Differential localization of acetylcholinesterase in relation to pre- and postsynaptic nicotinic receptors in the chick brain.

Immunofluorescence and confocal microscopy were combined to study the distribution of acetylcholinesterase in relation to the localization of the beta2 subunit of the nicotinic acetylcholine receptors in the chick brain. In several areas where the beta2 subunit is recognizably part of presynaptic receptors, the localization of acetylcholinesterase appeared not to overlap the localization of beta2. On the other hand, acetylcholinesterase and the beta2 subunit exhibited a strictly matching localization in areas where postsynaptic nicotinic receptors are known to be present. These data may represent a morphological substrate for possible differential actions of acetylcholinesterase at presynaptic and postsynaptic nicotinic sites.

Effects of cholinergic deafferentation upon the expression of the alpha2 subunit of the nicotinic acetylcholine receptors in the chick lateral spiriform nucleus.

Lesion, immunohistochemical, and immunoblotting methods were used to evaluate the effects of cholinergic deafferentation upon the expression of the alpha2 subunit of the nicotinic acetylcholine receptors in the lateral spiriform nucleus (SpL) of the chick brain. The expression of the alpha2 subunit in the SpL showed biphasic changes after lesion of its cholinergic source (nucleus semilunaris), with an increase after 2 days postlesion and a decrease after 3-7 days. Our results could represent a correlate of the phenomena of nicotinic receptor up- and down-regulation, induced by removal of the cholinergic input.

Distribution of the alfa2, alfa3, and alfa5 nicotinic acetylcholine receptor subunits in the chick brain

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors comprised of alpha and beta subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the alpha2, alpha3, and alpha5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the alpha5 nAChR subunit. The alpha2 and alpha3 subunits, on the other hand, exhibited a more restricted distribution than alpha5 and other subunits previously studied, namely alpha7, alpha8 and beta2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses.

Differential co-localization of nicotinic acetylcholine receptor subunits with calcium-binding proteins in retinal ganglion cells.

Immunohistochemistry was used to examine the co-occurrence of nicotinic acetylcholine receptor subunits with calcium-binding proteins in ganglion cells of the chick retina. The alpha3 subunit was rarely observed in ganglion cells containing calbindin, calretinin, or parvalbumin. On the other hand, the alpha8 subunit was more often co-localized with all calcium-binding proteins studied. These results may be related to the high calcium permeability of nicotinic receptors that contain the alpha8 subunit.

Distribution of the alpha 2, alpha 3, and alpha 5 nicotinic acetylcholine receptor subunits in the chick brain.

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors comprised of alpha and beta subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the alpha 2, alpha 3, alpha 5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the alpha 5 nAChR subunit. The alpha 2 and alpha 3 subunits, on the other hand, exhibited a more restricted distribution than alpha 5 and other subunits previously studies, namely alpha 7, alpha 8 and beta 2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses.

Presumptive presynaptic nicotinic acetylcholine receptors in the chick tectum: effects of lesions of the lateral spiriform nucleus.

There are indications that nicotinic acetylcholine receptor subunits in the superficial layers of the chick tectum (Cajal's layers 1-7) may be transported from the retina. However, nicotinic receptor subunits are detectable by immunohistochemistry in all layers of the optic tectum. In this study, we performed unilateral electrolytic lesions of the lateral spiriform nucleus, which projects to the deep layers of the tectum and contains high amounts of nicotinic receptors in its perikarya. Following lesions of the lateral spiriform nucleus, both the alpha 5 and the beta 2 subunits were markedly depleted in the neuropil of the deep layers of the ipsilateral optic tectum (layers 8-13). No changes were observed in somata that contain either subunit. The present results suggest that most of the nicotinic acetylcholine receptor subunits in the chick optic tectum occur in axonal systems and could then constitute presynaptic receptors.

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.

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.

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.

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.