effects of retinal lesions upon the distribution of ...the journal of comparative neurology...

THE JOURNAL OF COMPARATIVE NEUROLOGY 350:473-484 (1994)

Effects of Retinal Lesions Upon the Distribution of Nicotinic

Acetylcholine Receptor Subunits in the Chick Visual System

L.R.G. BRITTO, A.S. T O R R ~ O , D.E. HAMASSAKI-BRITTO, J. MPODOZIS, K.T. KEYSER, J.M. LINDSTROM, AND H.J. KARTEN

Departments of Physiology and Biophysics (L.R.G.B., A.S.T.) and Department of Histology and Embryology (D.E.H.-B.), Institute of Biomedical Sciences, University of SBo Paulo,

05508 S5o Paulo SP, Brazil; Department of Biology, College of Sciences, University of Chile, Santiago, Chile (J.M.); Department of Neurosciences, University of California San Diego,

La Jolla, California 92093-0608 (K.T.K., H.J.K.); and Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6074 (J.M.L.)

ABSTRACT 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 p2 receptor subunit, a major component of a-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 a-bungarotoxin- sensitive receptor subunits, 017 and a8, was only slightly affected after retinal lesions. Decreased neuropil staining for a7-like immunoreactivity was only observed in the nucleus of the basal optic root and layers 2-4 and 7 of the optic tectum. For a8-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 p2 subunit is transported from retinal ganglion cells to their central targets, whereas the a7 and a8 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 a7 and a8 subunits in all retinorecipient areas. In agreement with this hypothesis, the p2 subunit of the nicotinic acetylcholine receptors was not frequently found in perikarya of the same areas. 0 1994 Wiley-Liss, Inc.

Key words: a-bungarotoxin, ionotropic receptors, neurotransmitters, nicotine, visual pathways

In recent years, molecular biology techniques have revealed an unexpected diversity of the neuronal nicotinic acetylcholine receptors (nAChRs) as well as other members of the ligand-gated ion channel superfamily (reviewed by Schofield et al., 1990). Several subunits of the nAChRs have now been cloned and characterized. At least seven ligand- binding (or a) and three structural (non-a or p) nAChR subunits have been identified so far (reviewed by Lindstrom et al., 1987; Deneris et al., 1991; Role, 1992; Sargent, 1993). Recent studies have also disclosed the existence of a new subclass of nAChRs that bind a-bungarotoxin (a-Bgt) in much the same way as the muscle nicotinic acetylcholine receptors (Couturier et al., 1990; Schoepfer et al., 1990;

SBguela et al., 1993; reviewed by Clarke, 1992). This new subclass of nAChRs includes a7 and a8 nAChR subunits, possibly either as homoligomers (in case of the a7 subunit) or as heteroligomers (a7 plus a8). The complete subunit composition of these subtypes is not known, and they may contain uncharacterized structural subunits. Combina- tions of these two subunits with subunits of the non-a-Bgt-

Accepted June 7,1994. Address reprint requests to Luiz R.G. Britto, Department of Physiology

and Biophysics, Institute of Biomedical Sciences, University of Sgo Paulo, Av. Prof. Lineu Prestes 1524,05508-900 Slo Paulo SP, Brazil.

O 1994 WILEY-LISS, INC.

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sensitive type or other, as yet unknown, subunits may also exist (Clarke, 1992; Role, 1992; Sargent, 1993).

