Biomedical subjects

J M Lindstrom

Publications and source records attributed to J M Lindstrom.

At least 37 records · Page 2Linked to original sources

PubMed1996

Neurogenesis of cholinoceptive neurons in the chick retina.

Immunocytochemistry and [3H]thymidine autoradiography were combined in this study to determine the neurogenesis of cholinoceptive cells in the chick retina. After injections of [3H]thymidine between embryonic days 1 and 11, the time of birth of retinal neurons containing either the alpha 3 or the alpha 8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the alpha 3-positive neurons in the ganglion cell layer leave the cell cycle from E2 through E7, and those in the inner nuclear layer (amacrine and displaced ganglion cells) from E2 through E9. The alpha 8-positive cells in the ganglion cell layer were born from E1 through E7, and those in the inner nuclear layer (amacrine and bipolar cells) from E2 through E11. These data suggest that the time of birth of cholinoceptive neurons in the chick retina follows the general pattern of cell generation in the chick retina, and that alpha 8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the alpha 3-positive cells in the same layer.

Acetylcholine↗

PubMed1994

Effects of retinal lesions upon the distribution of nicotinic acetylcholine receptor subunits in the chick visual system.

Immunohistochemistry was used in this study to evaluate the effects of retinal lesions upon the distribution of neuronal nicotinic acetylcholine receptor subunits in the chick visual system. Following unilateral retinal lesions, the neuropil staining with an antibody against the beta 2 receptor subunit, a major component of alpha-bungarotoxin-insensitive nicotinic receptors, was dramatically reduced or completely eliminated in all of the contralateral retinorecipient structures. On the other hand, neuropil staining with antibodies against two alpha-bungarotoxin-sensitive receptor subunits, alpha 7 and alpha 8, was only slightly affected after retinal lesions. Decreased neuropil staining for alpha 7-like immunoreactivity was only observed in the nucleus of the basal optic root and layers 2-4 and 7 of the optic tectum. For alpha 8-like immunoreactivity, slight reduction of neuropil staining could be observed in the ventral geniculate complex, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, and nucleus suprachiasmaticus. Taken together with previous data on the localization of nicotinic receptors in the retina, the present results indicate that the beta 2 subunit is transported from retinal ganglion cells to their central targets, whereas the alpha 7 and alpha 8 subunit immunoreactivity appears to have a central origin. The source of these immunoreactivities could be, at least in part, the stained perikarya that were observed to contain alpha 7 and alpha 8 subunits in all retinorecipient areas. In agreement with this hypothesis, the beta 2 subunit of the nicotinic acetylcholine receptors was not frequently found in perikarya of the same areas.

Afferent Pathways↗

PubMed1994

Differential development of alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina.

The development of cells containing neuronal nicotinic receptors (nAChRs) in the chick retina was investigated by means of immunohistochemical techniques with antibodies directed against the alpha 3 and alpha 8 nAChR subunits. The alpha 3 subunit is one of the major alpha-bungarotoxin-insensitive nicotinic receptor subunits in the chick retina, whereas alpha 8 appears to be the most common alpha-bungarotoxin-sensitive subunit in the same structure, alpha 3-like immunoreactivity (alpha 3-LI) was first detected in cells of the vitreal margin, on the embryonic day 4.5 (E4.5). alpha 8-LI was first detected in the same type of cell almost a day later. However, the processes of alpha 8-LI cells developed much faster than those of alpha 3-LI cells, generating visible stained laminae in the prospective inner plexiform layer as early as E7. alpha 3-LI was only clearly seen in laminae of the inner plexiform layer by E12. By this date, both alpha 3 and alpha 8-LI were seen in the same types of cells as in the adult retina, i.e., amacrines, displaced ganglion cells, and cells of the ganglion cell layer for alpha 3-LI; and amacrines, bipolar cells, and cells of the ganglion cell layer for alpha 8-LI. These results reveal different patterns of development of cells containing the alpha 3 and alpha 8 nAChR subunits in the chick retina and indicate that those nAChR subunits are expressed in the chick retina before choline acetyltransferase-positive cells can be detected and well before synaptogenesis. These data also suggest that nAChRs may have a developmental function in the retina.

Animals↗

PubMed1993

Myasthenia gravis: effect on antibody binding of conservative substitutions of amino acid residues forming the main immunogenic region of the nicotinic acetylcholine receptor.

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR alpha-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence alpha 67-76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence alpha 67-76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure. Conservative substitutions of residues Asn68 and Asp71 greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp70 and Tyr72 moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position alpha 70 or alpha 71. Substitutions at residues Asp/Ala70 and Val/Ile70 between human and Torpedo alpha-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.

Amino Acid Sequence↗

PubMed1993

Amino acid residues within the sequence region alpha 55-74 of Torpedo nicotinic acetylcholine receptor interacting with antibodies to the main immunogenic region and with snake alpha-neurotoxins.

