- Email: [email protected]
The cholinergic system in retinal transplants in rats
Brain Research, 523 (1990) 156-160 Elsevier
156 BRES 24174
The cholinergic system in retinal transplants in rats L.S. Jen 1, D. Tsang 2, R.M.W.
C h a u 3 a n d W . Z . S h e n ~'*
Departments of tAnatomy and 2Biochemistry, The Chinese Universityof Hong Kong (Hong Kong) and ~Departmentof Anatomy, University of Hong Kong (Hong Kong) (Accepted 27 March 1990)
Key words: Retinal transplant; Acetylcholinesterase; Choline acetyltransferase; Rat
Retinas from embryonic day 13 or 14 Sprague-Dawley albino rats were transplanted to the brainstem of newborn rats with unilateral eye enucleation at birth. Two months after the transplantation, the activity and distribution of acetylcholinesterase and choline acetyltransferase were studied using histochemical and immunocytochemical methods respectively. Results obtained showed that the staining patterns of these two cholinergic enzymes in the retinal transplants were essentially the same as those observed in the retinas of normal rats and in the control retinas of the recipient animals. The similarities in the distribution of these two cholinergic enzymes in these retinas suggest that the cholinergic system in the retinal transplants is likely to be functional.
Intracranial transplantation of retina in the rat has become a useful model for studying factors involved in and conditions required for determining the survival, organization and functional connections of transplanted central neurons in mammals 4'7-9'11'12'14'21-23. Recent studies have focused mainly, however, on the interactions
between ganglion cells of the transplants and the visual centers of the host brain, while relatively little is known about intrinsic organization of the various transmitter systems in the transplants. Previously we have shown that the laminar organization of cytochrome oxidase activity in retinal transplants was comparable to that of host eyes and normal retinas 9. The present study examined the cholinergic system in retinal transplants and normal retinas using acetylcholinesterase (ACHE) histochemistry and choline acetyltransferase (CHAT) immunocytochemistry in an attempt to shed light on the functional organization of the cholinergic system in retinal transplants. In all mammalian retinas studied in recent years, including rat, acetylcholine (ACh) has been found and is known to play a functional role in visual transmission 3' 6,13,15,18,19,24; in particular, the cholinergic system in the amacrine and displaced amacrine cells is believed to be involved in the ON- and OFF-activities in the inner
retina 1,5,13,17-19,24. Five litters of Sprague-Dawley rats supplied by the Animal House of the Chinese University were used in this study. Retinas from embryonic day 13 to 14 old donor rats were dissected out in ice-cold Ham's F-10
medium (Gibco) and then injected into the brainstem of newborn rats as described in previous studies 8'9'22. The right eyes of the recipient rats were enucleated to enhance the ingrowth of the optic axons of the transplanted retinas to the partially denervated targets. In each litter, 2-4 animals were sham-operated and these served as normal controls. All animals were then returned to their respective litters and raised until 2 months old before they were sacrificed. All animals were anaesthetized deeply with 7.5% chloral hydrate (0.5 ml/100 g b. wt.) before they were perfused with physiological saline and then 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains containing retinal transplants were sectioned transversely (20-30/xm thickness) with a cryostat. Two to 3 series of sections were collected and processed for AChE histochemistry and ChAT immunocytochemistry. For A C h E staining, sections were incubated with acetylthiocholine iodide (0.5 mg/g, Sigma) for 3 h to overnight as described by Karnovsky and Roots ~°, while control sections were processed similarly but in the presence of 10-5 M of BW 284c51 (1,5-bis-(4-allyldimethylammoniumphenyl)-pentan3-one dibromide, Sigma), a specific inhibitor of ACHE. For the localization of ChAT activity in the retinas, sections were incubated with rat monoclonal anti-ChAT antibody (3 #g/ml, Boehringer, Mannheim) in Tris buffer containing normal rabbit serum, bovine serum albumin, Triton X-100 and sodium azide for 24 h. Sections were then processed according to the avidin-biotin (Vector Labs.)