The distribution of several of the nAChR subunits in the vertebrate brain has been studied with both immunohistochemistry (Swanson et al., 1983, 1987; Deutch et al., 1987; Sargent et al., 1989; Schroder et al., 1989; Britto et al., 1992b; Hill et al., 1993) and in situ hybridization experiments (Goldman et al., 1986, 1987; Boulter et al., 1987; Duvoisin et al,, 1989; Wada et al., 1989, 1990; Morris et al., 1990; Dineley-Miller and Patrick, 1992; Seguela et al., 1993). In general, the distribution of nAChR subunits, revealed by subunit-specific antibodies and nucleic acid probes, has agreed well with the results of ligand binding experiments (Hunt and Schmidt, 1978; Marks and Collins, 1982; Clarke et al., 1985; Swanson et al., 1987; Watson et al., 1988; Sorenson and Chiappinelli, 1992). Many nAChR subunits are detectable in central visual structures as well as in retinal neurons, including ganglion cells (Keyser et al., 1988, 1993; Sargent et al., 1989; Cauley et al., 1990; Hamassaki-Britto et al., 1991; Britto et al., 1992a; Hoover and Goldman, 1992). The presence of nAChRs in retinal ganglion cells and in their presumptive terminals in the brain, together with physiological data indicative of an effect of acetylcholine in the modulation of retinofugal transmission, has led to the suggestion that at least some of the nAChRs in central visual structures may represent presynaptic receptors that were transported from the retina (Brecha et al., 1979; Henley et al., 1986; Langdon and Freeman, 1987; Swanson et al., 1987; Keyser et al., 1988; Sargent et al., 1989; Cauley et al., 1990; King, 1990; Britto et al., 1992b). This hypothesis was supported by the observation that there was a marked depletion of nAChR immunolabeling in the rodent superior colliculus (Swanson et al., 1987), the frog optic tectum (Sargent et al., 19891, and the chick optic tectum (Swanson et al., 1987; Britto et al., 1992b) following contralateral retinal lesions. Interest- ingly, these lesions caused the depletion of binding of a radiolabeled antibody against the p2 nAChR subunit in the rodent superior colliculus but affected only slightly the distribution of a-Bgt binding sites (Swanson et al., 1987). This suggested that a-Bgt-sensitive and a-Bgt-insensitive nAChRs were differentially transported in the mammalian visual system. In the chick tectum, the distribution of both types of nAChRs appeared to be affected by retinal lesions, although the effects on the distribution of a-Bgt-insensitive nAChR subunits in the tectum appeared more pronounced (Swanson et al., 1987; Britto et al., 1992b). Other than the optic tectum, there is little information concerning the effects of retinal lesions on the distribution of nAChRs in other retinorecipient structures. There is, however, autoradiographic evidence that retinal lesions reduce immunolabeling for the p2 subunit in the ventral lateral geniculate nucleus, dorsal lateral geniculate nucleus, pretectal nuclei, and medial terminal nucleus of the accessory optic system in the rat (Swanson et al., 1987).

The present study was undertaken to verify the effects of retinal lesions upon the distribution of nAChRs in all of the retinorecipient structures of the chick brain. Special emphasis was given to the evaluation of the possibility that the distribution of a-Bgt-sensitive and a-Bgt-insensitive nAChRs in retinorecipient structures other than the optic tectum could be differentially affected by retinal lesions. To detect nAChRs, we have used monoclonal antibodies against the /32 nAChR subunit, which is believed to be a major component of nAChRs of the a-Bgt-insensitive type (Whiting et

L.R.G. BRITTO ET AL.

al., 1987; Role, 1992; Sargent, 1993) and antibodies against the a7 and a8 subunits that constitute a-Bgt-sensitive nAChRs (Couturier et al., 1990; Schoepfer et al., 1990).

MATERIALS AND METHODS Twelve 1-30-day-old chicks (Gallus gallus) obtained

from McIntyre Poultry (Lakeside, CA) were used in this study. Unilateral, total retinal lesions were performed on seven chicks that were anesthetized with ketamine (5 mg/100 g of body weight, i.m.1 and xylazine (1 mg/lOO g, i.m.). First, a corneal incision was performed, and the lens and vitreous were removed. Lidocaine was then injected into the eye. After 5-7 minutes, the neural retina was completely destroyed and removed with cotton swabs. Gelfoam was placed inside the eye, and the eyelids were sutured closed. Five other chicks were anesthetized in the same way and subjected to small retinal lesions performed with a 30-gauge needle (see Ehrlich et al., 1987). Survival times ranged from 5 to 30 days. Following the experimental period, the birds were deeply anesthetized with ketamine and xylazine and perfused through the heart with phosphate-buffered saline and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, PB). After 3-5 hours of postfix- ation, the brains were transferred to a 30% sucrose solution in PB to ensure cryoprotection. Coronal or sagittal sections (30 km) of the frozen brains were cut on a sliding micro- tome.

The immunohistochemical techniques used here to detect nAChR subunits have been previously described in detail (Britto et al., 1992b). Briefly, the brain sections were incubated free-floating with rat monoclonal antibodies (mAbs) against p2 (mAb270; Whiting et al., 1987) and a8 nAChR subunits (mAb305; Schoepfer et al., 1990) or a mouse monoclonal antibody against the a7 nAChR subunit (mAb306; Schoepfer et al., 1990) diluted 1500 to 1: 1,000 in PB containing 0.3% Triton X-100. Incubations with primary antibodies were conducted for 14-18 hours at 4°C. After three washes (15 minutes each) in PB, the sections were incubated with biotinylated rabbit anti-rat or anti- mouse sera (Vector, Burlingame, CAI diluted 1:200 in PB for 1 hour at room temperature. The sections were washed in PB again and incubated with avidin-biotin-peroxidase complex (ABC Elite; Vector). Following the reaction with 0.05% 3-3'-diaminobenzidine and a 0.01% solution of hydro- gen peroxide in PB and intensification with 0.05% osmium tetroxide in water, the sections were mounted on gelatin and chromoalumen-coated slides, dehydrated, cleared, and coverslipped with Permount (Fisher). The material was analyzed on a microscope equipped with differential interfer- ence contrast (Nomarski) optics and photographed with Technical Pan (Kodak). The main control for specificity of immunostaining was the omission of the primary antibodies from the procedure. In addition, in several experiments, the mAbs were replaced by normal serum from the same species (rat serum for mAbs270 and 305 and mouse serum for mAb306). Specific staining was abolished under either of those conditions. It should be recalled that the antibodies used in this study have been extensively tested for their specificity against nAChR subunits (Whiting et al., 1987, 1991; Schoepfer et al., 1990; McLane et al., 1992) and have been previously used to detect nAChR subunits in neural tissue (Keyser et al., 1988, 1993; Britto et al., 1992a,b).