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha-toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, Asp71, or Tyr72 had marginal effects. Substitutions within the region alpha 68-72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp60 substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp62 slightly but consistently affected binding of mAbs 6 and 22.

Amino Acid Sequence↗

PubMed1992

Neurons of the chick brain and retina expressing both alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis.

Immunohistochemical methods were used to study the possible co-localization of two alpha-bungarotoxin-sensitive (alpha 7 and alpha 8) and two alpha-bungarotoxin-insensitive (beta 2 and alpha 3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the alpha 7 subunit and the beta 2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the alpha 7 and alpha 8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain alpha 8 and alpha 3 subunits, whereas neurons in the ganglion cell layer were seen to contain alpha 7 and alpha 8 or, less frequently, alpha 7 and alpha 3 subunits. These results indicate that alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.

Animals↗

PubMed1992

Immunohistochemical localization of nicotinic acetylcholine receptor subunits in the mesencephalon and diencephalon of the chick (Gallus gallus).

Monoclonal antibodies against two alpha-bungarotoxin-binding subunits (alpha 7 and alpha 8) of the nicotinic acetylcholine receptors (nAChRs) were used as immunohistochemical probes to map their distribution in the chick diencephalon and mesencephalon. The distribution of the alpha 7 and alpha 8 nAChR subunits was compared to the distribution of immunoreactivity produced by a monoclonal antibody against the beta 2 structural subunit of the nAChRs. Structures that contained high numbers of alpha 7-like immunoreactive (LI) somata included the intergeniculate leaflet, nucleus intercalatus thalami, nucleus ovoidalis, organum paraventricularis, nucleus rotundus, isthmic nuclei, nucleus trochlearis, oculomotor complex, nucleus interstitio-pretecto-subpretectalis, stratum griseum centrale of the optic tectum, and nucleus semilunaris. Neuropil staining for alpha 7-LI was intense in the nucleus dorsomedialis hypothalami, nucleus geniculatus lateralis ventralis, griseum tecti, isthmic nuclei, nucleus lentiformis mesencephali, nucleus of the basal optic root, and stratum griseum et fibrosum superficiale of the tectum. High numbers of alpha 8-LI somata were found in the stratum griseum et fibrosum superficiale of the tectum and the nucleus interstitio-pretecto-subpretectalis, and intense neuropil staining for alpha 8-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griseum et fibrosum superficiale of the tectum. High numbers of beta 2-LI somata were found only in the nucleus spiriformis lateralis, whereas neuropil staining for beta 2-LI was intense in the nucleus geniculatus lateralis ventralis, nucleus suprachiasmaticus, nucleus lateralis anterior, nucleus habenularis lateralis, area pretectalis, griseum tecti, nucleus lentiformis mesencephalis, nucleus externus, and nucleus interpeduncularis, and in the stratum griseum centrale, stratum griseum et fibrosum superficiale, and stratum opticum of the tectum. These results indicate that there are major disparities in the localization of the alpha-bungarotoxin-binding alpha 7 and alpha 8 nAChR subunits and the beta 2 structural nAChR subunit in the chick diencephalon and mesencephalon. These nAChR subunits appear, however, to coexist in several regions of the chick brain.

Animals↗

PubMed1992

Epitope mapping of monoclonal antibodies to Torpedo acetylcholine receptor gamma subunits, which specifically recognize the epsilon subunit of mammalian muscle acetylcholine receptor.

Epitopes for four monoclonal antibodies (mAbs) to the gamma subunit of Torpedo nicotinic acetylcholine receptor (AChR), and one mAb crossreactive with the gamma and delta subunits of Torpedo AChR were mapped using overlapping synthetic peptides corresponding to the complete amino acid sequence of Torpedo gamma subunit. The epitopes for all mAbs were within a 50 residue sequence region, on the cytoplasmic surface of the AChR. Three mAbs crossreacted with mammalian muscle AChRs. Two of them specifically recognized the epsilon subunit of AChRs at adult neuromuscular junction. The epsilon-specific mAbs were used, in conjunction with mAbs specific for the alpha and beta subunits and anti-peptide antisera specific for the epsilon, gamma and delta subunits, to identify in Western blots the subunit complement of embryonic and adult bovine muscle AChRs.

Amino Acid Sequence↗

PubMed1992

Epitope mapping of polyclonal and monoclonal antibodies against two alpha-bungarotoxin-binding alpha subunits from neuronal nicotinic receptors.

Recently, cDNAs for alpha subunits of two different neuronal alpha-bungarotoxin-binding proteins (alpha BgtBP) were isolated from chick brain, designated alpha BgtBP alpha 1 and alpha BgtBP alpha 2. These are now also referred to as subunits alpha 7 and alpha 8, respectively. Expression studies in Xenopus oocytes have indicated that alpha 7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain alpha BgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences alpha 7(327-412) and alpha 8(293-435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequences are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified alpha BgtBPs and the fusion proteins indicates that sequence segments 290-420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured alpha BgtBPs isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbs characterized in these studies will provide useful probes for further studies of alpha BgtBP structure and histological localization.

