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Light-induced c-fos expression in amacrine cells in the rabbit retina
MOLECULAR BRAIN RESEARCH ELSEVIER
Molecular
Brain Research
Research
Light-induced
29 (1995) 53-63
report
c-f&s expression in amacrine cells in the rabbit retina Jari Koistinaho
a,b,*, Stephen
M. Sagar a
a Department of Neurology, University of California, and Department of Veterans Affairs Medical Center, San Francisco, CA, USA ’ Neural Injury Research Laboratory, Department of Biomedical Sciences, University of Tampere, Tampere, Finland Accepted
13 September
1994
Abstract Retinal neurons that express the immediate early gene c-fos after light exposure were characterized by neurotransmitter content using histochemical and immunocytochemical staining. In Northern blots the amount of c-fos mRNA peaked at 30 min, but remained detectable 60 min following light stimulation. Fos proteins were seen in the inner nuclear and ganglion cell layers, and the staining was most intense two and three hours after beginning the light exposure. In the ganglion cell layer 30-40% of Fos-immunoreactive cells were cholinergic displaced amacrine cells and 3-5% were ganglion cells. In the inner nuclear layer 24% of Fos-immunoreactive cells were Type I and 7% Type II NADPH-diaphorase-reactive (nitric oxide synthase) amacrine was seen cells, 11% were tyrosine hydroxylase-containing cells, and 10-Z% cholinergic amacrine cells. No Fos immunoreactivity in serotoninergic, somatostatinor VIP-immunoreactive cells, bipolar, horizontal or photoreceptor cells. Nicotine, kainic acid, NMDA and SCH 38393, a dopamine D, receptor agonist, induced Fos immunostaining in the inner nuclear and ganglion cell layers, but administration of the corresponding receptor blockers mecamylamine, kynuretic acid, MK-801, haloperidol and SCH 23990 did not prevent light-induced Fos expression. Keywords:
Amacrine
cell; Ganglion
cell; Nitric oxide synthase; Acetylcholine;
1. Introduction
Flashing light is commonly used as a physiological stimulus to the visual pathways in the mammalian nervous system. It produces an electrophysiological response in most retinal neurons, but the extent of depolarization varies from one type of neuron to another [6]. In the rabbit retina, two types of second-order neurons, rod bipolar and On cone bipolar cells, depolarize in response to light [4,6,39]. Input from depolarized bipolar cells is received by a large and heterogenous population of amacrine and ganglion cells in the inner retina. The rapid transsynaptic responses to photic stimulation initiate events such as alteration of neurotransmitter synthesis and release, change in the phosphorylation state of ligand-gated ion channels and activation of multiple second messenger pathways [6].
* Corresponding author. Neural Injury Research Laboratory, Department of Biomedical Sciences, University of Tampere, PO Box 607, FIN-33101 Tampere, Finland. Fax: (358) 31-215 6071. E-mail: [email protected]. 0169-328X/95/$09.50 0 1995 Elsewer SSDI 0169-328X(94)00218-5
Science
B.V. All rights reserved
Tyrosine hydroxylase; Gene induction
Some of the changes may be long-lasting and require gene induction and subsequent protein synthesis [S]. Immediate early genes, including some proto-oncogenes, are transiently induced in neurons following synaptic stimulation [1,2,10,18,20,22,27,40]. For example c-fos and its protein product Fos are thought to participate in transcriptional regulation of the genes needed for long-term alterations in neuronal activity [1,10,22,40]. Therefore, immunocytochemical localization of Fos has provided, with certain limitations, a method to perform functional neuroanatomic mapping at the cellular level [10,22,26,27]. In the rabbit ‘retina, light induces Fos in a subset of neurons and Fos expression, with a varying pattern, can be seen following administration of pharmacologically active agents [13,14,261. Using histochemical and immunocytochemical double-labeling methods we attempted in the present study to classify by neurotransmitter content the retinal neurons which express Fos in response to flashing light. We also tested the effect of glutamate, dopamine and nicotinic receptor agonists and antagonists on Fos expression.
.I. Koistinaho,
54
2. Material
S. M. Sagar / Molecular
and methods
2.1. Chemicals
Nicotinamide adenine dinucleotide phosphate (NADP), 4,6-diamidino-2-phenylindole (DAPI), 5,7_dihydroxytryptamine (5,7-DHTI, kainic acid, iv-methyl-D-aspartate (NMDA), 5-methyl-lO,ll-dihydro5H-dibenzocyclohepten-5,10-imine (MK-801), nicotine, mecamylamine and haloperidol were obtained from Sigma Chemical Co., St. Louis, MO. SCH 238393 and SCH 23390, a dopamine D, receptor agonist and antagonist, respectively, were purchased from RBI, Nitick, MA. Synthetic M peptide was obtained from Penisula Laboratories, Belmont, CA, and FluoroGold from FluoroChrome, Eaglewood, CO. Avidin-biotin-peroxidase (ABC) kit was from Vector Laboratories, Burlingame, CA. Fluorescein-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were from Boehringer Mannheim, Indianapolis, IN. A pSP65-foslA plasmid was generously provided by Dr. Tom Curran, Roche Institute of Molecular Biology, Nutley, NJ. 2.2. Primary antibodies An affinity-purified polyclonal rabbit antiserum (RlB61, raised to a synthetic peptide corresponding to residues 132-154 of Fos [13,26] was used at dilutions of 1 :50-l : 100. A monoclonal anti-tyrosine hydroxylase (Boehringer-Mannheim) and a monoclonal anti-ChAT (choline acetyltransferase, Boehringer-Mannheim) were used at 1: loo-250 dilutions. A rabbit polyclonal anti-VIP (vasoactive intestinal peptide, Chemicon, Temecula, CA) was used at 1:200-l : 300 dilutions. A monoclonal anti-somatostatin [25] (IE9, generously provided by Dr. Linda Chun, Massachusetts General Hospital and Harvard University, Boston, MA) was used at dilution of 1 :3000.