The intensity of nAChR subunit immunoreactivity in the neuropil was subjectively rated from absent to very intense,

RETINAL LESIONS AND NICOTINIC RECEPTORS

and the immunoreactive perikarya were counted in every tenth coronal section of the optic tectum and every fourth coronal section for most of the other retinorecipient structures. In the case of very small structures such as the nucleus suprachiasmaticus, pars lateralis, every section was used for the cell counts. The task of counting labeled cells and estimating their percentage in relation to the total number of cells in the same structure was greatly facilitated by counterstaining the sections with Giemsa (Ifiiguez et al., 1985). In some cases, estimates of the total number of cells in the retinorecipient structures of the chick brain were obtained from adjacent cresyl violet-stained sections. No attempt was made to quantify the intensity of somata staining. Labeled cells were also subjectively characterized, and the counted cells were only those that stained well above background. These procedures were essentially iden- tical to those used in a previous mapping of the distribution of the same nAChR subunits in the chick mesencephalon and diencephalon (Britto et al., 199213). Because the variability of somata staining with nAChR antibodies was manifest, no statistics were applied to verify if the number of labeled cells in the control side was different from that in the deafTerented side. It should be stressed that the number of stained somata and the intensity of neuropil staining were evaluated by at least two independent observers, and the degree of consistency between the results was always high both within the same experiment and between experiments.

The identification of the meso- and diencephalic retinorecipient structures of the chick brain was based on the stereotaxic atlases for the chick (Kuenzel and Masson, 1988) and the pigeon (Karten and Hodos, 1967) brains as well as on some reports on retinorecipient areas of both species (Ehrlich and Mark, 1984; Gamlin and Cohen, 1988; Britto et al., 1989; Hamassaki and Britto, 1990; Gunturkun and Karten, 1991). The layers of the optic tectum were numbered according to Cajal’s scheme (see Kuenzel and Masson, 1988).

4 75

RESULTS Distribution of pZ, a7, and a8 nAChR subunits

in retinorecipient areas of the chick brain Every retinorecipient area of the chick brain contained

immunoreactivity for all three subunits examined in this study with varying degrees of intensity. Both neuropil and somata staining were easily discernible in retinorecipient structures of the chick brain. These results confirmed the data from a previous mapping study using the same antibodies (Britto et al., 1992b) and are in agreement with the data from a recent nicotine and neurotoxin binding study in the chick brain (Sorenson and Chiappinelli, 1992).

Table 1 shows the distribution of nAChRs in the neuropil of retinorecipient areas of the chick brain. Intense or very intense neuropil staining for PZ-like immunoreactivity (PP-LI) was observed in the nucleus geniculatus lateralis, pars dorsalis principalis, nucleus geniculatus lateralis, pars ventralis, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, nucleus of the basal optic root, pars dorsalis, layer 7 of the optic tectum, nucleus opticus principalis thalami, pars laterodorsalis, nucleus posterodorsalis of the pretectum, nucleus pregeniculatus tecti optici, and nucleus suprachiasmaticus, pars lateralis. Neuropil staining for a7-LI was intense or very intense in the nucleus geniculatus lateralis, pars dorsalis principalis,

nucleus geniculatus lateralis, pars ventralis, nucleus lentiformis mesencephali, nucleus of the basal optic root, pars principalis and pars lateralis, and layers 2-4 and 7 of the optic tectum. Neuropil staining for a8-LI was intense or very intense in the nucleus geniculatus lateralis, pars dorsalis principalis, nucleus geniculatus lateralis, pars ventralis, griseum tecti, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, nucleus opticus principalis thalami, and the nucleus pregeniculatus tecti optici.

The numbers of perikarya that stained for p2-LI, a7-LI, and a8-LI in the chick visual system have been published before (Britto et al., 1992b) and are not presented here, because we could not detect major changes in the numbers of stained somata following retinal lesions (see below). However, it should be stressed that, as in our previous mapping paper, we found many cells containing PZ-LI, a7-LI, and a8-LI in the chick retinorecipient structures (Britto et al., 1992b). The numbers of a7-LI and a8-LI neurons in the chick visual system were found to be much higher than the numbers of p2-LI somata. The numbers of a8-LI somata were slightly higher than the numbers of u7-LI somata.

