Kent T. Keyser

Assembly of Human Neuronal Nicotinic Receptor α5 Subunits with α3, β2, and β4 Subunits

Nicotinic acetylcholine receptors formed from combinations of α3, β2, β4, and α5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human α3, β2, β4, and α5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of α3β2, α3β4, α3β2α5, and α3β4α5 subunits but not for other combinations of one, two, or three of these subunits. α5 subunits assembled inside the oocyte with β2 but not with α3 subunits or other α5 subunits. α5 subunits coassembled very efficiently with α3β2 or α3β4 combinations. The presence of α5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also α3 and β2 or α3 and β4 subunits. The presence of α5 subunits increased the rate of desensitization of both receptors containing also α3 and β2 or α3 and β4 subunits. In the case of receptors containing α3 and β4 subunits, the addition of α5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing α3 and β2 subunits, the addition of α5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 μM and the EC50 for nicotine from 6.8 to 1.9 μM, while increasing the efficacy of nicotine from 50% on α3β2 receptors to 100% on α3β2α5 receptors. Both α3β2 and α3β2α5 receptors expressed in oocytes sedimented at the same 11 S value as native α3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma α3, β2, and α5 subunits were co-assembled, and 56% of the receptor subtypes containing α3 subunits also contained β2 subunits. The β2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from α3 and β2 or α3, β2, and α5 subunits rather than the low affinity exhibited by receptors formed from α3 and β4 or α3, β4, and α5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing α3 subunits in SH-SY5Y cells. The affinities of α3β2 receptors expressed in oocytes were similar to the affinities of native α3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Three subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors are expressed in chick retina

A recent report described the isolation of cDNA clones encoding alpha 7 and alpha 8 subunits of alpha-bungarotoxin-sensitive nicotinic ACh receptors (alpha BgtAChRs) from chick brain and demonstrated that they were related to, but distinct from, the alpha subunits of nicotinic ACh receptors (nAChRs) from muscles and neurons. Monoclonal antibodies against the two alpha BgtAChR subunits were used to demonstrate that at least two subtypes are present in embryonic day 18 chicken brain. The predominant brain subtype contains alpha 7 subunits, while a minor subtype contains both alpha 7 and alpha 8 subunits. Both subtypes may also contain other subunits. Here we report the results of immune precipitation studies and immunohistochemical studies of alpha BgtAChRs in the chick retina. In addition to the two subtypes found in brain, a new alpha BgtAChR subtype that contains alpha 8 subunits, but not alpha 7 subunits, was identified and was found to be the major subtype in chick retina. This subtype has a lower affinity for alpha-bungarotoxin (alpha Bgt) than does the subtype containing only alpha 7 subunits. Small amounts of this alpha 8 subtype were also detected in brain by labeling with higher concentrations of 125I-alpha Bgt than had been used previously. The subtype containing only alpha 7 subunits comprised 14% of the alpha BgtAChRs in hatchling chick retina. The subtype containing alpha 8 subunits (but no alpha 7 subunits) accounted for 69%, and the alpha 7 alpha 8 subtype accounted for 17%. Amacrine, bipolar, and ganglion cells displayed alpha 8 subunit immunoreactivity, and a complex pattern of labeling was evident in both the inner and outer plexiform layers. In contrast, only amacrine and ganglion cells exhibited alpha 7 subunit immunoreactivity, and the pattern of alpha 7 subunit labeling in the inner plexiform layer differed from that of alpha 8 subunit labeling. These disparities suggest that the alpha BgtAChR subunits are differentially expressed by different populations of retinal neurons. In addition, the distribution of alpha BgtAChR subunit immunoreactivity was found to differ from that of alpha-Bgt- insensitive nAChR subunits.

Effects of the destruction of starburst-cholinergic amacrine cells by the toxin AF64A on rabbit retinal directional selectivity

The effects of intraocular injections of ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of choline uptake, on the rabbit retina were assessed electrophysiologically, pharmacologically, anatomically, and behaviorally. Survival times from 1 day to 30 days were investigated. After 24 h, the shortest time tested, the directional selectivity of On-Off responding ganglion cells having the characteristic morphology of On-Off directionally selective directionally selective (DS) ganglion cells, as revealed by intracellular dye injection, was significantly reduced, both by an apparent decrease of preferred direction responses and an increase in responses to null-direction movement. No toxin-mediated changes in the dendritic trees of these cells were noted. Cells in AF64A-affected retinas having the DS morphology did not respond significantly to GABAergic or cholinergic agents such as picrotoxin and eserine, but did respond to nicotine. Recordings from a small random sample of other ganglion cell classes in the same retinas yielded no obvious changes in response properties. The direct effects on starburst (cholinergic) amacrine cells, which were identified by intraocular injection of the fluorescent dye DAPI with the AF64A, were investigated by intracellular injections of Lucifer yellow, and by immunohistochemical staining with antibodies to choline acetyltransferase (ChAT). Although starburst amacrine cell somas survived the AF64A treatment for at least several days, the dendrites could not be visualized by fluorescent dye injection in affected retinas due to dye leakage of the injected fluorescent dye from either the soma or proximal dendritic region. ChAT staining revealed a sequence in which ChAT-positive cells were undetectable first in the inner nuclear layer, and then in the ganglion cell layer. Cholinergic amacrine cells in the central retina were also affected before those in the periphery. The electrophysiological changes observed typically preceded the loss of ChAT activity. Behavioral tests for optokinetic nystagmus responses also revealed a lack of such responses in the affected eyes.

Synaptic Connections of Starburst Amacrine Cells and Localization of Acetylcholine Receptors in Primate Retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human α3‐ and α5‐containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210‐immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways. J. Comp. Neurol. 461:76–90, 2003.