* W.Z. Shen is a visiting research fellow on leave from the Department of Anatomy, Jinan University, Guangzhou, People's Republic of China. Correspondence: L.S. Jen, Department of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
157 c o m p l e x m e t h o d as described by H e n d r y et al. 6. Control sections were incubated with n o r m a l rat serum instead of the p r i m a r y antibody, and no positive reactions were o b s e r v e d in these control sections. O n e series of sections was c o u n t e r s t a i n e d with Cresyl violet or haematoxylin and eosin in addition to A C h E histochemistry or C h A T i m m u n o c y t o c h e m i s t r y for cytoarchitectonic studies. For c o m p a r i s o n , retinas of s h a m - o p e r a t e d rats of similar ages and the left eyes of the host animals were similarly processed to reveal the n o r m a l distribution patterns of
A C h E and CHAT. In retinal sections stained for A C h E activity, distinct positive reaction was o b s e r v e d t h r o u g h o u t the inner plexiform layer (IPL) (Fig. 1 A - C ) . In a g r e e m e n t with a previous study on n o r m a l rat retina 19, the A C h E activity in the normal retinas was not uniformly distributed in the IPL, and is divided into two m a j o r zones (Fig. 1B,C). The inner and thinner zones of the I P L bordering the ganglion cell layer ( G C L ) was invariably d a r k e r stained than the o u t e r and wider zone. In most of the retinal
Fig. 1. Photomicrographs illustrating AChE staining of a normal retina and a retinal transplant. The sections were lightly counterstained with Cresyl violet to reveal the laminar organization of the retinal tissues. A: a portion of a normal retina. The AChE staining concentrates in the inner plexiform layer (IPL). The transition region between the IPL and the inner nuclear layer is marked with arrows. Note that sublamination of IPL could be observed in certain parts of the IPL. (B) and (C) show different parts of the same retina. While sublamination in the outer part of IPL as revealed by AChE staining is not clear in B, it is clearly observed in C. D: a retinal transplant located between the two inferior coiliculi (IC). The transplant is outlined by asterisks, and the arrows indicate region bordering IPL and inner nuclear layer. The region marked by the rectangle is shown in E which shows sublamination of the AChE positive region in IPL. A and D, x200; B, C and E, x600.
158
(A)
~r
4¢
(B)
!
Fig. 2. Photomicrographs showing a retinal transplant located near the left superior colliculus (SC) which was partially denervated at birth by eye enucleation. The transplant in A is outlined by the asterisks. B: a portion of the transplant marked by the rectangle in A at higher magnification. The outer part of the photoreceptor layer is indicated by arrows. The ChAT-immunoreactive neurons are distributed in the inner nuclear layer and ganglion cell layer (GCL), respectively. Note also the bilaminar pattern of ChAT-immunoreactive bands in the inner plexiform layer (IPL). MGN, medial geniculate nucleus. A ×120; B ×600.
sections e x a m i n e d , the outer zone can be divided into 4 thin A C h E - r e a c t i v e bands with the o u t e r m o s t one located at the inner margin of the inner nuclear layer (INL) (Fig. 1C). In general, the A C h E staining pattern observed in all retinal transplants was similar to those observed in the remaining host eyes and control retinas regardless of the
graft sites in the brainstem. In all cases, the ACHEreactive zone was always restricted to the IPL, and a sublamination of the A C h E - s t a i n i n g region was clearly and consistently observable. A n example is shown in Fig. 1D which reveals a retinal transplant situated b e t w e e n the two inferior colliculi and the cerebellum. It can be seen that the inner part of the IPL a d j a c e n t to the
159 AChE histochemistry and ChAT immunocytochemistry in retinal transplants is essentially the same as that in the normal retinas. This finding, together with our previous study on cytochrome oxidase distribution in retinal transplants 9, supports the conclusion that the structural and functional organization of retinas transplanted to a foreign environment are similar to those of the normal retinas. As the distribution pattern of the two populations of ChAT-immunoreactive neurons as well as their processes in the IPL of the retinal transplants and normal retinas are so similar, it is tempting to conclude that the development and organization of the cholinergic system in the retinal transplants is quite normal. In normal mammalian retinas, the cholinergic amacrine and displaced amacrine cells in the IPL are believed to be involved in the ON- and OFF-activities in the inner retina 1'5'13"17-19'24, thus the present finding suggests that the ON- and OFF-visual channels in the transplanted retinas may be functional. Comparison of the AChE and ChAT staining patterns showed that the AChE activity is more widely distributed than that of CHAT, and that some of the AChE-reactive bands have no counterparts in preparations processed for ChAT immunocytochemistry. This is not surprising as AChE is known to be present in both neuronal and non-neuronal tissues. In addition, it has recently been suggested that the AChE is not exclusively used as an enzyme for inactivation of the cholinergic neurotransmission, but probably as a neuropeptide processing enzyme as well 2'2°. Whether AChE also functions in a similar manner in the rat retina is unclear at present. Though it is known that AChE is not a specific marker for cholinergic neurons, the present finding that distinct AChE patterns can be observed in both normal and transplanted retinas suggests that AChE histochemistry can be used as a cellular marker for revealing the structural, and possibly functional, organization of the IPL even in retinas transplanted to an ectopic region or foreign environment.