Effects of retinal lesions on the distribution of p2, a7, and a8 nAChR subunits in

retinorecipient areas of the chick brain As expected, the small retinal lesions produced localized

effects on the distribution of PZ-LI, a7-LI, and a8-LI in the chick visual system. These effects were always in perfect retinotopic register with the site of the retinal lesion as previously shown for the optic tectum (Britto et al., 1992b). Complete retinal lesions, on the other hand, generated much more striking effects on the distribution of nAChR immunoreactivity associated with a reduction in size of several retinorecipient structures, especially the optic tectum and the nucleus of the basal optic root. There were no major differences in the effects of retinal lesions at the various survival times examined. After the shorter survival times (5-7 days), the effects on p2-LI, a7-LI, and as-LI were less manifest than those observed after 12-15 days postlesion. The changes appeared to stabilize after 2 weeks of the lesions. After the longest of the survival times examined, however, there was a marked reduction of the size of several retinorecipient structures on the deafferented side. This shrinkage was taken into account when analyzing the effects of retinal lesions upon the neuropil staining with nAChR antibodies. For example, the shrinkage in some structures could generate the impression of a higher density of staining in some visual areas. In these cases, examination of the brains of birds subjected to small retinal lesions were used as “controls,” because, in these cases, no substantial shrinkage could be noted. Table 1 includes the description of the effects of retinal lesions on nAChR neuropil immunoreactivity for the different retinorecipient structures. All the data in Table 1 were taken from brains of birds that were allowed to survive for 2-3 weeks after the retinal lesions.

The retinal lesions produced marked effects on the neuropil staining for p2-LI in the chick retinorecipient areas on the contralateral side. There was a marked depletion or complete elimination of p2-LI in the vast majority of retinorecipient structures of the chick brain. However, there were no noticeable changes in p2-LI in layers 4-6 and 8-15 of the optic tectum. It should be stressed that layers 1-7 constitute the retinorecipient zone

476

of the avian tectum, whereas layers 8-15 are nonretinore- cipient (Ehrlich and Mark, 1984; Gamlin and Cohen, 1988). In area pretectalis, the reduction of the staining for p2-LI appeared to be only slight. Figure 1 illustrates the effects of retinal lesions upon the distribution of p2-LI in some visual structures of the chick brain.

The effects of retinal lesions upon the staining for a7-LI and a8-LI were always less pronounced than those for 62-LI. However, we could detect slight reductions of staining for 1x7-LI in the nucleus of the basal optic root, pars principalis and pars lateralis, and layers 2-4 and 7 of the optic tectum (Fig. 2). For aS-LI, slight reduction of staining could be observed in the nucleus geniculatus lateralis, pars dorsalis principalis, nucleus geniculatus lateralis, pars ventralis, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, nucleus pregeniculatus tecti optici, and nucleus suprachiasmaticus, pars lateralis (Fig. 3).

Only layer 7 of the optic tectum exhibited decreases of neuropil staining for all three types of immunoreactivity examined here. In several other structures, we observed decreases in the staining for one or two of the nAChR subunits, even if to different degrees. Layer 4 of the optic tectum was the only structure in which we could see the effects of retinal lesions for both the a-Bgt-sensitive nAChR subunits (a7 and a8) but not for the p2 nAChR subunit. The expression of the latter subunit was changed in several visual areas in which modification of the staining for a7-LI and/or a8-LI after retinal lesions could not be detected. Depletion of p2-LI and reduction of a7-LI was found only in the nucleus of the basal optic root, pars principalis, and pars lateralis. Depletion of PB-LI and reduction of a8-LI was observed in the nucleus geniculatus lateralis, pars dorsalis principalis, nucleus geniculatus lateralis, pars ventralis, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, pars magnocellularis, nucleus pregeniculatus tecti optici, and nucleus suprachiasmaticus, pars lateralis (Table 1).