Amino Acid Sequence↗

PubMed1991

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 microns. Some double-labeled cells ranging from 7-21 microns were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells.

Acetylcholine↗

PubMed1991

Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain.

The relationship between neuronal alpha-bungarotoxin binding proteins (alpha BGTBPs) and nicotinic acetylcholine receptor function in the brain of higher vertebrates has remained controversial for over a decade. Recently, the cDNAs for two homologous putative ligand binding subunits, designated alpha BGTBP alpha 1 and alpha BGTBP alpha 2, have been isolated on the basis of their homology to the N terminus of an alpha BGTBP purified from chick brain. In the present study, a panel of overlapping synthetic peptides corresponding to the complete chick brain alpha BGTBP alpha 1 subunit and residues 166-215 of the alpha BGTBP alpha 2 subunits were tested for their ability to bind 125I-alpha BGT. The sequence segments corresponding to alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were found to consistently and specifically bind 125I-alpha BGT. The ability of these peptides to bind alpha BGT was significantly decreased by reduction and alkylation of the Cys residues at positions 190/191, whereas oxidation had little effect on alpha BGT binding activity. The relative affinities for alpha BGT of the peptide sequences alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were compared with those of peptides corresponding to the sequence segments Torpedo alpha 1-(181-200) and chick muscle alpha 1-(179-198). In competition assays, the IC50 for alpha BGTBP alpha 1-(181-200) was 20-fold higher than that obtained for the other peptides (approximately 2 versus 40 microM). These results indicate that alpha BGTBP alpha 1 and alpha BGTBP alpha 2 are ligand binding subunits able to bind alpha BGT at sites homologous with nAChR alpha subunits and that these subunits may confer differential ligand binding properties on the two alpha BGTBP subtypes of which they are components.

Acetylcholine↗

PubMed1991

Expression of nicotinic acetylcholine receptor subtypes in brain and retina.

Neuronal nicotinic acetylcholine receptors (AChRs) are composed of two types of subunits: ACh-binding (termed alpha 2, alpha 3, alpha 4 ...) and structural (termed beta 2, beta 3, beta 4 ...). AChR subtypes composed of combinations of subunits of these two types encoded by several related genes are expressed in different parts of the nervous system, where they presumably serve different functional roles. Here we identify the ACh-binding subunit of the most prominent chicken brain AChR subtype by N-terminal amino acid sequence and show that it corresponds to the alpha 4 gene. Previously we identified the structural subunit for this AChR subtype from chicken brain as beta 2 by N-terminal amino acid sequence. Thus, this identifies both genes which encode subunits of the major nicotinic AChR subtype in avian brains. By immunoprecipitation, immunohistochemistry, and northern blot analysis we show that alpha 3 (or a very closely related sequence) is expressed at low levels in the brain and relatively high levels in the retina, while alpha 4 is expressed at high levels in the brain and lower levels in the retina. This differential expression indicates that alpha 3-containing 'ganglionic-type' AChRs may be an important AChR subtype in avian retina.

Amino Acid Sequence↗

PubMed1990

Affinity purification of nicotinic acetylcholine receptor from rat brain.

Using chromatography on DE-52 and acetylcholine-Affi-Gel columns, nicotinic acetylcholine receptor was purified to approximately 10,000 fold from Lubrol extract of rat brain with a recovery of 15%. The purified preparation contained no cholinesterase activity. alpha-Bungarotoxin did not inhibit [3H]acetylcholine binding to the purified preparation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 4 major protein bands with apparent molecular weights of 53,000, 67,000, 80,000 and 108,000. When nicotinic acetylcholine receptor was eluted with either carbachol or nicotine from the affinity column, these major bands were found on SDS-PAGE gels. Immunoblot analysis showed that the Mr 80,000 protein was an acetylcholine-binding subunit and that the Mr 48,000 protein, a minor band on SDS-PAGE gel, was a structural subunit.

Acetylcholine↗

PubMed1989

Expression of the four subunits of the Torpedo californica nicotinic acetylcholine receptor in Saccharomyces cerevisiae.

Yeast expression vectors were constructed containing complementary DNA encoding the alpha-, beta-, gamma-, and delta-subunits of the Torpedo californica nicotinic acetylcholine receptor under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter. All four plasmids were integrated into the yeast genome of a single yeast cell. The resulting yeast strain synthesized polypeptides novel to yeast that had the molecular weights and antigenic properties similar to the authentic T. californica receptor alpha-, gamma, and delta-subunits. The beta-subunit polypeptide could not be detected in this yeast strain, even though the poly(A)+ RNA from this strain contained all the information necessary for the expression of functional acetylcholine receptors in Xenopus laevis oocytes. The replacement of the beta-subunit mRNA 5'-untranslated leader and its N-terminal signal sequence by the corresponding alpha-subunit sequences, however, resulted in the expression of the beta-subunit polypeptide in yeast grown at 5 degrees C.