ganglion cell layer was homogeneously stained for AChE activity, and several thin AChE reactive bands separated by lighter clefts were also frequently observed in the outer and wider part of the IPL (Fig. 1E). The AChE staining in the outer zone of the IPL bordering the GCL, and some of the sublaminae in the outer part of the INL appeared to be slightly darker in this particular specimen than the corresponding parts in most of the normal retinas and retinal transplants. As slight variations in the staining intensity in the IPL were observed in normal as well as transplanted retinas, this may reflect variations in AChE activity in the IPL region. No difference in ChAT staining intensity and distribution pattern was observed between retinas of normal and host rats and retinal transplants, and the pattern observed is similar to that described previously by Voigt in the rat 24. In the present study, two populations of distinctly immunoreactive cells which organized in a mirror-symmetrical fashion as described in rabbit retinas 5 were also detected in the inner part of normal retinas and retinal transplants (Fig. 2). One population of the ChAT-immunoreactive cells had their somata located mainly in the innermost regions of the INL. Although some of the immunoreactive cells in this population were located in the more outer part of the INL, the number was small and the pattern was certainly different from the population of cholinergic neurons observed in lower vertebrates such as chicken 15. The somata of the other population of ChAT-immunoreactive cells were distributed primarily in the G C L (Fig. 2B). In addition to these two populations of labelled cells, two distinct ChAT-immunoreactive bands were also found in the IPL. Closer examination of the cells in the IPL revealed that the cellular processes of some of the two populations of ChAT-immunoreactive cells which flanked the IPL also contributed to the ChAT reactive bands, but it is not certain whether some of these cells may send their processes to both bands. Comparison of the location and pattern of the ChAT-rich bands with the A C h E reactive bands revealed that these two CHATimmunoreactive bands correspond to the second and fourth sublaminae of the AChE-rich sublaminae. This finding is in good agreement with the results observed in the normal retinas of the tree shrew 3. The main finding in this study in the rat is that the organization of the cholinergic system as revealed by
The authors would like to thank Prof. R.D. Lund for providing the opportunity to L.S.J. to perfect the transplantation techniques, and Ms. Suki Wong and Yuen Shan Tsang for technical assistance. This study was supported by grants from the Hong Kong University and Polytechnic Grants Committee (221400030), the Croucher Foundation (360-031-0792-4F), and the United College, The Chinese University of Hong Kong.
1 Bloomfield, S.A. and Miller, R.E, A functional organization of ON and OFF pathways in the rabbit retina, Neuroscience, 6 (1986) 1-13. 2 Chubb, I.W., Ranieri, E., White, G.H. and Hodgson, A.J., Acetylcholinesterase hydrolyses substance P, Neuroscience, 5 (1980) 2065-2072. 3 Conley, M., Fitzpatrick, D. and Lachica, E.A., Laminar
asymmetry in the distribution of choline acetyltransferase immunoreactive neurons in the retina of the tree shrew (Tupaia belangeri), Brain Research, 399 (1986) 332-338. 4 Craner, S.L., Radel, J.D., Jen, L.S. and Lund, R.D., Lightevoked cortical activity produced by illumination of intracranial retinal transplants: experimental studies in rats, Exp. Neurol., 104 (1989) 93-100.
