Possible induction of [Met]enkephalin-Arg6-Gly7-Leu8 immunoreactivity in neurons of the rat superior colliculus following eye enucleation

Neuroscience Letters, 120 (1990) 74-79 Elsevier Scientific Publishers Ireland Ltd.

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Possible induction of [Met]enkephalin-Arg6-Gly7-Leu 8 immunoreactivity in neurons of the rat superior colliculus following eye enucleation S. Okamoto I, H. Okamura 2, Y. Akagi l, N. Yanaihara 3 and Y. Ibata 2 Departments of 1Ophthalmology and eAnatomy, Kyoto Prefectural Universityof Medicine, Kyoto (Japan) and SLaboratory of Bioorganic Chemistry, Department of Pharmacology, Universityof Shizuoka, Sizuoka (Japan) (Received 8 February 1990; Revised version received 10 August 1990; Accepted 10 August 1990)

Key words." Met-Enkephalin-Arg6-GlyT-Leu8; Eye enucleation; Superior colliculus; Immunocytochemistry; Rat The distribution of [Met]enkephalin-Argr-GlyT-Leu 8 (MEAGL)-immunoreactive (-IR) neurons and its modification after enucleation have been investigated in the rat superior colliculus. In normal rats and on the ipsilateral side of monocular-enucleated rats, small sized vertically elongated fusiform-shaped weakly immunostained neurons were dispersed throughout the sublamina of the stratum griseum superficiale (SGS). In bilaterally enucleated rats and on the contralateral side of monocular-enucleated rats, many small strongly immunoreactive MEAGL-containing neurons, projecting processes horizontally or obliquely toward the surface, appeared in the deepest part of the SGS and the superficial part of the stratum opticum (SO), in contrast to the disappearance of the fusiform-shaped weakly stained neurons in the SGS. MEAGL-IR fibers increased in density throughout the sublamina of the SGS, being densest in the deep SGS, accompanying their increase in the neighboring SO. Sporadically found MEAGL-IR neurons in the deep SO and the stratum griseum intermediale did not show the detectable change of immunoreactivity. These results indicate that enkephalin biosynthesis is undergone.by different type of neurons in the normal and the ocular-deprived superior colliculus, and suggest that some neurons in the adult superior colliculus have a potentiality to express the peptidergic phenotype.

The information from the presynaptic terminal is presumed to be crucial for induction of transmitter biosynthesis of postsynaptic neurons in the peripheral tissue [2, 18], but there seems to be little knowledge about this phenomenon in the central nervous system. The superior colliculus (SC) of the rat brain is an excellent site to examine how presynaptic retinal influence changes the biochemical properties of postsynaptic neurons, because of the sharp laminar distribution of strong retinal afferents, and the existence of several types of peptidergic neurons [11]. In the present study, we examined the immunoreactive change of [Met]enkephalin-Arg6-Gly7-Leu 8 (MEAGL), a peptide derived from proenkephalin A [13], after eye enucleation by an immunocytochemical method. Twenty-two male Wistar rats were used in this study. They were bilaterally (n=8) and unilaterally (n=4) enucleated at 8 weeks of age, and were housed in a normal light-dark cycle (06.00-18.00 h lights on, 18.00-6.00 h lights off) for 11 weeks. For the control, unoperated rats (n = 8) of the same age were housed under the same conditions. All the animals were first deeply anesthesized Correspondence: H. Okamura, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

with pentobarbital and then perfused with 0.1 M phosphate-buffered saline (pH 7.4) and then with an ice-cold fixative containing 4% paraformaldehyde, 0.2% picric acid, and 0.35% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and further fixed with the same fixative without glutaraldehyde for 2 days at 4°C. Coronal sections (24/~m in thickness) were cut on a cryostat. The sections were incubated with antiMEAGL serum (code no. R0171; 1:5000 dilution) for 4 days at 4°C, and then washed and incubated with antirabbit IgG (Immuno-Tech) for 2 h, then with rabbit peroxidase-antiperoxidase (Dako) which was finally revealed by 3,3'-diaminobenzidine [17]. Immunopositive cells were plotted with camera lucida. Borders of each layer were determined consulting adjacent Cresyl violet stained sections. In normal and unilaterally enucleated rats, the number of immunopositive cells at the central level of SC (bregma - 6 . 5 ram; 1 mm wide from the mid point of each SC) of each section was counted according to the depth (10/~m each) from the surface of SC. Cell counts were cumulated from 5 consecutive sections for each rat (n = 4). The specificity of the anti-MEAGL serum had been described in a previous report [12]. Preabsorption of the antiserum with 1/~g/ml of diluted antigen resulted in no immunostaining. In normal rats, small (long diameter, 9.8 + 1.9 /~m