Brain Research
29 (1995)
53-63
lated using guanidinium/lithium chloride method. 5 pg RNA was electrophoresed through a 1.5% agarose gel and transferred to a Nylon membrane (Nytran, Scheicher and Schuell, NH) by capillary blotting. c-fos riboprobe was prepared from c-fos cDNA (cloned into pSP65-foslA plasmid) by using SP6 polymerase and 13’P]CTP. Hybridization buffer contained 50% formamide, 1.5 X SSPE, 1% SDS, 0.5% non-fat dry milk, 0.2 mg/ml sheared denatured salmon testis DNA, and 0.5 mg/ml yeast RNA. The membranes were hybridized at 65°C washed at the same temperature for 30 min each twice with 2x SSC/O.l% SDS, 0.5 x SSC/O.l% SDS and 0.2~ SSC/O.l% SDS, and exposed to Kodak SB5 X-ray film with intensifying screens at ~ 70°C overnight. 2.6. Injection of neuron
markers
For retrograde labelling of ganglion cells rabbits were sedated two days prior the experiment with ketamine (Aveco, Fort Dodge, IA, 25 mg/kg) and xylazine (Anaced, Lloyd Laboratories, Shenandoah, IA, 5 mg/kg), and the right optic nerve was exposed intraorbitally. The nerve was crushed 2 times for 30 s and 2 ~1 2% FluoroGold was injected into the nerve. Care was taken not to disturb the retinal blood supply. The left optic nerve was only exposed without crushing and used as a control. The wounds were closed and the animals allowed to recover. For intravitreal DAPI injections, the rabbits were sedated one day prior to the experiment with pentobarbital (40 mg/i.v.), and the sclera was topically anesthetized with 1% proparicaine ophthalmic solution. Injections of DAPI (0.15 pg in 50 ~1 distilled water) were made at multiple locations in the vitreous cavity of one eye through a 30 gauge needle. The opposite eye of each rabbit received 50 ~1 saline as a control. 5,7-DHT (20 Fg in 50 ~1 distilled water containing 1% ascorbic acid) was injected similar to DAPI, but was administered in the dark 3 h prior to the light exposure.
2.3. Animals 2.7. Histochemistry Adult male New Zealand white rabbits were kept in a central animal care facility on a 12: 12 h light-dark cycle with free access to food and water. On the day prior to the experiment, rabbits were transported to the laboratory and dark-adapted for 24 h. For study of dark-adapted retinas, animals were sacrificed by injection of pentobarbital, 150 mg/kg iv., under infrared illumination with the aid of night vision goggles (Litton Industries, Tempe, AZ). For light exposure, animals were placed in restraining cage and were exposed to flashing light for 2 h. Three Hz, 60 ms approximately 300 lux flashes of white light were generated with a bank of 4 fluorescent lamps driven by a special driver designed to produce a square wave pattern of light intensity (Ralph Gerbrands Co., Arlington, MA). The lights were turned off and the rabbits were sacrificed. Each experiment was repeated at least three times. 2.4. Drug administration 7.5 nmol of kainic acid, 10 nmol of NMDA, 50 Fg of nicotine or 0.5 nmol of SCH 38393 were injected in 50 ~1 of water intravitreally to the right eye of urethane (4.5 g/kg, i.p.1 anesthetized dark-adapted rabbits. Our previous studies have shown that urethane does not induce or block activity-induced neuronal Fos expression in the rabbit retina. MK-801 (1.5-5 mg/kgJ, haloperidol (5 mg/kg), mecamylamine (6 mg/kg), kynuretic acid (100 mg/kgl and SCH 23390 (6 mg/kg) were administred intraperitoneally 30 min prior light exposure.
2.5. Northern
blotting
Dark-adapted retinas and retinas exposed to flashing light for varying period of time were homogenized and total RNA was iso-
and immunocytochemistv
After light exposure eyes were removed in the light and were hemisected at about the equator. The posterior eye cups were immersed in freshly prepared PLP (0.05 M sodium phosphate buffer, pH 7.4, 0.1 M L-lysine, 0.01 M sodium m-periodate, 1.5% paraformaldehyde) prepared according to McLean and Nakane [17], for 3 h at 4°C. After washing in 0.1 M sodium phosphate buffer, pH 7.4 (PB), retinas were dissected, washed in PB, and processed for NADPH-diaphorase histochemistry or directly for immunocytochemistry. A modification of the method of Scherer-Singler et al. [29] was used to demonstrate NADPH diaphorase [14,25]. Tissue was incubated in 0.1 M Tris-maleate, pH 8.0, 15 mM malic acid, 1 mM MnCI,, 1 mM NADP, 0.2 mM nitro blue tetrazolium, and 0.2% Triton X-100 for l-2 h at 37°C. After washing in PB, the tissue was further processed for Fos immunostaining. For immunostaining retinas were incubated in Fos antibody diluted in PB containing 0.1% bovine serum albumin, 0.2% Triton X-100, and 2% normal goat serum (PB-G) at 4°C with gentle agitation for 48-72 h. The tissue was washed in PB, and sites of antibody binding was visualized with FITC-conjugated secondary antibody or the avidin-biotin-peroxidase (ABC) procedure using a Vectastain Kit and diaminobenzidine as chromogen. In some double-staining experiments a rhodamine-conjugated secondary antibody was used. Secondary antiserum and ABC reagent were diluted in PB-G; incubation times were 2-3 h at room temperature. After washing in PB for 2 h, the retinas were subsequently double labelled with monoclonal antibodies. Rabbit polyclonal antibodies were diluted in PB-G and monoclonal antibodies in PB-H (PB containing 0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal horse serum). Binding sites of the antibodies were visualized by incubating
.I. Koistinaho, S.M. Sagar /Molecular retinas in FITC-conjugated secondary antibodies (diluted 1 : 40-100 in PB-G or PB-H) at 37°C for 2 h. After washing in PB, the retinas were flat mounted on gelatin-subbed slides and coverslipped in glycerol-PB mixture (3 : 1).