160 5 Famiglietti Jr., E.V., 'Starburst' amacrine cells and cholinergic neurons: mirror-symmetric ON and OFF amacrine cells of rabbit retina, Brain Research, 261 (1983) 138-144. 6 Hendry, S.H.C., Jones, E.G., Killackey, H.P. and Chalupa, L.M., Choline acetyltransferase-immunoreactive neurons in fetal monkey cerebral cortex, Dev. Brain Res., 37 (1987) 313-317. 7 Hankin, M.H. and Lund, R.D., Specific target-directed axonal outgrowth from transplanted embryonic rodent retinae into neonatal rat superior colliculus, Brain Research, 408 (1987) 344-348. 8 Hankin, M.H. and Lund, R.D., Role of the target in the directing the outgrowth of retinal axons: transplants reveal surface-related and surface-independent cues, J. Comp. Neurol., 263 (1987) 455-466. 9 Jen, L.S., Chau, R.M.W. and Tsang, D., Cytochrome oxidase activity in retinas transplanted to the brainstem in rats, Neurosci. Lett., 105 (1989) 275-280. 10 Karnosky, M.J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, J. Histochem. Cytochem., 12 (1964) 219-221. 11 Klassen, H. and Lund, R.D., Retinal transplants can drive a pupillary reflex in host rat brains, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6958-6960. 12 Lund, R.D., Rao, K., Kunz, H.W. and Gill III, T.J., Instability of neural xenografts placed neonatal rat brains, Transplantation, 46 (1988) 216-223. 13 Masland, R.H. and Mills, J.W., Autoradiographic identification of acetylcholine in the rabbit retina, J. Cell Biol., 83 (1979) 159-178. 14 McLoon, S.C. and Lund, R.D., Specific projections of retina transplanted to rat brain, Exp. Brain Res., 40 (1980) 273-282. 15 Millar, T.J., Ishimoto, I., Chubb, I.W., Epstein, M.L., Johnson,
16
17 18 19
20 21 22
23
24
C.D. and Morgan, I.G., Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study, Neuroscience, 21 (1987) 725-742. Nelson, R., Famiglietti, E.V. and Kolb, H., IntraceUular staining reveals different levels of stratification for ON- and OFF-center ganglion cells in cat retina, J. Neurophysiol., 41 (1978) 472-483. Perry, V.H., Evidence for an amacrine cell system in the ganglion cell layer of the rat retina, Neuroscience, 6 (1981) 931-944. Pourcho, R.G. and Osman, K., Acetylcholinesterase localization in cat retina: a comparison with choline acetyltransferase, Exp. Eye Res., 43 (1986) 585-594. Ross, C.D., Dunning, D.D., Juengel, L.I. and Godfrey, D.A., Laminar distributions of choline acetyltransferase and acetylcholinesterase activities in the inner plexiform layer of rat retina, J. Neurochem., 44 (1985) 1091-1099. Small, D.H., Acetylcholinesterase: zymogens of neuropeptide processing enzymes?, Neuroscience, 29 (1989) 241-249. Sefton, A.J. and Lund, R.D., Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex, J. Cornp. Neurol., 269 (1988) 548-564. Sefton, A.J., Lund, R.D. and Perry, V.H., Target regions enhance the outgrowth and survival of ganglion cells in embryonic retina transplanted to cerebral cortex in neonatal rats, Dev. Brain Res., 33 (1987) 145-149. Simons, D.J. and Lund, R.D., Fetal retinae transplanted over tecta of neonatal rats respond to light and evoke patterned neuronal discharges in the host superior colliculus, Dev. Brain Res., 21 (1985) 156-159. Voigt, T., Cholinergic amacrine cells in the rat retina, J. Comp. Neurol., 248 (1986) 19-35.