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Fig. 1. Immunocytochemistry of [Met]enkephalin-Arg6-GlyT-Leu s (MEAGL) in the superior colliculus of normal (A, C) and bilaterally eye-enucleated (B, D) rats. (C) and (D) are high power view of (A) and (B), respectively. Arrows in (A) indicate weakly stained neurons. (E-I): MEAGLimmunoreactive neurons found in the SGS (E, F), the border of SGS and the SO (G), the deep SO (H) and the SGI (I) in normal (E, F, H, I) and bilaterally eye-enucleated (G) rats. Note immunoreactive neurons of the normal rats project fibers vertically from apical ends of elongated cell bodies (C, E, F), but those of enucleated rats project them obliquely (D) or horizontally (G) toward the surface. SZ, stratum zonale; SGS, stratum griseum superficiale; SO, stratum opticum; SGI, stratum griseum intermediale. Bars = 100/an in A-D; 20 gm in E-I.

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(mean + S.D.); short diameter, 6.9 + 0.9/zm, n = 75) fusiform-shaped MEAGL-immunoreactive (IR) neurons were distributed throughout the sublamina of the stratum griseum superficiale (SGS) (Fig. 1A,C,E,F). Most of them had weakly immunostained vertically elongated cell bodies with ascending and descending processes (Fig. I C). Very sporadically, medium to large (long diameter, 21-28/lm) neurons were detected in deeper layers (the deep part of stratum opticum (SO) and the stratum griseum intermediale (SGI)) (Fig. 1H, I). The density of immunoreactive fibers of the normal rats was moderate in the stratum zonale (SZ), SO and SGI, but sparse in the SGS. After bilateral eye enucleation, the MEAGL-immunoreactivity changed markedly in the SC, especially in the SGS. Many strongly immunoreactive small (long diameter, 8.7+1.3 /~m, short diameter, 7.3-1-0.9 /~m, n = 79) neurons appeared in the SO and the deep sublamina of SGS, projecting processes horizontally or obliquely toward the surface (Fig. 1B,D,G). In contrast, weakly immunostained fusiform-shaped neurons that had been detected in the control rats disappeared in the SGS of bilaterally enucleated rats. Increase in the density of immunoreactive fibers was noted throughout the SGS and SO, but no prominent change of immunoreactivity was detected in the SZ, deep SO and SGI. The number or the density of immunoreactive neurons were never changed in the deep SO and SGI, and in the interpeduncular nucleus or the midbrain central gray.

In the rats that had undergone unilateral enucleation, the above alteration of MEAGL-immunoreactivity was found in the contralateral side of the superior colliculus, without affecting that of ipsilateral one. The denserimmunoreactive cells were observed in the contralateral side of the deep SGS and the superficial part of SO from the rostral to the caudal pole (Fig. 2). The location of immunoreactive neurons were plotted according to the depth from the surface of SC (Fig. 3). In the normal and the ipsilateral side of the unilaterally enucleated rats, most of MEAGL-IR cells were located in 40--260/~m depth throughout the SGS. In the contralateral side of the unilaterally enucleated rats, most of immunoreactive neurons were found in 190-310 pm depth with highest density in the 240-290/~m depth, which correspond to bordering area between the SGS and SO. The calculated total number of immunopositive cells from the surface to the 310/lm depth, was almost doubled (97:193) in the contralateral side as compared with the ipsilateral side. Enkephalin-containing neurons have been reported in the cat SC [5]. In the rat SC, there have been no extensive studies of the enkephalinergic neurons (see refs. 7, 12). In the present study, by using anti-MEAGL serum which detects MEAGL and its N-terminally extended form of MEAGL [12], we found small enkephalinergic neurons in the SGS and larger neurons in the deep SO and SGI of the rat. Many of SGS-dispersed neurons resembled the pyramidal or piriform cells (type B) of Cajal [3], and narrow field vertical cells of Langer and

Fig. 2. Camera lucida plotting of the MEAGL-IR neurons in the superior colliculus in frontal sections following unilateral eye enucleation. Frontal sections ((A) to (D)) are aligned in rostro-caudal order. Contralateral superior colliculus is presented on the right hand side of the figures.