3. Results 3.1. Light-induced Fos expression Retinas from dark-adapted untreated or salinetreated eyes displayed no or little detectable nuclear Fos-immunostaining as described previously [ 13,14,26]. Two days after crushing the optic nerve or 3 h after DAPI or 5,7-DHT injections, no Fos-immunostaining was observed in dark-adapted retinas. Flashing light induced a mosaic of Fos-IR cells in the ganglion cell layer and at the proximal border of the inner nuclear layer, as described previously [13,26] (Fig. 1). The num-
Fig. 1. Fos immunostaining Fos-immunoreactive nuclei Bar = 30 Wm.
Brain Research 29 (1995) 53-63
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ber of Fos-IR cells appeared to be greater near the visual streak than in the periphery of the retina. In the inner nuclear layer there were always two or three times as many Fos-IR cells as in the ganglion cell layer; this difference was obvious throughtout the retina. Northern blots demonstrated no c-fos mRNA in dark-adapted retinas. After the onset of flashing light, the expression of c-fos mRNA peaked at 30 min, stayed high at 60 min and was back to the control level at 120 min (Fig. 2). 3.2. Effects of drug administration SCH 38393, kainic acid, NMDA and nicotine all induced nuclear Fos-staining in cells in the INL and GCL of dark-adapted retinas (Fig. 3). The density of immunostained neurons appeared to be greatest in nicotine-treated retinas and lowest in NMDA-treated
in a cross-section (A) and whole-mount (B) (focus are seen in both the inner nuclear layer and ganglion
on the inner nuclear layer) of a light-stimulated retina. cell layer. in, inner nuclear layer; on, outer nuclear layer.
J. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
56
c
0.5
1
retinas. Dopamine receptor antagonists (haloperidol and SCH 23390), nicotine receptor antagonists (mecamylamine) or glutamate receptor antagonists (MK-801 and kynuretic acid) did not block the light-induced Fos-expression, when administred 30 min prior to light exposure (Fig. 4). However, the receptor blockers markedly or completely prevented Fos expression induced by corresponding agonists (not shown).
2
3.3. Fos expression in different neuron populations
Fig 2. Northern blot of 5 pg per lane of total cellular RNA extracted fro) m a dark-adapted retina (C) and retinas exposed to flashing light for 0.5. 1 and 2 h. Arrow heads ooint to 18s and 2% bands. c-fos band is indicated with an arrow. _I
Ganglion cells of varying size displayed yellow-fluorescent cytoplasmic staining two days following intraorbital FluoroGold injection into the optic nerve. All FluoroGold-positive cells were located in the GCL. Only 3% of Fos-positive cells in the GCL were
Fig. 3. Fos-immunostained retinal flat mounts 2 h after administration of SCH 38393, a D, receptor agonist (A), kainate (B), nicotine (C) or NMDA CD). Focus is in the level of the inner nuclear layer. Immunoreactive nuclei were detected with a fluorescein-conjugated secondary antibody and are seen as bright dots. All the drugs induce Fos both in the ganglion and inner nuclear layers. The density of immunostained nuclei is greatest in the nicotine-treated retina. Bar = 35 km.
_I.Koistinaho, S.M. Sagar /Molecular
Brain Research 29 (1995) 53-63
Fig. 4. Fos-immunostained retinal flat mounts. Rabbits were dark-adapted and the retinas treated with haloperidol, a dopamine receptor antagonist (B), mecamylamine, a nicotinic receptor antagonist (C), MK-801, an NMDA-receptor antagonist CD), and kynuretic acid, a kainate receptor antagonist (E). The antagonists were administered 30 min prior light exposure. Light-induced Fos in saline-treated control retina is shown in A. Immunoreactive nuclei were detected with a fluorescein-conjugated secondary antibody. Bar = 35 pm.
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.I. Koistinaho, S.M. Sagar / Molecular Brain Research 29 (1995) 53-63
Fig. 5. Fos-immunostained retinal flat mounts after light exposure. A: FluoroGold (FG)-labelled ganglion cells. B: Fos immunostaining in the same field as in A, arrows point to a cell double-labelled with FG and Fos. C: Fos-immunostained (fluorescein) retina reacted for NADPHd (dark precipitation); two NADPHd-reactive cells are double-labelled with Fos. Bar = 35 pm (A,B) and 25 pm (C).
double-labelled with FluoroGold after light exposure. The great majority of FluoroGold-positive cells did not display Fos-IR (Fig. 5A,B). NADPH diaphorase (NADPHd) stained two populations of amacrine cells near the inner border of the INL as has been described previously: large densely staining (Type I> and more numerous, smaller and lightly staining (Type II) cells [25] (Fig. 5C). 24% of Fos-IR cells in the INL were Type I NADPHd reactive cells and 7% were Type II NADPHd reactive cells. On the other hand, 65% of Type I NADPHd reactive cells showed Fos-IR. TH antibody strongly stained a sparse population of large amacrine cells with a dense network of neuronal processes. 11% of Fos-IR cells in the INL were TH-IR, and 100% of TH-IR cells showed Fos expression after light stimulation (Fig. 6A,B). Fos immunostaining in these cells was always relatively light. Cholinergic amacrine cells, which comprise to 12% of amacrine cells in the INL and 80% of amacrice cells in the GCL [37,39] were labelled either with DAPI or
ChAT antibody (Fig. 7). Double labelling with Fos immunostaining demonstrated that lo-15% of Fos-IR cells were DAPI-positive in the INL after light exposure. In the GCL 30-40% of Fos-IR stained with DAPI. Since DAPI is taken up by amacrine cells other than cholinergic amacrines, even though to a lesser extent, double-labelling with ChAT and Fos-antibodies was also performed. Twelve percent in the INL and 30% in the GCL of ChAT-immunoreactive amacrine cells was found to express Fos proteins. 5,7-DHT is taken up by five different populations of amacrice cells, two of which are located in the GCL and three in the INL [22,28]. 5,7-DHT demonstrated strongly yellow fluorescent populations of cells both in the INL and GCL 3 h after injection. Double-labelling with Fos antibody did not reveal any 5,7-DHT-positive cell with Fos-IR after light exposure (Fig. 6C,D). Somatostatin-IR cells were seen in the GCL and the vast majority of the cells were located in the inferior half of the retina. None of these cells expressed Fos proteins after light exposure. Amacrine cells expressing
J. Koistinaho, SM. Sagar/Molecular
another neuropeptide, VIP, were also devoid of Fosstaining after light exposure (Fig. 8).