Neither the complete retinal lesions nor the small lesions produced any dramatic effect on staining of the somata in visual areas. However, it should be stressed that we did not attempt to quantify the staining of perikarya for p2-LI, a7-LI, and US-LI, and there was a clear variability in somata staining for all three antibodies. The numbers of cells considered labeled in the deafferented side always appeared to be similar to the control side, at least in terms of percentage of labeled cells in relation to the total number of neurons in the same location. In some instances, the stained cells in the deafferented side exhibited signs of degenerative changes such as smaller sizes and altered shapes. These effects were not analyzed in detail, as the emphasis of this study was related to the question of transport of nAChRs from retinal ganglion cells. The lesions actually unmasked the presence of p2-LI perikarya in some retinorecipient structures of the chick brain. This appeared to occur for the nucleus geniculatus lateralis, pars dorsalis principalis, nucleus geniculatus lateralis, pars ventralis, griseum tecti, nucleus lentiformis mesencephali, pars magnocellularis, nucleus pregeniculatus tecti optici, and nucleus suprachiasmaticus, pars lateralis (Fig. 4). Careful inspection of Giemsa-counterstained sections in control and deafferented nuclei revealed that the heavy neuropil label in those structures precluded the prompt identification of stained perikarya in noncounterstained, control sections. Their visualization was much easier after the retinal lesions and the drastic reduction of neuropil

L.R.G. BRITTO ET AL.

staining that ensued, but the actual percentage of labeled cells remained basically the same as in the control side.

DISCUSSION The effects of retinal lesions on the distribution of p2-LI,

a7-LI, and a8-LI in the chick visual system are in agreement with the localization of those three subunits in ganglion cells of the chick retina. Many ganglion cells have been shown to contain p2-LI (Keyser et al., 1988) and a8-LI (Keyser et al., 1993), whereas a smaller number of ganglion cells appear to contain a7-LI (Keyser et al., 1993). Presum- ably, the ganglion cells exhibiting those three types of immunoreactivity are the cells that transport nAChRs to their central targets, where they may function as presynaptic receptors. The possibility exists, however, that at least part of the loss of immunoreactivity in retinorecipient areas of the chick brain could be due to loss of postsynaptic structures. Deafferentation-induced loss of postsynaptic elements has indeed been shown in the amphibian tectum (Ostberg and Norden, 1979). The results of the present experiments also imply that the cholinoceptive ganglion cells project to a variety of different central targets, as the effects of retinal lesions were detectable on several of those targets. The nucleus of the basal optic root poses a special problem in this regard. We detected a slight reduction of a7-LI in the contralateral nucleus of the basal optic root after retinal lesions. However, no a7-LI has been found in displaced ganglion cells (Britto et al., 1992a; Keyser et al., 19931, which represent the sole source of retinal afferents to the nucleus of the basal optic root in birds (Britto et al., 1989). The significance of this finding is not clear. It is possible that retinal lesions induced a loss of a7-LI in processes of neurons of the nucleus of the basal optic root but did not change appreciably the staining of their perikarya.

The reduction of p2-LI after retinal lesions was by far the most dramatic, although, in some structures, reduction of a7 and a8 immunoreactivity was evident as well. This was the case, for example, in layer 7 of the optic tectum, where we observed reduction of all three types of immunoreactivity. Retinal ganglion cells projecting to the tectal layer 7 could therefore contain p2-LI, a7-LI, and a8-LI. A number of other structures exhibited changes for two types of nAChR subunit immunoreactivities, that were either p2-LI and a7-LI, p2-LI and a8-LI, or a7-LI and a8-LI. In all of the structures where p2-LI was affected by retinal lesions together with a7-LI andlor aS-LI, the presumptive presynaptic nAChRs could be either of the a-Bgt-sensitive or the a-Bgt-insensitive types.

In that both the numbers of p2-LI and a8-LI ganglion cells in the chick retina are large (Keyser et al., 1988; 1993) and the lesion-induced depletion of p2-LI was much more impressive than that of aS-LI, it appears that a-Bgt- insensitive and a-Bgt-sensitive nAChRs are differentially transported from retinal ganglion cells to retinorecipient structures of the chick brain. This conclusion is supported by the fact that a-Bgt-insensitive and a-Bgt-sensitive nAChRs colocalize in a t least some ganglion cells of the chick retina (Britto et al., 1992a). The different types of nAChRs could then have different impacts on retinofugal transmission by acting as presynaptic receptors. The possibility that p2-LI, a7-L1, and a8-LI in the neuropil of visual nuclei could represent “spurious” transport from the retinal ganglion cells cannot, however, be excluded at the present. According to that hypothesis, retinal ganglion cells

RETINAL LESIONS AND NICOTINIC RECEPTORS 477

Fig. 1. Effects of retinal lesions (survival time: 1 week) on the distribution of 62-LI in the chick visual system. The photomicrographs on the left (A-C) depict the control side, whereas the ones on the right (A'-C') show the deafferented side. The numbers on the left of each photomicrograph in A and A' indicate the tectal layers. The asterisk in

C indicates p2-LI in optic tract axons. GLdp, nucleus geniculatus lateralis, pars dorsalis principalis; GLv, nucleus geniculatus lateralis, pars ventralis; nBOR, nucleus of the basal optic root (d, pars dorsalis; p, pars principalis); TeO, tectum opticum; TrO, tractus opticus. Scale bars = 30 pm in A,A', 100 W r n in B,B', 150 pm in C,C'.

