by their glial cell sheath and by bundles of collagen fibrils, interganglionic primary afferent fibres are likely to be less exposed to deformation.

Morphological relationships of choleragenoid horseradish peroxidase-labeled spinal primary afferents with myenteric ganglia and mucosal associated lymphoid tissue in the cat esophagogastric junction.

The goal of the present study was to gain insight into the environmental factors influencing the activity of primary spinal afferent fibers in the different layers of the esophagogastric junction of the cat and, thus, to analyze the relationships of these afferents with various cellular components. Spinal primary afferent fibers were selectively labeled by anterogradely transported choleragenoid horseradish peroxidase conjugate (B-HRP). B-HRP was injected into the thoracic dorsal root ganglion at the T8-T13 levels. 6-Hydroxydopamine-induced sympathectomy was performed prior to B-HRP injection in order to prevent otherwise unavoidable labeling of sympathetic fibers in the gut wall. Numerous labeled fibers ran between, around, and within the myenteric ganglia. Others crossed the muscle layers directly and entered the mucosa, where some ran near granulocytes and around or through solitary lymphoid follicles. Labeled fibers were observed in the squamous esophageal epithelium but not in the fundic glandular epithelium. The fibers in the myenteric area are probably connected to the muscular tension receptors that have been detected by electrophysiologic techniques. This assumption is based on the observation that only a few fibers appear to terminate in muscle layers and on the fact that the myenteric area is very narrow and subject to powerful forces. Fibers in the myenteric ganglia could be involved in local efferent functions. Fibers in the mucosa could act as nociceptors and might be involved in local immunological responses.