4. Discussion Fos, a protein product of the c-fos immediate early gene, is rapidly induced by depolarization and agents that activate intracellular protein kinase C or elevate calcium and CAMP levels [1,20]. It forms a heterodimeric protein complex with a member of Jun family, binds then to the AP-1 binding site, and regu-
Brain Research 29 (1995) 53-63
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lates transcription of genes containing this DNA element 11,201. It is therefore thought that Fos expression marks the activated neurons that undergo changes in gene expression after synaptic stimulation [10,20,27]. In vivo and in vitro experiments have shown that, in addition to Fos, several members of Jun family and protein products of other transcription factors than Fos and Jun families, are induced by depolarization [l]. Therefore, we cannot rule out the possibility that many Fos-negative retinal neurons ungergo changes in gene expression that exclusively require transcription factors other that Fos.
Fig. 6. Fos-immunostained retinal flat mounts after light exposure double-labelled for tyrosine hydroxylase (TH) (A,B) or serotonin (SER) (CD). In A, arrows point dark Fos-positive amacrine cells that show TH immunoreactivity when the same field is photographed under UV light (B). C shows fluorescent Fos-positive cells in a retina which was double-labelled with a fluorescent serotonin-derivative CD). None of the serotonin cells shows Fos expression. Bar = 25 pm.
hO
.I. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
Light induces Fos expression in dopaminergic, cholinergic and NOS-containing amacrines cells. Even though ON bipolar cells and serotoninergic amacrine cells depolarize in response to light [4,28,32,35,37,39], no Fos induction was seen in these cells. This indicates that light-stimulated synaptic input does not induce changes in Fos-regulated gene expression in all depolarized retinal neurons. Retinal neurons vary in the extent they synthesize Fos, possibly depending on their degree of depolarization. A detectable Fos immunos-
taining would be expected to be seen only in those neurons that respond to flashing light by a sustained depolarization. It is also possible that the identity of intracellular signal tranduction systems that are activated determines the degree of c-fos expression. Flashing light may trigger the second messengers that activate c-fos only in a proportion of retinal neurons. Administration of nicotine, glutamate receptor and dopamine receptor agonists mimicked the overall pattern of light-evoked Fos immunostaining, but their
double-labelled with either Fos-antiserum Fig. 7. A retinal flat mount double-labelled with Fos-antiserum (A) and DAPI (B), and cross-sections (Cl and DAPI (D) or Fos-antiserum (El and ChAT-antibody (Fl. Fos-immunostained nuclei in A and C and the corresponding DAPI-labelled nuclei in B and D are shown with white arrows. In E, black arrows point to faintly and strongly labelled Fos-positive nucleus in the ganglion cell layer. F shows the same corresponding ChAT-immunolabelled cells (black arrows) in the same field. IN, inner nuclear layer; ON, outer nuclear layer. Bar = 25 pm.
.I. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
antagonists were unable to block the light-evoked Fos immunostaining. Previous studies have indicated changes in the release of acetylcholine [15,21], glutamate [16] and dopamine [5,9,11,121 in light-stimulated mammalian retina. All these neurotransmitters may be involved in retinal Fos induction, even though none of them alone is required for light-stimulated Fos induction in rabbit amacrine cells. Virtually all dopaminergic neurons in the retina displayed Fos staining in response to flashing light. Since tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis, has an AP-1 binding site in the promoter region of its gene [34], it is likely to be among the genes that are regulated by light in these cells. A long-lasting activation of TH by light requires an increased protein synthesis and gene expression to provide sufficient enzyme to synthesize dopamine, which is secreted in the light. Genes encoding other neurotransmitter, second messenger and metabolic enzymes in dopaminergic amacrine cells may also be regulated by Fos. In the rabbit retina there are two populations of cholinergic neurons: a population that releases acetylcholine in response to lights OFF is located in the INL, and a population that releases acetylcholine in response to lights ON is located in the GCL [6,21,39]. In the present study, a proportion of both populations of the cells displayed Fos staining in response to flashing
Fig. 8. Fos-immunostained retinal flat mounts fluorescein-conjugated secondary antibodies. Fos-induction in VIP- or somatostatin-positive
61
light, as can be expected. Even though alternative splicing can produce several types of mRNA for choline acetyltrasferase, the rate-limiting enzyme of acetylcholine synthesis, the enzyme has an AP-1 binding site in the promoter region of the gene [19]. It is therefore likely that Fos is among the transcription factors that regulate acetylcholine synthesis, but that factors other than Fos are required to determine the mRNA level of choline acetyltransferase in different populations of cholinergic amacrine cells. The vast majority of Type I NADPHd reactive amacrine cells were Fos-immunoreactive after light exposure. NADPHd histochemistry stains the neuronal type of nitric oxide synthase (NOS) [3,14]. Because neuronal NOS is a Ca2+-sensitive enzyme [3], and Fos expression was induced by light-evoked synaptic input which presumably involves Ca2+ influx, it is possible that light stimulates NOS in these amacrine cells causing NO release in the retina. NO is well known to increase cGMP levels in its target cells by stimulating soluble guanylyl cyclase [3,141. We have previously shown that cGMP levels are increased in On cone bipolar cells after administration of nitric oxide donors to the retina [14]. In addition, others have shown that light increases cGMP levels in ON bipolar cells in a NO-dependent manner [32]. The present results taken together with previous studies suggest that NOS is stimulated by light in NADPHd reactive amacrine cells
after light exposure double-labelled with VIP (A) or somatostatin Fos-positive nuclei are seen as dark shadows (pointed with cells. Bar = 25 pm.