156 BRES 24174
The cholinergic system in retinal transplants in rats L.S. Jen 1, D. Tsang 2, R.M.W.
C h a u 3 a n d W . Z . S h e n ~'*
Departments of tAnatomy and 2Biochemistry, The Chinese Universityof Hong Kong (Hong Kong) and ~Departmentof Anatomy, University of Hong Kong (Hong Kong) (Accepted 27 March 1990)
Key words: Retinal transplant; Acetylcholinesterase; Choline acetyltransferase; Rat
Retinas from embryonic day 13 or 14 Sprague-Dawley albino rats were transplanted to the brainstem of newborn rats with unilateral eye enucleation at birth. Two months after the transplantation, the activity and distribution of acetylcholinesterase and choline acetyltransferase were studied using histochemical and immunocytochemical methods respectively. Results obtained showed that the staining patterns of these two cholinergic enzymes in the retinal transplants were essentially the same as those observed in the retinas of normal rats and in the control retinas of the recipient animals. The similarities in the distribution of these two cholinergic enzymes in these retinas suggest that the cholinergic system in the retinal transplants is likely to be functional.
Intracranial transplantation of retina in the rat has become a useful model for studying factors involved in and conditions required for determining the survival, organization and functional connections of transplanted central neurons in mammals 4'7-9'11'12'14'21-23. Recent studies have focused mainly, however, on the interactions
between ganglion cells of the transplants and the visual centers of the host brain, while relatively little is known about intrinsic organization of the various transmitter systems in the transplants. Previously we have shown that the laminar organization of cytochrome oxidase activity in retinal transplants was comparable to that of host eyes and normal retinas 9. The present study examined the cholinergic system in retinal transplants and normal retinas using acetylcholinesterase (ACHE) histochemistry and choline acetyltransferase (CHAT) immunocytochemistry in an attempt to shed light on the functional organization of the cholinergic system in retinal transplants. In all mammalian retinas studied in recent years, including rat, acetylcholine (ACh) has been found and is known to play a functional role in visual transmission 3' 6,13,15,18,19,24; in particular, the cholinergic system in the amacrine and displaced amacrine cells is believed to be involved in the ON- and OFF-activities in the inner
retina 1,5,13,17-19,24. Five litters of Sprague-Dawley rats supplied by the Animal House of the Chinese University were used in this study. Retinas from embryonic day 13 to 14 old donor rats were dissected out in ice-cold Ham's F-10
medium (Gibco) and then injected into the brainstem of newborn rats as described in previous studies 8'9'22. The right eyes of the recipient rats were enucleated to enhance the ingrowth of the optic axons of the transplanted retinas to the partially denervated targets. In each litter, 2-4 animals were sham-operated and these served as normal controls. All animals were then returned to their respective litters and raised until 2 months old before they were sacrificed. All animals were anaesthetized deeply with 7.5% chloral hydrate (0.5 ml/100 g b. wt.) before they were perfused with physiological saline and then 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains containing retinal transplants were sectioned transversely (20-30/xm thickness) with a cryostat. Two to 3 series of sections were collected and processed for AChE histochemistry and ChAT immunocytochemistry. For A C h E staining, sections were incubated with acetylthiocholine iodide (0.5 mg/g, Sigma) for 3 h to overnight as described by Karnovsky and Roots ~°, while control sections were processed similarly but in the presence of 10-5 M of BW 284c51 (1,5-bis-(4-allyldimethylammoniumphenyl)-pentan3-one dibromide, Sigma), a specific inhibitor of ACHE. For the localization of ChAT activity in the retinas, sections were incubated with rat monoclonal anti-ChAT antibody (3 #g/ml, Boehringer, Mannheim) in Tris buffer containing normal rabbit serum, bovine serum albumin, Triton X-100 and sodium azide for 24 h. Sections were then processed according to the avidin-biotin (Vector Labs.)