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Fig. 3. Depth profiles of the numbers of MEAGL-IR cells in normal (A) and monocular-deprived superior colliculus (B, C). The counts represent the number of stained neurons in contiguous rectangles 1 mm wide and 10/am deep through one side of normal (A), ipsilateral side (B) and contralateral side (C) of the eye enucleation. Cell counts are the summation of 5 serial sections from each rat (n=4). Layers are determined by matching the MEAGL-stained sections with adjacent Cresyl violet-stained sections. Abbreviations are given in Fig. 1.

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Lund [10]. The presence of enkephalinergic neurons in both the superficial and deep layers of SC of cat and rat suggests that the enkephalins are involved both in visual and sensorimotor information processing in the SC. After bilateral eye enucleation, strongly immunoreactive neurons with horizontally oriented processes appeared numerous in the deep SGS and the superficial SO, in contrast to the disappearance of weakly immunoreactive SGS-dispersed neurons. The category of neurons which become labeled after enucleation is unknown, because only cell bodies and initial dendrites were immunostained. However, their location and the direction of initial processes were in concordance with those of wide field vertical cells of the Golgi study by Langer and Lund [10], although newly labeled MEAGL neurons of this study were much smaller than the wide field vertical cells (15-25/~m [10]). The immunocytochemical change of MEAGL was especially noticeable in the area deprived of retinal terminals, because, (1) in the monocular enucleated rats, the change was noticeable only on the contralateral side without affecting ipsilateral side of the SC, (2) no prominent change of immunostaining was observed in MEAGL-IR neurons located in the deeper layers (deep SO and SGI), or in neurons located outside of the SC (interpeduncular nucleus and central gray of the midbrain). The presynaptic control of biochemical properties of postsynaptic neurons is best known in the developing [2] and adult [18] superior cervical ganglion, where cholinergic presynaptic information regulates synthesis of tyrosine hydroxylase in ganglion cells. In the retinal projection areas, we recently reported that the blindness [14] or the darkness [17] induced increase and decrease of the immunoreactivity of vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus and the SC, respectively. These results suggest that some peptide biosyntheses in neurons of visual target areas are regulated by retinal information. There are two possible explanations about the origin of the small strongly immunoreactive MEAGL neurons. One possibility is that weakly immunostained vertically elongated fusiform-shaped cells of the intact SC transformed to strongly-immunostained neurons with horizontally oriented processes after denervation. Monocular-deprivation is known to induce change of cell morphology such as cell-shrinkage accompanying regression of dendrites in the lateral geniculate nucleus [4, 6]. However, by this explanation, it is impossible to explain the change of cell localization and the increase of MEAGLIR cell number. Complete difference in localization and cell type of MEAGL-IR neurons before and after eye enucleation supports the other possibility that strongly immuno-

stained neurons are completely different in origin. Fusiform-shaped MEAGL-IR neurons may be dead or become unable to synthesize MEAGL above the level of detection, and neurons with horizontally or obliquely oriented processes located in the SO and the deep SGS might increase or obtain the ability to synthesize enkephalins. Retinal afferents may suppress the MEAGL synthesis of the latter neurons, and the elimination of retinal afferents increases the enkephalin synthesis, as in the case of striatal enkephalinergic neurons after 6-hydroxydopamine-induced catecholamine lesions [16]. In the normal condition, enkephalin traits may not be expressed, or only very slightly expressed in the neurons with horizontally oriented processes, because intraventricular colchicine treatment to untreated rats for accumulating MEAGL in cell bodies could not reveal such type of immunoreactive cells (Okamura, unpublished observation). In the peripheral nervous system, it is known that, under appropriate conditions, fully differentiated sympathetic neurons can express cholinergic traits [15], and conversely, parasympathetic neurons can express adrenergic traits [1, 9]. In the adult central nervous system such example is rarely known except hypothalamic corticotropin releasing factor containing neurons which begin to synthesize vasopressin after adrenectomy [8]. The present result suggests that some neurons in the adult SC are also plastic in transmitter choice. Since it does not refute the possibility that some of the observed changes shown in this study result from the severe manipulation that an eye removal constitutes, further studies are needed whether a limited retinal lesion by photocoagulation could induce the observed effect only in the retinotopically corresponding region in the SC.

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