(B). Peptides were stained with an arrow in B). There are no
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in the rabbit retina. However, there is no evidence suggesting that Fos is a transcription factor for the NOS gene in neurons. A,, amacrine cells are glycinergic narrow-field neurons that receive their major input from rod bipolar cells [5,32,37-391. Rabbit An cells respond to light with a triphasic potential, characterized by a depolarizing transient at light ON, followed by a sustained plateau phase, and finally by a hyperpolarizing transient at light OFF [4,5,6,35,37-391. These cells can be labelled in vivo with nuclear yellow, which stains the nuclei of A,, cells yellow [37]. We were unable to combine this staining method with Fos immunostaining, since the yellow fluorescence did not last through immunocytochemical procedure. Another way of demonstrating these cells is glycine immunocytochemistry, which requires inclusion of glutaraldehyde into fixation solution. Because the Fos antibody used in the study is sensitive to aldehydes, we could not use this approach either, and the relationship between An amacrine cells and Fos induction after light exposure remains unsolved. Considering that A,, amacrine cells constitute up to 11% of amacrine cells in the INL and that they depolarize at light ON [4,5,35,37-391, it is highly possible that some A,, amacrine cells are among the 50% of Fos-positive cells that were not characterized in the present study. VIP [24] and somatostatin [23] are neuropeptides present in the specific populations of amacrine cells in the rabbit retina. Both VIP gene and preprosomatostatin gene have an AP-1 binding site in their promoter regions [7,33], indicating their protein synthesis as potentially regulated by Fos transcription factor. VIP-positive cells have their cell bodies in the INL and are more numerous in the central than in the peripheral retina [24]. Even though the distribution and morphology of VIP-positive cells have been described in the mammalian retina, the function and target cells of the peptide have remained obscure. VIP stimulates adenylate cyclase and glycogenolysis in the rabbit retina and VIP has been suggested to act on Miiller cells rather than to have a role in retinal neurotransmission [30,31]. Flashing light did not induce Fos in VIP-positive cells in the rabbit retina, suggesting that VIP-positive cells are not strongly stimulated by light in the rabbit retina. Somatostatin-immunoreactive cells in the rabbit retina are displaced amacrine cells in the GCL, but extend long axon-like processes in the IPL [271. Their cell bodies are almost exclusively localized in the inferior half of the retina 1271. No Fos-expressing somatostatin-immunoreactive cells were seen after exposure to flashing light. The function of somatostatin-immunoreactive cells in the retina is still unclear, but a role in modulation of global retinal visual function, such as light sensitivity, or metabolic or trophic func-
tion, and generation of direction selectivity, has been suggested previously 123,411. The few ganglion cells that expressed Fos after light exposure had a large cell body and were presumably cx ganglion cells. These cells belong to ON ganglion cells and depolarize in response to light [6,39]. Since the density of Fos-stained ganglion cells was very low, only a small proportion of ON ganglion cells respond to light stimulation by Fos gene induction. Fos immunocytochemistry is only an indirect indication of physiological activation of neurons. Although no statement can be rigorously made about neurons that fail to express c-fos in response to an experimental stimulus, the preponderance of the evidence is that neurons that do express the gene have undergone potent synaptic excitation [1,10,34,40]. The neuronal localization of Fos permits the detailed anatomic dissection of physiologic pathways through the visual system. The experiments reported here employed the most general and potent visual stimulus, bright flashing light. Whether the same approach can be applied to more refined visual stimuli, such as moving targets or color remains to be demonstrated. Our finding in vivo support previous evidence from neurotransmitter release studies in vitro that light exposure excites dopaminergic and cholinergic amacrine cells [5,6,9,11,12,15,21,39]. Our most novel finding is that presumptive NOS amacrine cells are excited by light. The morphology of these cells, especially the Type I NADPHd amacrine cells that have multiple very long axon-like processes in the rabbit IPL [14,25,36], suggests that light induces a diffuse release of NO in the IPL. The presumed target of this NO is a soluble guanylyl cyclase in ON cone bipolar cells, which contain cGMP-gated cation channel [ 14,321. Illumination would thereby stimulate cGMP synthesis diffusely in ON cone bipolar cells. Focal light exposure would then act by a G protein-coupled stimulation of cGMP hydrolysis to provide contrast detection. This hypothesis is testable electrophysiologically, as selective NOS inhibitors are readily available.
Acknowledgements This study was supported by the National Institute of Neurology Disease and Stroke (NS27488) and Department of Veterans Affairs Merit Review Program.
References [l] Bartel, D.P., Sheng, M., Lau, L.F. and Greenberg, M.E., Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction, Genes Del,.. 3 (1989) 304-313.