478 L.R.G. BRITTO ET AL.

Fig. 2. Effects of retinal lesions (survival time: 4 weeks) on the distribution of a7-LI in the chick visual system. The photomicrographs on the left ( A X ) depict the control side, whereas the ones on the right (A'-C') show the dederented side. Abbreviations as in Figure 1. Scale bars = 30 pm in A,A, 100 km in B,B', 150 pm in C,C'.

RETINAL LESIONS AND NICOTINIC RECEPTORS

Fig. 3. Effects of retinal lesions (survival time: 4 weeks) on the distribution of a8-LI in the chick visual system. The photomicrographs on the left (A-C) depict the control side, whereas the ones on the right (A'-C') show the deafferented side. Abbreviations as in Figure 1. Scale bars = 30 Frn in A,A', 100 Fm in B,B', 150 Frn in C,C'.

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Intensity of neuropil staining: AP, area pretectalis; GLdp, nucleus geniculatus lateralis, pars dorsalis princi. palis; GLv, nucleus geniculatus lateralis, pars ventralis; GT, griseum tecti; IGL, intergeniculate leaflet; LA, nucleus lateralis anterior thalami; LMmc, nucleus lentiformis mesencephali, pars magnocellularis; nBORp,I, nucleus of the basal optic root, pars principalis and pars lateralis; nBORd, nucleus of the basal optic root, pars dorsalis; OPT, nucleus opticus principalis thalami; OPTId, nucleus opticus principalis thalami, pars laterodorsalis; PD, nucleus posterodorsalis of the pretedum; PTO, nucleus pregenieulatus tecti optici; SCNI, nucleus suprachiasmaticus, pars lateralis; TeO, optic tectum; a-Bgt, alpha-hungarotoxin.

could synthesize those proteins to be used in their retinal circuitry and the immunoreactivity that we detect in the neuropil of the central visual system could just mean that the molecules are also transported to the axon terminals of the ganglion cells. The intense cytoplasm staining for nAChR immunoreactivity could be interpreted in the same way (see Britto et al., 1992b; Sargent, 1993). Nevertheless, the abundance of nAChRs in the chick visual system is in agreement with the important cholinergic afferentation of most of the retinorecipient structures of the chick brain (Sorenson et al., 1989). This suggests that nAChR subunits could indeed exist in presynaptic sites in the visual system and could actually constitute functional nAChRs. Presump-

tive presynaptic nAChRs have already been revealed by both physiological and anatomical studies (Wonnacott et al., 1990). In the mammalian interpeduncular nucleus, at least one type of nAChR occurs on the terminals of habenu- lointerpeduncular neurons (Clarke et al., 1986; Mulle et al., 1991; LBna et al., 1993). Nigrostriatal neurons also appear to have nAChRs in their terminals (Schulz and Zigmond, 1989). In the visual system, there is already evidence of presynaptic nAChRs in the cat visual cortex and superior colliculus (Prusky et al., 1987; Prusky and Cynader, 1988) and the optic tectum of the chick (Swanson et al., 1983, 1987; Britto et al., 1992b1, frog (Sargent et al., 19891, and goldfish (Henley et al., 1986; Cauley et al., 1990). Recently,


Fig. 4. Unmasking of p2-LI neurons in the nucleus suprachiasmaticus, pars lateralis (SCNl), following contralateral retinal lesions (survival time: 2 weeks). A: Dederented side. B: Control side. SCN1, nucleus suprachiasmaticus, pars lateralis; TrO, tractus opticus; VLT, nucleus ventrolateralis thalami. Scale bar = 25 km.

electrophysiological evidence has also become available on the existence of presynaptic nAChRs in the chick nucleus geniculatus lateralis, pars ventralis (McMahon et al., 1994). The data from the present lesion study indicated that the 62 nAChR subunits could be part of presynaptic nAChRs in the nucleus geniculatus lateralis, pars ventralis; they appeared to be transported by retinal axons terminating in that nucleus. The a8 nAChR subunit could also possibly be found as a component of some presynaptic nAChRs in the nucleus geniculatus lateralis, pars ventralis; retinal lesions appeared to produce a small reduction of the neuropil staining for a8-LI in that visual nucleus.

Transport of receptor molecules and presynaptic localization in the visual system has already been suggested for other receptor systems as well. For example, y-aminobu- tyric acid (GABA)-A receptors have also been immunohisto- chemically detected in retinal ganglion cell axons (Hughes et al., 1989, 1991). Also, both opiate-and melatonin-binding sites appear to be depleted in visual structures following retinal lesions (Giolli et al., 1990; Krause et al., 1992).

