Biomedical subjects

M Conrath

Publications and source records attributed to M Conrath.

32 records · Page 2Linked to original sources

PubMed1992

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.

Animals↗

PubMed1991

Noradrenaline-like terminals in the cat nucleus ventralis posterior of the thalamus.

Noradrenaline-like immunoreactivity in the cat nucleus ventralis posterior of the thalamus was investigated using an indirect immunocytochemical technique. Specific antinoradrenaline antibodies, raised in rabbits, were used. It was first verified that these antibodies recognize noradrenaline cells bodies of the locus coeruleus and their ascending axons in the ascending noradrenergic tract. In the nucleus ventralis posterior itself, noradrenaline-like fibers were observed. They were either randomly distributed or grouped around nonlabeled cell bodies. These neurons were generally oblong and measured 60-80 microns. With electron microscopy, preliminary results showed immunoreactive fibers in close apposition to unlabeled cell bodies or dendrites. The precise nature of these profiles was sometimes difficult to ascertain, since experiments were done in presence of detergent. In some cases symmetric synapses might be observed between immunoreactive axon terminals and unlabeled dendrites. The specificity of the reaction is discussed in the light of several control experiments.

Animals↗

PubMed1991

Cholecystokinin-like immunoreactivity in the rat spinal cord: effects of thoracic transection.

A study of cholecystokinin-like immunoreactivity in the lumbar (L1-L5) spinal cord segments of rats was realised 24-48 hours after complete thoracic transection (T6-T8). A comparison was made with corresponding spinal cord segments from control and sham-operated animals. The immunocytochemical study with light microscopy showed cholecystokinin-like immunoreactive cell bodies in laminae VII and X at L1-L5, caudal to the transection. In addition, the immunoreactivity was greatly enhanced in bundles of the dorsolateral funiculus compared to sham-operated animals. Our results suggest that part of cholecystokinin-like cell bodies of laminae VII and X send projections to supraspinal sites. Some of these supraspinal projections would go through the dorsolateral funiculus. In the lumbar dorsal horn of operated animals, the immunoreactivity was greatly enhanced in lamina I, while it was slightly decreased in lamina II, compared to control animals. Using electron microscopy, in lamina I, the immunoreactivity localized in different neurites was generally very intense. Moreover, axon terminals showed swelling: their mean size was 0.8-1.8 microns (0.5-1.2 in control animals). This result suggests that some cholecystokinin-like neurons also project to lamina I of rostral cervical segments. In lamina II, numerous degenerating axons were observed (24 hours after thoracic spinal transection). This would suggest that part of descending cholecystokinin-like projections terminate in lamina II.

Animals↗

PubMed1990

Evidence for Pro-dermorphin processing products in rat tissues.

Dermorphin (Tyr-D.Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D.Met-Phe-His-Leu-Met-Asp-NH2), two powerful opioid peptides issued from a common biosynthetic precursor, were recently isolated from the skin of the frog phyllomedusa sauvagii. Since many amphibian's skin secretory peptides or their homologues are found in the mammalian central nervous system and gastrointestinal tract, dermorphin and dermenkephalin may have counterparts in mammals. We have prepared antibodies directed against dermorphin, dermenkephalin and the spacer sequence lying between them in the progenitor form and developed sensitive enzyme immunoassays that detect under the picogram level to verify the occurrence of these peptides in rat tissues. Immunocytochemical studies of rat brain sections revealed a similar distribution of immunoreactivities both at the regional and the cellular levels when either one of these three anti-sera was used. Characterization of the immunoreactive peptides was achieved with molecular sieve filtration followed with Reverse Phase High Performance Liquid Chromatography of various rat tissues extracts. Identification was achieved by immunological analysis and chromatographic comparison with synthetic peptides. Immunoreactive materials corresponding to dermorphin, dermenkephalin and the spacer peptide were detected in either brain, stomach or intestine, indicating processing of the dermorphin precursor in these tissues. Immunoreactive species of higher Mr were also detected in all three tissues and may represent extended forms or homologous peptides.

Amino Acid Sequence↗

PubMed1990

Use of anti-idiotypic antibodies as probes for in vitro and in vivo identification of substance P receptor.

In order to develop immunological tools for studying the receptor of the neuropeptide substance P (SP), anti-idiotypic antibodies (anti-Id abs) were produced by immunization with anti-SP antibodies whose specificity was close to that of the SP receptor. Immunological studies revealed structural similarity between some anti-Id abs and SP itself. As a consequence, these anti-Id abs were able to bind to mammalian SP receptors. These antibodies were used in immunocytochemistry to label SP receptors both in the rat spinal cord and in rat and guinea pig peripheral tissues (parotid gland and trachea, respectively). Like SP, anti-Id abs were able to trigger protein secretion by isolated rat parotid gland cells. Finally, it was shown that anti-Id abs in vivo modulated reactivity to chemical stimuli. These antibodies therefore appear to be promising tools for further biochemical, cytochemical, and pharmacological characterization of SP receptors.