J. Koistinaho,
S.M. Sagar/Molecular
[2] Bhat, R.V., Worley, P.F., Cole, A.J. and Baraban, J.M., Activation of the zinc finger encoding gene krox-20 in adult rat brain: comparison with zif268, Mol. Brain Rex, 13 (1989) 263-266. [3] Bredt. D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snider, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615-624. [4] Dacheux, R.F. and Raviola, E., The rod pathway in the rabbit retina: depolarizing bipolar and amacrine cells, J. Neurosci., 6 (1986) 331-345. [5] Dowling, J.E. and Ehringer, B., Synaptic organization of the dopaminergic neurons in the rabbit retina, J. Camp. Neural., 180 (1978) 203-220. [6] Dowling, J.E., The Retina, Harvard University Press, Cambridge, MA, 1987. [7] Fink, J.S., Verhave, M., Walton, K., Mandel, G. and Goodman, R.H., Cyclic AMP- and phorbol ester-induced transcriptional activation are mediated by the same enhancer element in the human vasoactive intestinal peptide gene, J. Biol. Chem., 266 (1991) 3882-3887. [B] Hadjiconstantinou, M., Rossetti, Z.L., Krajnc, D. and Neff, N.H., Aromatic L-/amino acid decarboxylase activity of the rat retina in modulated in vivo by environmental light, J. Neurochem., 51 (1988) 1560-1564. [9] Hokoc, J.N. and Mariani, A.P., Synapses from bipolar cells onto dopaminergic amacrine cells in cat and rabbit retinas, Brain Res., 461 (1988) 17-26. [lo] Hunt, S.P., Pini, A. and Evan, G., Induction of c-fos like protein in spinal cord neurones following sensory stimulation, Nature, 328 (1987) 632-634. [ll] Jensen, R.J., Mechanism and site of action of a dopamine Dl antagonist in the rabbit retina, vis. Neurosci., 3 (1989) 573-585. [ 121 Jensen, R.J. and Daw, N.W., Effect of dopamine antagonists on receptive fields of brisk cells and directionally selective cells in the rabbit retina, J. Neurosci., 4 (1984) 2972-2985. [13] Koistinaho, J., Hicks, K. and Sagar, S.M., Tetrodotoxin enhances light-induced c-fos expression in the rabbit retina, Mol. Brain Res., 17 (1993) 179-183. [14] Koistinaho, J., Swanson, R.A., de Vente, S. and Sagar, S.M., NAPDH-diaphorase (nitric oxide synthase) reactive amacrine cells of rabbit retina: putative target cells and stimulation by light, Neuroscience, 57 (1993) 587-597. [15] Masland, R.H. and Livingston, C.J., Effect of stimulation with light on synthesis and release of acetylcholine by an isolated mammalian retina, J. Neurophysiol., 39 (1976) 1210-1219. [16] Massey, S.C., Cell types using glutamate as a neurotransmitter in vertebrate retina. In N. Osborne and J. Chader (Eds.), Progress in Retinal Research, Vol. 9, Pergamon, Oxford, 1990, pp. 399-425. [17] McLean, I.W. and Nagane, P.K., Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. [18] Millbrandt, J., A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor, Science, 238 (1987) 797-799. 1191 Misawa, H., Ishii, K. and Deguchi, T., Gene expression of mouse choline acetyltransferase, J. Biol. Chem., 267 (1992) 20392-20399. [20] Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-197.
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29 (1995) 53-63
63
[21] O’Malley, D.M., Sandell, J.H. and Masland, R.H., Co-release of acetylcholine and GABA by the starburst amacrine cells, J. Neurosci., 12 (1992) 1394-1408. [22] Osborne, N.N. and Beaton, D.W., Direct histochemical localization of 5,7_dihydroxytryptamine and the uptake of serotonin by a subpopulation of GABA neurones in the rabbit retina, Brain Res., 382 (1986) 158-162. [23] Sagar, S.M., Somatostatin-like immunoreactive material in the rabbit retina: Immunohistochemical staining using monoclonal J. Camp. Neural., 266 (1987) 291-299. antibodies, [24] Sagar, S.M., Vasoactive intestinal polypeptide (VIP) immunohistochemistry in the rabbit retina, Brain Res., 426 (1987) 1577163. [25] Sagar, S.M., NADPH-diaphorase reactive neurons of the rabbit retina: differential sensitivity to excitotoxins and unusual morphologic features, J. Camp. Neural., 300 (1990) 309-319. [26] Sagar, SM. and Sharp, F.R., Light induces a Fos-like nuclear antigen in retinal neurons, Mol. Brain Rex, 7 (1990) 17-21. [27] Sagar, S.M., Sharp, F.R. and Curran, T., Expression of the c-fos protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. [28] Sandell, J.H., Masland, R.H., Raviola, E. and Dacheux, R.F., Connections of indolamine-accumulating cells in the rabbit retina, J. Neurosci., 6 (1989) 3331-3347. [29] Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229-234. [30] Schoderet, M., Sovilla, J.-Y. and Magisstretti, P.J., The effects of VIP on cyclic AMP and glycogen levels in vertebrate retina, Peptides, 5 (1984) 295-298. [31] Schoderet, M., Sovilla, J.-Y. and Magisstretti, P.J., VIP and glucagon-induced formation of cyclic AMP in intact retinae in vitro, Eur. J. Pharmacol., 71 (1981) 131-133. [32] Shieldls, R. and Falk, G., Retinal on bipolar cells contain a nitric oxide sensitive guanylate cyclase, NeuroReport, 3 (1992) 845-848. [33] Smith, S.E., Papavassilou, A.G. and Bohmann, D., Differential TRE-related elements are distinguished by sets of DNA-binding proteins with ovelapping sequence specificity, Nucl. Acids Res., 21 (1993) 1581-1985. [34] Sonnenberg, J.L., Rauscher, III F.J., Morgan, J.I. and Curran, T., Regulation of proenkephalin by Fos and Jun, Science, 246 (1989) 1622-1625. [35] Strettoi, E., Dacheux, R.F. and Raviola, E., Synaptic connections of rod bipolar cells in the inner plexiform layer of the rabbit retina, J. Camp. Neurol., 295 (1990) 449-466. [36] Vaney, D.I. and Young, H.M., GABA-like immunoreactivity in NADPH-diaphorase amacrine cells of the rabbit retina, Brain Res., 240 (1988) 380-385. [37] Vaney, D.I., Young, H.M. and Gynther, I.C., The rod circuit in the rabbit retina, I/is. Neurosci., 7 (1991) 141-154. [38] Voigt, T. and WBssle, H., Dopaminergic innervation of AI1 amacrine cells in mammalian retina, J. Neurosci., 7 (1987) 41154128. [39] Wgssle, H. and Boycott, B.B., Functional architecture of the mammalian retina, Physiol. Reu., 71 (1991)141-154. [40] Wisden, W., Errington, M.L., Williams, S., Dunnet, S.B., Waters, C., Hitchcock, D., Evan, G., Bliss, T.V.P. and Hunt, S.P., Differential expression of immediate early genes in the hippocampus and spinal cord, Neuron, 4 (1990) 603-614. 1411 Zalutsky, R.A. and Miller, A., The physiology of somatostatin in the rabbit retina, J. Neurosci., 10 (1990) 383-393.