This study found the p2 subunit of the nAChRs to be more likely part of presynaptic nAChRs; most of the immunochemical neuropil staining for this subunit disap- peared from visual areas after retinal lesions. This finding is consistent with the presence of that subunit on ganglion cell terminals that degenerate after retinal lesions. The a-Bgt-sensitive subunits (a7 and a8), on the other hand, were less affected by retinal lesions, suggesting that a7 or 1x8-containing nAChRs are found only in a portion of ganglion cell terminals in retinorecipient structures of the chick brain. Therefore, the two a-Bgt-sensitive subunits could be more frequently found as postsynaptic nicotinic receptors on neurons of the chick visual system. However, there are no reports of physiological responses to acetylcholine or nicotine in the visual system that are blocked by a-Bgt (Clarke, 1992; Role, 1992; Sargent, 1993). One possible explanation for this finding could be related to the rapid desensitization of a-Bgt-type nAChRs as shown in oocytes (Couturier et al., 1990; Seguela et al., 1993), rat hippocampus (Alkondon and Albuquerque, 1993), and ciliary ganglion neurons (Zhang et al., 1994). Also, we cannot exclude the possibility that those a-Bgt-sensitive nAChR subunits could be presynaptic to other fiber systems in the visual system. Alternatively, some other unusual property of the a-Bgt-sensitive nAChRs or the possibility of coassem-

bly of the a7 and a8 subunits with other uncharacterized nAChR subunits has to be considered. The coassembly with other subunits could alter or somehow mask the property of blockade by a-Bgt (see, for example, Listerud et al., 1991). Yet another explanation for the difficulty of obtaining reliable a-Bgt-blockable responses in the central nervous system concerns the finding that at least part of nAChRs actually occur in extrasynaptic sites, as demonstrated with binding experiments for the goldfish retina (Zucker and Yazulla, 1982) and the chick ciliary ganglion (Jacob and Berg, 1983; Loring et al., 1985) and immunocytochemically for the frog optic tectum (Sargent et al., 1989) and cardiac ganglion (Sargent and Pang, 1989). Ultrastructural studies with the antibodies used in the present study are needed to verify if that is the case for the chick visual system.

The nAChR subunits studied here are also expressed by neurons of the retinorecipient structures themselves; we found perikarya stained for p2-LI, a7-LI, and a8-LI in most of those regions. The finding that perikarya also stain for nAChR-LI in visual areas agrees with in situ hybridization studies that showed nAChR gene expression by cells of the optic tectum and other visual structures (Matter et al., 1990; Morris et al., 1990). We were not able to observe any differences in somata staining with nAChR antibodies after retinal lesions, which could imply that the postsynaptic neurons continue to synthesize nAChRs even after deafferentation. However, the immunohistochemistry technique employed here does not have enough sensitivity to permit conclusive inferences about this problem. In situ hybridization experiments are underway in our laboratories to discern if nAChR gene expression changes after retinal lesions. The number of neurons that exhibited p2-LI in the chick visual system was much smaller than the number of somata that exhibited a7-LI and a8-LI (Britto et al., 199213; this study). This is consistent with the moderate reduction of neuropil staining for a7-LI and a8-LI that was observed in the present retinal lesion experiments. The neuropil staining that persisted after those lesions probably origi- nates in the somata that contained those two types of immunoreactivity. The effect of retinal lesions on p2-LI neuropil staining was much more dramatic, probably because most of the PB-LI is derived from retinal axons. Indeed, very few cells in chick visual nuclei contained p2-LI. In other vertebrates, nAChR genes are also expressed by neurons of retinorecipient areas (Wada et al., 1989; Cauley


et al., 1990), and neuropil staining with antibodies against nAChR protein products in those areas has already been demonstrated (Swanson et al., 1987; Sargent et al., 1989).

As was mentioned above for p2, a7, and 018, the a3 nAChR subunit has also been localized to ganglion cells and displaced ganglion cells of the chick retina (Hamassaki- Britto et al., 1991; Whiting et al., 1991). However, no neuropil staining for a3-LI is detectable in central retinorecipient structures but only in perikarya and proximal processes of a few brainstem cell groups (Britto, Keyser, Karten, and Lindstrom, unpublished). This could be interpreted to mean that the a3 nAChR subunit is not transported by retinal ganglion cell axons to the brain, but could instead be only shipped to the ganglion cell dendrites within the retinal inner plexiform layer. In this synaptic retinal layer, dendrites of a 3 nAChR-bearing retinal ganglion cells are in close register with dendrites of cholinergic retinal neurons (Hamassaki-Britto et al., 1991; Whiting et al., 1991). Another interpretation for the lack of neuropil staining for a3-LI in visual structures could be related to the specific properties of the antibodies used to detect a3-LI. We have recently obtained similar findings for the a5 nAChR subunit, which is also present in retinal ganglion cells but not in presumptive axons in the central visual system (Britto, Torriio, Keyser, Karten, and Lindstrom, unpublished). Further studies are needed to verify if the a 3 and a5 nAChR subunits are used in retinal ganglion cell circuitry but are not shipped to the central nervous system. Interestingly, the a3 and a5 nAChR subunits appear together in at least some receptor complexes in chick brain and ciliary ganglia (Conroy et al., 1992). If the a3 and a5 are not exported along retinal ganglion cell axons, it is possible that the pZ nAChR subunit in retinal terminals is coas- sembled with a4 and/or a2 subunits. This is consistent with the notion that at least half of the brain nAChR subtypes with high affinity for nicotine have an a4p2 composition and that the a2p2 subtype is also very abundant (Whitinget al., 1991; Sargent, 1993).