* W.Z. Shen is a visiting research fellow on leave from the Department of Anatomy, Jinan University, Guangzhou, People's Republic of China. Correspondence: L.S. Jen, Department of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
157 c o m p l e x m e t h o d as described by H e n d r y et al. 6. Control sections were incubated with n o r m a l rat serum instead of the p r i m a r y antibody, and no positive reactions were o b s e r v e d in these control sections. O n e series of sections was c o u n t e r s t a i n e d with Cresyl violet or haematoxylin and eosin in addition to A C h E histochemistry or C h A T i m m u n o c y t o c h e m i s t r y for cytoarchitectonic studies. For c o m p a r i s o n , retinas of s h a m - o p e r a t e d rats of similar ages and the left eyes of the host animals were similarly processed to reveal the n o r m a l distribution patterns of
A C h E and CHAT. In retinal sections stained for A C h E activity, distinct positive reaction was o b s e r v e d t h r o u g h o u t the inner plexiform layer (IPL) (Fig. 1 A - C ) . In a g r e e m e n t with a previous study on n o r m a l rat retina 19, the A C h E activity in the normal retinas was not uniformly distributed in the IPL, and is divided into two m a j o r zones (Fig. 1B,C). The inner and thinner zones of the I P L bordering the ganglion cell layer ( G C L ) was invariably d a r k e r stained than the o u t e r and wider zone. In most of the retinal
Fig. 1. Photomicrographs illustrating AChE staining of a normal retina and a retinal transplant. The sections were lightly counterstained with Cresyl violet to reveal the laminar organization of the retinal tissues. A: a portion of a normal retina. The AChE staining concentrates in the inner plexiform layer (IPL). The transition region between the IPL and the inner nuclear layer is marked with arrows. Note that sublamination of IPL could be observed in certain parts of the IPL. (B) and (C) show different parts of the same retina. While sublamination in the outer part of IPL as revealed by AChE staining is not clear in B, it is clearly observed in C. D: a retinal transplant located between the two inferior coiliculi (IC). The transplant is outlined by asterisks, and the arrows indicate region bordering IPL and inner nuclear layer. The region marked by the rectangle is shown in E which shows sublamination of the AChE positive region in IPL. A and D, x200; B, C and E, x600.
158
(A)
~r
4¢
(B)
!
Fig. 2. Photomicrographs showing a retinal transplant located near the left superior colliculus (SC) which was partially denervated at birth by eye enucleation. The transplant in A is outlined by the asterisks. B: a portion of the transplant marked by the rectangle in A at higher magnification. The outer part of the photoreceptor layer is indicated by arrows. The ChAT-immunoreactive neurons are distributed in the inner nuclear layer and ganglion cell layer (GCL), respectively. Note also the bilaminar pattern of ChAT-immunoreactive bands in the inner plexiform layer (IPL). MGN, medial geniculate nucleus. A ×120; B ×600.
sections e x a m i n e d , the outer zone can be divided into 4 thin A C h E - r e a c t i v e bands with the o u t e r m o s t one located at the inner margin of the inner nuclear layer (INL) (Fig. 1C). In general, the A C h E staining pattern observed in all retinal transplants was similar to those observed in the remaining host eyes and control retinas regardless of the
graft sites in the brainstem. In all cases, the ACHEreactive zone was always restricted to the IPL, and a sublamination of the A C h E - s t a i n i n g region was clearly and consistently observable. A n example is shown in Fig. 1D which reveals a retinal transplant situated b e t w e e n the two inferior colliculi and the cerebellum. It can be seen that the inner part of the IPL a d j a c e n t to the
159 AChE histochemistry and ChAT immunocytochemistry in retinal transplants is essentially the same as that in the normal retinas. This finding, together with our previous study on cytochrome oxidase distribution in retinal transplants 9, supports the conclusion that the structural and functional organization of retinas transplanted to a foreign environment are similar to those of the normal retinas. As the distribution pattern of the two populations of ChAT-immunoreactive neurons as well as their processes in the IPL of the retinal transplants and normal retinas are so similar, it is tempting to conclude that the development and organization of the cholinergic system in the retinal transplants is quite normal. In normal mammalian retinas, the cholinergic amacrine and displaced amacrine cells in the IPL are believed to be involved in the ON- and OFF-activities in the inner retina 1'5'13"17-19'24, thus the present finding suggests that the ON- and OFF-visual channels in the transplanted retinas may be functional. Comparison of the AChE and ChAT staining patterns showed that the AChE activity is more widely distributed than that of CHAT, and that some of the AChE-reactive bands have no counterparts in preparations processed for ChAT immunocytochemistry. This is not surprising as AChE is known to be present in both neuronal and non-neuronal tissues. In addition, it has recently been suggested that the AChE is not exclusively used as an enzyme for inactivation of the cholinergic neurotransmission, but probably as a neuropeptide processing enzyme as well 2'2°. Whether AChE also functions in a similar manner in the rat retina is unclear at present. Though it is known that AChE is not a specific marker for cholinergic neurons, the present finding that distinct AChE patterns can be observed in both normal and transplanted retinas suggests that AChE histochemistry can be used as a cellular marker for revealing the structural, and possibly functional, organization of the IPL even in retinas transplanted to an ectopic region or foreign environment.