Animals↗

PubMed1990

Cholecystokinin-like immunoreactivity in the dorsal horn of the rat spinal cord: an attempt to analyse contradictory results between immunocytochemistry and radioimmunoassay.

This study investigated the nature of the cholecystokinin-like immunoreactivity (CCKLI) in the dorsal horn of the rat spinal cord. Indeed, using the same antiserum, no variation of radioimmunoassayable CCKLI in the dorsal horn of rats treated neonatally with capsaicin could be seen, whereas the density of immunoreactive CCKLI fibres was greatly reduced in immunocytochemistry (IC). By comparing the technical conditions used in IC and radioimmunoassay (RIA), it could be concluded that the contradictory results obtained by the two techniques were very unlikely due to the fixation of the tissues, but rather to the concentration of the antiserum. First, CCKLI levels in extracts from fresh or 4% paraformaldehyde fixed tissues were not significantly different. Second, glomerular terminals, which are typical terminals of the fine primary afferents were immunoreactive at the surface of the block but unlabelled in its depth, where the concentration of antiserum might gradually reach a value closer to that used in radioimmunoassay. High pressure liquid chromatography analyses of acidic extracts of the dorsal part of the lumbar spinal cord of control rats revealed the presence, besides two major peaks coeluting with sulphated and non-sulphated CCK8, of two minor peaks '1' and '2' accounting for 23% of total CCKLI. Capsaicin treatment resulted in the disappearance of peak 2 and a marked reduction in the proportion of CCKLI in peak 1. Reported data suggested that the CCKLI material present in capsaicin-sensitive neurons, accounting for only about 10% of radioimmunoassayable CCKLI but for a much higher proportion of that detected in IC, was distinct from both genuine CCK and CGRP.

Animals↗

PubMed1989

Effect of DSP4, a neurotoxic agent, on attentive behaviour and related electrocortical activity in cat.

Six behaving cats were administered N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) (i.p. 10 mg/kg), a neurotoxic agent known to destroy noradrenergic endings in the CNS. They were placed, both before (control) and after treatment in 3 different situations, each time for 90 min: (1) a 'neutral' one (N) with no significant stimuli; (2) another one eliciting focused attention (F); (3) a third one, creating a situation of 'expectancy of an event to occur' (E), with the animal usually displaying a posture of quiet waking. Simultaneously, the electrocorticogram (ECoG) was recorded from the sensorimotor and parietal cortex through implanted electrodes. We already knew and were able to confirm that the normal animals (i.e. before treatment) displayed distinct behavioural patterns depending on the situation and distinct accompanying parietofrontal ECoG activities, with a dominance of drowsiness and sleep during N, that of a 36-Hz 'beta' rhythms in condition F, and of 14-Hz 'mu' rhythms in condition E. It was shown that the prevailing attitude of the animals after treatment was now, in all 3 situations, that of 'quiet waking and/or expectancy-like watching', with a large if not exclusive dominance of only one ECoG pattern, namely mu. These changes were considered as due to a release of the mu system from a noradrenergic modulatory blockade, in accordance with some of our previous data. An immunohistochemical study with anti-tyrosine hydroxylase antibody was also performed; it confirmed that after DSP4 treatment there were substantial alterations in the immunoreactivity of locus coeruleus cells, the structure which is likely to be involved in this NAergic control of the mu rhythms and of its concomitant behaviour.

Animals↗

PubMed1988

Immunocytochemical study of Met-enkephalin-like cell bodies in the cat hypothalamus.

The distribution of methionine-enkephalin-like cell bodies in the hypothalamus of the cat was studied using an indirect immunoperoxidase technique. Previously, intraventricular or intratissular injections of colchicine were carried out. Met-enkephalin-like neurons were located in all hypothalamic nuclei. The densest clusters of immunoreactive perikarya were observed in the nucleus arcuatus, median eminence, regio praeoptica and perifornical region, whereas the nucleus supraopticus and hypothalamus anterior had the lowest density. In other hypothalamic nuclei the density of methionine-enkephalin (Met-enkephalin) cell bodies was intermediate.

Animals↗

PubMed1988

Anti-idiotypic antibodies as a tool for cytochemical identification of substance P receptors in the central nervous system.