Distribution of parvalbumin immunoreactivity in the vertebrate retina

Parvalbumin, a calcium-binding protein thought to buffer intracellular calcium, is expressed in selected neuronal and non-neuronal cell populations. We used a well-characterized antibody directed against parvalbumin to investigate the distribution of parvalbumin in the retina of twelve vertebrate species to evaluate patterns of cellular expression for recurrent functional features. Parvalbumin immunoreactivity was displayed by subpopulations of ganglion, amacrine, bipolar and horizontal cells in different species-specific combinations. In the pigeon retina, subpopulations of amacrine, ganglion and bipolar cells were immunoreactive for parvalbumin. Parvalbumin immunoreactive bipolar cells in this species were mostly confined to the temporal dorsal region of the retina. In the owl, no immunoreactive amacrine cells were found, but many bipolar cells displayed parvalbumin immunoreactivity. In the teleost retina, amacrine and ganglion cells were found to be immunoreactive for parvalbumin. A high degree of species-specific variation was encountered in the mammalian retina. The most consistent finding within this class was that subpopulations of parvalbumin-immunoreactive amacrine cells were consistently observed in every species. In the rabbit, horizontal and ganglion cells displaying parvalbumin immunoreactivity were also seen. In rodents (hamster, ground squirrel), parvalbumin immunoreactivity was displayed by subpopulations of amacrine cells and, in the squirrel, by some ganglion cells as well. In the cat and in the baboon retina, parvalbumin immunoreactivity was found in horizontal cells, ganglion cells and a subpopulation of amacrine cells. The distribution of parvalbumin immunoreactive neurons in the vertebrate retinae studied showed no systematic correlation with phylogenetic proximity. The expression of parvalbumin within the systems of retinal neurons may therefore reflect the functional needs of different visual behaviors.

Amacrine, ganglion, and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors.

Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three beta and eight alpha subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain beta2 subunits, some of which are in combination with alpha3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 microm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 microm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.

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

Abstract Immunohistochemical methods were used to study the possible co-localization of two α-bungarotoxin-sensitive (α7 and α8) and two α-bungarotoxin-insensitive (β2 and α3) 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 α7 subunit and the β2 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 α7 and α8 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 α8 and α3 subunits, whereas neurons in the ganglion cell layer were seen to contain α7 and α8 or, less frequently, α7 and α3 subunits. These results indicate that α-bungarotoxin-sensitive and α-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.

Projection of the nucleus pretectalis to a retinorecipient tectal layer in the pigeon (Columba livia)

The avian optic tectum is composed of at least 15 separate laminae that are distinguishable on the basis of their morphological features and patterns of afferent and efferent connectivity. Layer 5b, a major retinorecipient layer, exhibits dense, dust‐like, neuropeptide Y‐positive (NPY+) immunoreactive labeling, whereas sparse, larger caliber NPY+ fibers are found in laminae 4 and 7. Anterograde and retrograde labeling techniques, immunohistochemistry, and retinal lesion studies were used to determine the source of this tectal NPY+ labeling.

Parvalbumin immunoreactivity in the rat retina

The distribution of the Ca2+ binding protein parvalbumin was studied in the rat retina with immunocytochemistry using a mouse monoclonal antibody. Specific parvalbumin immunoreactivity was identified within a subpopulation of ganglion cells and a subpopulation of amacrine cells. The topographical data provided by the present study may serve as a basis for a functional characterization of parvalbumins role in the nervous system.

Neurotransmitters, receptors, and neuropeptides in the accessory optic system: An immunohistochemical survey in the pigeon (Columba livia)

Immunohistochemical techniques were used to survey the distribution of several conventional transmitters, receptors, and neuropeptides in the pigeon nucleus of the basal optic root (nBOR), a component of the accessory optic system. Amongst the conventional neurotransmitters/modulators, the most intense labeling of fibers/terminals within the nBOR was obtained with antisera directed against glutamic acid decarboxylase (GAD) and serotonin (5-HT). Moderately dense fiber plexuses were seen to label with antibodies directed against tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). GAD-like immunoreactivity (GAD-LI) was found in many small and medium-sized perikarya within the nBOR. Some of the medium-sized cells were occasionally positive for ChAT-LI. Cell body and dendritic staining was also commonly seen with the two tested antisera against receptors-anti-GABA-A receptor and anti-nicotinic acetylcholine receptor. The antisera directed against various neuropeptides produced only fiber labeling within the nBOR. The densest fiber plexus staining was observed with antiserum against neuropeptide Y (NPY-LI), while intermediate fiber densities were seen for substance P (SP-LI) and cholecystokinin (CCK-LI). A few varicose fibers were labeled with antisera against neurotensin (NT), leucine-enkephalin (L-ENK), and the vasoactive intestinal polypeptide (VIP). Unilateral enucleation produced an almost complete elimination of TH-LI in the contralateral nBOR. SP-LI and CCK-LI were also decreased after enucleation. No apparent changes were seen for all other substances. These results indicate that a wide variety of chemically-specific systems arborize within the nBOR. Three of the immunohistochemically defined fiber systems (TH-LI, SP-LI, and CCK-LI fibers) were reduced after removal of the retina, which may indicate the presence of these substances in retinal ganglion cells. In contrast, the fibers exhibiting ChAT-LI, GAD-LI, 5-HT-LI, NPY-LI, NT-LI, L-ENK-LI, and VIP-LI appear to be of nonretinal origin. Two different populations of nBOR neurons exhibited GAD-LI and ChAT-LI. However, these two populations together constituted only about 20% of the nBOR neurons.