Molecular
Brain Research
Research
Light-induced
29 (1995) 53-63
report
c-f&s expression in amacrine cells in the rabbit retina Jari Koistinaho
a,b,*, Stephen
M. Sagar a
a Department of Neurology, University of California, and Department of Veterans Affairs Medical Center, San Francisco, CA, USA ’ Neural Injury Research Laboratory, Department of Biomedical Sciences, University of Tampere, Tampere, Finland Accepted
13 September
1994
Abstract Retinal neurons that express the immediate early gene c-fos after light exposure were characterized by neurotransmitter content using histochemical and immunocytochemical staining. In Northern blots the amount of c-fos mRNA peaked at 30 min, but remained detectable 60 min following light stimulation. Fos proteins were seen in the inner nuclear and ganglion cell layers, and the staining was most intense two and three hours after beginning the light exposure. In the ganglion cell layer 30-40% of Fos-immunoreactive cells were cholinergic displaced amacrine cells and 3-5% were ganglion cells. In the inner nuclear layer 24% of Fos-immunoreactive cells were Type I and 7% Type II NADPH-diaphorase-reactive (nitric oxide synthase) amacrine was seen cells, 11% were tyrosine hydroxylase-containing cells, and 10-Z% cholinergic amacrine cells. No Fos immunoreactivity in serotoninergic, somatostatinor VIP-immunoreactive cells, bipolar, horizontal or photoreceptor cells. Nicotine, kainic acid, NMDA and SCH 38393, a dopamine D, receptor agonist, induced Fos immunostaining in the inner nuclear and ganglion cell layers, but administration of the corresponding receptor blockers mecamylamine, kynuretic acid, MK-801, haloperidol and SCH 23990 did not prevent light-induced Fos expression. Keywords:
Amacrine
cell; Ganglion
cell; Nitric oxide synthase; Acetylcholine;
1. Introduction
Flashing light is commonly used as a physiological stimulus to the visual pathways in the mammalian nervous system. It produces an electrophysiological response in most retinal neurons, but the extent of depolarization varies from one type of neuron to another [6]. In the rabbit retina, two types of second-order neurons, rod bipolar and On cone bipolar cells, depolarize in response to light [4,6,39]. Input from depolarized bipolar cells is received by a large and heterogenous population of amacrine and ganglion cells in the inner retina. The rapid transsynaptic responses to photic stimulation initiate events such as alteration of neurotransmitter synthesis and release, change in the phosphorylation state of ligand-gated ion channels and activation of multiple second messenger pathways [6].
* Corresponding author. Neural Injury Research Laboratory, Department of Biomedical Sciences, University of Tampere, PO Box 607, FIN-33101 Tampere, Finland. Fax: (358) 31-215 6071. E-mail: [email protected]. 0169-328X/95/$09.50 0 1995 Elsewer SSDI 0169-328X(94)00218-5
Science
B.V. All rights reserved
Tyrosine hydroxylase; Gene induction
Some of the changes may be long-lasting and require gene induction and subsequent protein synthesis [S]. Immediate early genes, including some proto-oncogenes, are transiently induced in neurons following synaptic stimulation [1,2,10,18,20,22,27,40]. For example c-fos and its protein product Fos are thought to participate in transcriptional regulation of the genes needed for long-term alterations in neuronal activity [1,10,22,40]. Therefore, immunocytochemical localization of Fos has provided, with certain limitations, a method to perform functional neuroanatomic mapping at the cellular level [10,22,26,27]. In the rabbit ‘retina, light induces Fos in a subset of neurons and Fos expression, with a varying pattern, can be seen following administration of pharmacologically active agents [13,14,261. Using histochemical and immunocytochemical double-labeling methods we attempted in the present study to classify by neurotransmitter content the retinal neurons which express Fos in response to flashing light. We also tested the effect of glutamate, dopamine and nicotinic receptor agonists and antagonists on Fos expression.
.I. Koistinaho,
54
2. Material
S. M. Sagar / Molecular
and methods
2.1. Chemicals
Nicotinamide adenine dinucleotide phosphate (NADP), 4,6-diamidino-2-phenylindole (DAPI), 5,7_dihydroxytryptamine (5,7-DHTI, kainic acid, iv-methyl-D-aspartate (NMDA), 5-methyl-lO,ll-dihydro5H-dibenzocyclohepten-5,10-imine (MK-801), nicotine, mecamylamine and haloperidol were obtained from Sigma Chemical Co., St. Louis, MO. SCH 238393 and SCH 23390, a dopamine D, receptor agonist and antagonist, respectively, were purchased from RBI, Nitick, MA. Synthetic M peptide was obtained from Penisula Laboratories, Belmont, CA, and FluoroGold from FluoroChrome, Eaglewood, CO. Avidin-biotin-peroxidase (ABC) kit was from Vector Laboratories, Burlingame, CA. Fluorescein-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were from Boehringer Mannheim, Indianapolis, IN. A pSP65-foslA plasmid was generously provided by Dr. Tom Curran, Roche Institute of Molecular Biology, Nutley, NJ. 2.2. Primary antibodies An affinity-purified polyclonal rabbit antiserum (RlB61, raised to a synthetic peptide corresponding to residues 132-154 of Fos [13,26] was used at dilutions of 1 :50-l : 100. A monoclonal anti-tyrosine hydroxylase (Boehringer-Mannheim) and a monoclonal anti-ChAT (choline acetyltransferase, Boehringer-Mannheim) were used at 1: loo-250 dilutions. A rabbit polyclonal anti-VIP (vasoactive intestinal peptide, Chemicon, Temecula, CA) was used at 1:200-l : 300 dilutions. A monoclonal anti-somatostatin [25] (IE9, generously provided by Dr. Linda Chun, Massachusetts General Hospital and Harvard University, Boston, MA) was used at dilution of 1 :3000.