Anti-idiotypic antibodies may serve as valuable probes for cytological identification of peptide receptors in the CNS. We have previously described the preparation of anti-substance P (SP) anti-idiotypic antibodies (anti-Id Ab) and have shown that they recognize SP receptors. These anti-Id Ab can be used in cytology to label SP receptors in CNS. We chose rat cervical spinal cord as a model because SP is present in large amounts in the dorsal and ventral horns, where it is implicated in pain and in motor function, respectively. After application of an indirect immunoperoxidase technique to tissue sections from perfused animals, immunolabeling was seen in the two superficial layers of the dorsal horn, the area surrounding the central canal, extending along the white matter in lamina VII, and in part of the ventral horn. This localization is in accordance with the classical distribution of SP receptors as seen by autoradiography with labeled SP. In the light of control experiments, as well as of biochemical and pharmacological arguments, we discuss the specificity of the immunolabeling. We conclude that anti-Id Ab recognize NK-P receptors, although crossreaction with NK-A or NK-B receptors cannot be totally ruled out.

Animals↗

PubMed1987

Monoclonal antibodies to substance P: production, characterization of their fine specificities, and use in immunocytochemistry.

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.

Animals↗

PubMed1986

Immunocytochemical study of enkephalin-like cell bodies in the thalamus of the cat.

Using an indirect immunoperoxidase technique, the localization of enkephalin-like cell bodies in the thalamus of the cat was carried out. Enkephalin-like cell bodies are widely distributed in the cat thalamus. However, immunoreactive cells may be regrouped in 4 clusters which do not exactly correlate with the anatomical subdivisions of the thalamus. One is located in the dorsocaudal aspect of the thalamus, another in the midline area, and the others are formed by the nuclei geniculatum mediale and laterale.

Animals↗

PubMed1986

Distribution of Met-enkephalin immunoreactive fibres in the thalamus of the cat.

The Met-enkephalin-like immunoreactivity was studied in the thalamus of the cat using an indirect immunoperoxidase method. The densest network of immunoreactive fibres and terminals was observed in the epithalamus and the intralaminar nuclei, particularly those located along the midline nuclei interanteromedialis, submedius, rhomboidens and reuniens. The nuclei parafascicularis and centrum medianum contained also numerous immunoreactive fibres and terminals, whereas the lamina medullaris externa had a lower density of immunoreactive terminals. Enkephalin fibres were almost totally absent in the lateral nuclei of the thalamus, and in the posterior group only the magnocellular part of the corpus geniculatum mediale contained some immunoreactive fibres.

Animals↗

PubMedDate not supplied

Immunocytochemical study of enkephalin-like cell bodies in the thalamus of the rat.

The distribution of enkephalin-like cell bodies in the thalamus of the rat was studied by means of intratissular injections of colchicine and using an indirect immunoperoxidase technique. The densest clusters of immunoreactive perikarya were observed in the nuclei geniculatum lateralis ventralis, medialis dorsalis, centralis lateralis, centralis medialis and anterior ventralis. Whereas the nuclei praetectalis lateralis, lateralis posterior, habenularis lateralis, parataenialis (its caudal part), parafascicularis, centrum medianum, reuniens and ventralis medialis had the lowest density. In other thalamic nuclei geniculatum mediale, paraventricularis and parataenialis (its rostral part) the density of enkephalin-like cell bodies was intermediate. These results suggest that the intratissular injection of colchicine is the better way of administration of the drug in order to study the distribution of peptidergic cell bodies in the mammalian CNS. The similarities and differences found in the distribution of enkephalinergic cell populations in the thalamus of different mammals are discussed.

Animals↗

PubMedDate not supplied

Immunocytochemical evidence for calcitonin gene-related peptide-like neurons in the dorsal horn and lateral spinal nucleus of the rat cervical spinal cord.

Calcitonin gene-related peptide (CGRP) in the dorsal horn of the rat spinal cord was assumed until now to be principally of primary afferent origin. It is shown here, on the basis of both light and electron microscopic immunocytochemical evidence, that some cell bodies of the dorsal horn and lateral spinal nucleus (LSn) of the rat cervical spinal cord contain a CGRP-like immunoreactivity. At the light microscopic level, immunoreactive cell bodies were observed in animals pretreated with colchicine injected intraventricularly, CGRP-like cell bodies were morphologically heterogeneous and distributed in the three superficial layers of the dorsal horn. They were very rare in lamina I and more numerous in laminae II and III. A group of immunoreactive cell bodies was also observed in the LSn. Using electron microscopic techniques, a few immunoreactive cell bodies were observed even in control animals. In addition, relatively numerous immunoreactive dendrites were observed in lamina II. The specificity of the reaction and the physiological implications of the results are discussed.

Animals↗