ganglion cell layer was homogeneously stained for AChE activity, and several thin AChE reactive bands separated by lighter clefts were also frequently observed in the outer and wider part of the IPL (Fig. 1E). The AChE staining in the outer zone of the IPL bordering the GCL, and some of the sublaminae in the outer part of the INL appeared to be slightly darker in this particular specimen than the corresponding parts in most of the normal retinas and retinal transplants. As slight variations in the staining intensity in the IPL were observed in normal as well as transplanted retinas, this may reflect variations in AChE activity in the IPL region. No difference in ChAT staining intensity and distribution pattern was observed between retinas of normal and host rats and retinal transplants, and the pattern observed is similar to that described previously by Voigt in the rat 24. In the present study, two populations of distinctly immunoreactive cells which organized in a mirror-symmetrical fashion as described in rabbit retinas 5 were also detected in the inner part of normal retinas and retinal transplants (Fig. 2). One population of the ChAT-immunoreactive cells had their somata located mainly in the innermost regions of the INL. Although some of the immunoreactive cells in this population were located in the more outer part of the INL, the number was small and the pattern was certainly different from the population of cholinergic neurons observed in lower vertebrates such as chicken 15. The somata of the other population of ChAT-immunoreactive cells were distributed primarily in the G C L (Fig. 2B). In addition to these two populations of labelled cells, two distinct ChAT-immunoreactive bands were also found in the IPL. Closer examination of the cells in the IPL revealed that the cellular processes of some of the two populations of ChAT-immunoreactive cells which flanked the IPL also contributed to the ChAT reactive bands, but it is not certain whether some of these cells may send their processes to both bands. Comparison of the location and pattern of the ChAT-rich bands with the A C h E reactive bands revealed that these two CHATimmunoreactive bands correspond to the second and fourth sublaminae of the AChE-rich sublaminae. This finding is in good agreement with the results observed in the normal retinas of the tree shrew 3. The main finding in this study in the rat is that the organization of the cholinergic system as revealed by
The authors would like to thank Prof. R.D. Lund for providing the opportunity to L.S.J. to perfect the transplantation techniques, and Ms. Suki Wong and Yuen Shan Tsang for technical assistance. This study was supported by grants from the Hong Kong University and Polytechnic Grants Committee (221400030), the Croucher Foundation (360-031-0792-4F), and the United College, The Chinese University of Hong Kong.
1 Bloomfield, S.A. and Miller, R.E, A functional organization of ON and OFF pathways in the rabbit retina, Neuroscience, 6 (1986) 1-13. 2 Chubb, I.W., Ranieri, E., White, G.H. and Hodgson, A.J., Acetylcholinesterase hydrolyses substance P, Neuroscience, 5 (1980) 2065-2072. 3 Conley, M., Fitzpatrick, D. and Lachica, E.A., Laminar
asymmetry in the distribution of choline acetyltransferase immunoreactive neurons in the retina of the tree shrew (Tupaia belangeri), Brain Research, 399 (1986) 332-338. 4 Craner, S.L., Radel, J.D., Jen, L.S. and Lund, R.D., Lightevoked cortical activity produced by illumination of intracranial retinal transplants: experimental studies in rats, Exp. Neurol., 104 (1989) 93-100.