Previous studies indicated that a-Bgt-insensitive nAChRs (at least those containing p2 subunits) were depleted from several visual regions of the rat brain following retinal lesions. These include the superior colliculus, dorsal and ventral geniculate nuclei, pretectal nuclei, and medial terminal nucleus of the accessory optic system (Swanson et al., 1987). The effect on the distribution of a-Bgt binding sites was very slight. The significance of that finding is not clear, although some interspecies variations might be involved. In the same study, Swanson et al. (1987) found a depletion of a-Bgt binding sites in the chick optic tectum following retinal lesions, which is in agreement with the present results. The possibility exists that in the rat the a-Bgt- sensitive nAChRs are present in small amounts in the retinal terminals and thus may not influence retinofugal transmission in any substantial way. On the other hand, perhaps both major types of nAChRs, a-Bgt-sensitive and a-Bgt-insensitive, could influence retinofugal transmission in the chick, although at different levels. In the frog, a-Bgt-insensitive nAChR subunits are also depleted from the optic tectum following retinal lesions (Sargent et al., 19891, but there is little information on the effects of retinal lesions on a-Bgt-sensitive nAChRs or a-Bgt binding sites in that species.

The functional significance of the occurrence of nAChRs in retinal terminals is not completely clear. There is evidence that retinotectal transmission is decreased by

d-tubocurarine in the goldfish (Langdon and Freeman, 19871, but, in the same study, no major effects of nicotine or a-Bgt on retinotectal transmission could be detected. How- ever, eserine and carbachol appeared to increase the ampli- tude of field potentials elicited in response to optic nerve stimulation (Langdon and Freeman, 1987). The cholinergic agonists nicotine, carbachol, and cytisine have been shown directly to depolarize retinal terminals and enhance retinofugal transmission in the goldfish tectum (King, 1990). In the chick, nicotinic receptor activation produced an increase of GABA spontaneous activity in the nucleus geniculatus ventralis, pars lateralis, an effect that was not blocked by tetrodotoxin (McMahon et al., 1994). All of the above pharmacological/physiological findings, taken together with the present data, indicate that presynaptic nAChRs may have an important role in the functional organization of the visual system. Clearly, additional experiments are needed to characterize how the retinofugal transmission can be modified by a-Bgt-insensitive and/or a-Bgt-sensitive nAChRs in the different retinorecipient structures of the vertebrate brain.

CONCLUSIONS This study indicates that both a-Bgt-insensitive and

a-Bgt-sensitive nAChR subunits are transported from retinal ganglion cells to the central retinorecipient structures of the chick brain. The marked effects of retinal lesions on the expression of p2-LI in the visual system suggests that the p2 subunit is an important component of the nAChRs that are transported in retinal axons and possibly contrib- ute to a presynaptic modulation of retinofugal transmission. On the other hand, the a7 and a8 nAChR subunits appear to have important intrinsic sources in the visual system and could perhaps be mainly found as components of postsynaptic nAChRs.

ACKNOWLEDGMENTS The authors thank Agnieszka Brzozowska-Prechtl

(UCSD, La Jolla) and Adilson Alves (USP, Siio Paulo, Brazil) for technical assistance. Thanks are also due to Kevin Cox (UCSD, La Jolla) for technical assistance and for reviewing the manuscript. This study was supported by NIH grants EY07845 (K.T.K.), EY06890 (H.J.K.), NS24560 (H.J.K.), and NS11323 (J.M.L.); Council for Smokeless Tobacco Research (J.M.L.); Muscular Dystrophy Associa- tion (J.M.L.); Council for Tobacco Research (J.M.L.); Cali- fornia Tobacco-Related Disease Research Program (3RT- 0157; K.T.K.); and FAPESP and CNPq (Brazil; L.R.G.B., D.E.H.B.). J.M. was supported by Fondecyt (Chile, Project 1081). A.S.T. was the recipient of a fellowship from FAPESP (Brazil).

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