Animals↗

PubMed1989

Nicotinic acetylcholine receptor-like molecules in the retina, retinotectal pathway, and optic tectum of the frog.

Forty-two monoclonal antibodies (mAbs) generated against nicotinic acetylcholine receptors (AChRs) from electric organ were tested for their ability to cross-react in the optic tectum of the frog Rana pipiens. Twenty-eight of the mAbs tested (67%) bound to the optic neuropil of the tectum as revealed by immunoperoxidase cytochemistry. The pattern of peroxidase stain for cross-reacting mAbs corresponded in position to a subset of the retinotectal projections. Electron microscopic examination revealed that peroxidase reaction product was associated with the surface of vesicle-containing profiles but not with synaptic sites. Removal of one retina resulted in the loss of immunoreactivity in the contralateral tectum. AChR-like immunoreactivity was also associated with the optic tract and optic nerve and with retinal ganglion cells. These results indicate that some classes of retinal ganglion cells bear AChR-like molecules on their surface. The existence of these molecules on ganglion cell axons and terminals seems the most likely explanation for the AChR-like immunoreactivity present in the tectum.

Animals↗

PubMed1988

Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata).

We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors (MAb 35 of Tzardos et al., J. Biol. Chem., 250: 8635-8645, '81; and MAb 270 of Whiting and Lindstrom: J. Neurosci. 6: 3061-3069, '86). Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei. Lobus parolfactorius and nucleus vestibularis medialis were labelled by only MAb 270, whereas only MAb 35 labelled nucleus laminaris and the medial and lateral pontine nuclei. These data extend previous reports of cholinergic participation in the song system (Ryan and Arnold: J. Comp. Neurol. 202: 211-219, '81) to suggest that the zebra finch song system may contain several closely related nicotinic receptors. In several brain nuclei it appeared that certain anatomical portions of a nucleus or a certain class of neurons were specifically labelled. Furthermore, in certain cases, the labelling appeared to be clustered around Nissl-stained cell nuclei, thus suggesting that the receptors are concentrated on or in somata.

Animals↗

PubMed1988

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes.

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.

Animals↗

PubMed1988

Cholinoceptive neurons in the retina of the chick: an immunohistochemical study of the nicotinic acetylcholine receptors.

Monoclonal antibodies directed against nicotinic acetylcholine receptors (nAChRs) were used to identify and characterize cholinoceptive neurons in the chick retina. Two monoclonal antibodies (mAbs), mAb 210 and mAb 270, stained many neurons in both the inner nuclear layer (INL) and ganglion cell layer (GCL). A class of large labeled cells in the inner INL were positioned at the INL/IPL (inner plexiform layer) border and resembled displaced ganglion cells (DGCs). Their identity was confirmed with injections of rhodamine-labeled microspheres into the ventral tectum and nucleus of the basal optic root (nBOR). Four days after the injection, large nAChR-positive neurons in the inner INL were labeled with beads. The distribution of these cells matched that reported for DGCs in the chicken and pigeon (Reiner et al., 1979; Fite et al., 1981). Many smaller cells in the INL also exhibited nAChR immunoreactivity. These cells were not retrogradely labeled after bead injections into retinal recipient areas. Their processes entered IPL where they arborized in a band comprised of the inner leaflet of lamina 1 and all of lamina 2. In some instances, a process continued inward to lamina 4. These neurons were tentatively identified as amacrine cells because of their position and branching pattern. Approximately 12-18% of the cells in the GCL exhibited nAChR immunoreactivity. Many of these cells could be classified as ganglion cells as their axons were also labeled following exposure to nAChR antibodies. Their distribution mirrored that of all ganglion cells with a higher density of cells in the central retina than in the periphery (Ehrlich, 1981). A "double label" technique was used to compare the distribution of nAChR-positive neurons with that of the choline acetyltransferase-positive (ChAT), cholinergic neurons in the chick retina. The two antigens were visualized with two different fluorophores: FITC and RITC. We were unable to find any cells in either the INL or GCL that exhibited both ChAT- and nAChR-like immunoreactivity. The nAChR-positive cells and the ChAT-positive cells both arborized in two bands within the IPL. The patterns were in perfect register in the inner IPL (lamina 4). But, in the outer IPL, the nAChR-positive dendrites were observed in the inner leaflet of lamina 1 and in all of lamina 2 while the ChAT-positive dendrites did not extend into the innermost portion of lamina 2.

Acetylcholine↗