160 5 Famiglietti Jr., E.V., 'Starburst' amacrine cells and cholinergic neurons: mirror-symmetric ON and OFF amacrine cells of rabbit retina, Brain Research, 261 (1983) 138-144. 6 Hendry, S.H.C., Jones, E.G., Killackey, H.P. and Chalupa, L.M., Choline acetyltransferase-immunoreactive neurons in fetal monkey cerebral cortex, Dev. Brain Res., 37 (1987) 313-317. 7 Hankin, M.H. and Lund, R.D., Specific target-directed axonal outgrowth from transplanted embryonic rodent retinae into neonatal rat superior colliculus, Brain Research, 408 (1987) 344-348. 8 Hankin, M.H. and Lund, R.D., Role of the target in the directing the outgrowth of retinal axons: transplants reveal surface-related and surface-independent cues, J. Comp. Neurol., 263 (1987) 455-466. 9 Jen, L.S., Chau, R.M.W. and Tsang, D., Cytochrome oxidase activity in retinas transplanted to the brainstem in rats, Neurosci. Lett., 105 (1989) 275-280. 10 Karnosky, M.J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, J. Histochem. Cytochem., 12 (1964) 219-221. 11 Klassen, H. and Lund, R.D., Retinal transplants can drive a pupillary reflex in host rat brains, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6958-6960. 12 Lund, R.D., Rao, K., Kunz, H.W. and Gill III, T.J., Instability of neural xenografts placed neonatal rat brains, Transplantation, 46 (1988) 216-223. 13 Masland, R.H. and Mills, J.W., Autoradiographic identification of acetylcholine in the rabbit retina, J. Cell Biol., 83 (1979) 159-178. 14 McLoon, S.C. and Lund, R.D., Specific projections of retina transplanted to rat brain, Exp. Brain Res., 40 (1980) 273-282. 15 Millar, T.J., Ishimoto, I., Chubb, I.W., Epstein, M.L., Johnson,
16
17 18 19
20 21 22
23
24
C.D. and Morgan, I.G., Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study, Neuroscience, 21 (1987) 725-742. Nelson, R., Famiglietti, E.V. and Kolb, H., IntraceUular staining reveals different levels of stratification for ON- and OFF-center ganglion cells in cat retina, J. Neurophysiol., 41 (1978) 472-483. Perry, V.H., Evidence for an amacrine cell system in the ganglion cell layer of the rat retina, Neuroscience, 6 (1981) 931-944. Pourcho, R.G. and Osman, K., Acetylcholinesterase localization in cat retina: a comparison with choline acetyltransferase, Exp. Eye Res., 43 (1986) 585-594. Ross, C.D., Dunning, D.D., Juengel, L.I. and Godfrey, D.A., Laminar distributions of choline acetyltransferase and acetylcholinesterase activities in the inner plexiform layer of rat retina, J. Neurochem., 44 (1985) 1091-1099. Small, D.H., Acetylcholinesterase: zymogens of neuropeptide processing enzymes?, Neuroscience, 29 (1989) 241-249. Sefton, A.J. and Lund, R.D., Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex, J. Cornp. Neurol., 269 (1988) 548-564. Sefton, A.J., Lund, R.D. and Perry, V.H., Target regions enhance the outgrowth and survival of ganglion cells in embryonic retina transplanted to cerebral cortex in neonatal rats, Dev. Brain Res., 33 (1987) 145-149. Simons, D.J. and Lund, R.D., Fetal retinae transplanted over tecta of neonatal rats respond to light and evoke patterned neuronal discharges in the host superior colliculus, Dev. Brain Res., 21 (1985) 156-159. Voigt, T., Cholinergic amacrine cells in the rat retina, J. Comp. Neurol., 248 (1986) 19-35.