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lated using guanidinium/lithium chloride method. 5 pg RNA was electrophoresed through a 1.5% agarose gel and transferred to a Nylon membrane (Nytran, Scheicher and Schuell, NH) by capillary blotting. c-fos riboprobe was prepared from c-fos cDNA (cloned into pSP65-foslA plasmid) by using SP6 polymerase and 13’P]CTP. Hybridization buffer contained 50% formamide, 1.5 X SSPE, 1% SDS, 0.5% non-fat dry milk, 0.2 mg/ml sheared denatured salmon testis DNA, and 0.5 mg/ml yeast RNA. The membranes were hybridized at 65°C washed at the same temperature for 30 min each twice with 2x SSC/O.l% SDS, 0.5 x SSC/O.l% SDS and 0.2~ SSC/O.l% SDS, and exposed to Kodak SB5 X-ray film with intensifying screens at ~ 70°C overnight. 2.6. Injection of neuron
markers
For retrograde labelling of ganglion cells rabbits were sedated two days prior the experiment with ketamine (Aveco, Fort Dodge, IA, 25 mg/kg) and xylazine (Anaced, Lloyd Laboratories, Shenandoah, IA, 5 mg/kg), and the right optic nerve was exposed intraorbitally. The nerve was crushed 2 times for 30 s and 2 ~1 2% FluoroGold was injected into the nerve. Care was taken not to disturb the retinal blood supply. The left optic nerve was only exposed without crushing and used as a control. The wounds were closed and the animals allowed to recover. For intravitreal DAPI injections, the rabbits were sedated one day prior to the experiment with pentobarbital (40 mg/i.v.), and the sclera was topically anesthetized with 1% proparicaine ophthalmic solution. Injections of DAPI (0.15 pg in 50 ~1 distilled water) were made at multiple locations in the vitreous cavity of one eye through a 30 gauge needle. The opposite eye of each rabbit received 50 ~1 saline as a control. 5,7-DHT (20 Fg in 50 ~1 distilled water containing 1% ascorbic acid) was injected similar to DAPI, but was administered in the dark 3 h prior to the light exposure.
2.3. Animals 2.7. Histochemistry Adult male New Zealand white rabbits were kept in a central animal care facility on a 12: 12 h light-dark cycle with free access to food and water. On the day prior to the experiment, rabbits were transported to the laboratory and dark-adapted for 24 h. For study of dark-adapted retinas, animals were sacrificed by injection of pentobarbital, 150 mg/kg iv., under infrared illumination with the aid of night vision goggles (Litton Industries, Tempe, AZ). For light exposure, animals were placed in restraining cage and were exposed to flashing light for 2 h. Three Hz, 60 ms approximately 300 lux flashes of white light were generated with a bank of 4 fluorescent lamps driven by a special driver designed to produce a square wave pattern of light intensity (Ralph Gerbrands Co., Arlington, MA). The lights were turned off and the rabbits were sacrificed. Each experiment was repeated at least three times. 2.4. Drug administration 7.5 nmol of kainic acid, 10 nmol of NMDA, 50 Fg of nicotine or 0.5 nmol of SCH 38393 were injected in 50 ~1 of water intravitreally to the right eye of urethane (4.5 g/kg, i.p.1 anesthetized dark-adapted rabbits. Our previous studies have shown that urethane does not induce or block activity-induced neuronal Fos expression in the rabbit retina. MK-801 (1.5-5 mg/kgJ, haloperidol (5 mg/kg), mecamylamine (6 mg/kg), kynuretic acid (100 mg/kgl and SCH 23390 (6 mg/kg) were administred intraperitoneally 30 min prior light exposure.
2.5. Northern
blotting
Dark-adapted retinas and retinas exposed to flashing light for varying period of time were homogenized and total RNA was iso-
and immunocytochemistv
After light exposure eyes were removed in the light and were hemisected at about the equator. The posterior eye cups were immersed in freshly prepared PLP (0.05 M sodium phosphate buffer, pH 7.4, 0.1 M L-lysine, 0.01 M sodium m-periodate, 1.5% paraformaldehyde) prepared according to McLean and Nakane [17], for 3 h at 4°C. After washing in 0.1 M sodium phosphate buffer, pH 7.4 (PB), retinas were dissected, washed in PB, and processed for NADPH-diaphorase histochemistry or directly for immunocytochemistry. A modification of the method of Scherer-Singler et al. [29] was used to demonstrate NADPH diaphorase [14,25]. Tissue was incubated in 0.1 M Tris-maleate, pH 8.0, 15 mM malic acid, 1 mM MnCI,, 1 mM NADP, 0.2 mM nitro blue tetrazolium, and 0.2% Triton X-100 for l-2 h at 37°C. After washing in PB, the tissue was further processed for Fos immunostaining. For immunostaining retinas were incubated in Fos antibody diluted in PB containing 0.1% bovine serum albumin, 0.2% Triton X-100, and 2% normal goat serum (PB-G) at 4°C with gentle agitation for 48-72 h. The tissue was washed in PB, and sites of antibody binding was visualized with FITC-conjugated secondary antibody or the avidin-biotin-peroxidase (ABC) procedure using a Vectastain Kit and diaminobenzidine as chromogen. In some double-staining experiments a rhodamine-conjugated secondary antibody was used. Secondary antiserum and ABC reagent were diluted in PB-G; incubation times were 2-3 h at room temperature. After washing in PB for 2 h, the retinas were subsequently double labelled with monoclonal antibodies. Rabbit polyclonal antibodies were diluted in PB-G and monoclonal antibodies in PB-H (PB containing 0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal horse serum). Binding sites of the antibodies were visualized by incubating
.I. Koistinaho, S.M. Sagar /Molecular retinas in FITC-conjugated secondary antibodies (diluted 1 : 40-100 in PB-G or PB-H) at 37°C for 2 h. After washing in PB, the retinas were flat mounted on gelatin-subbed slides and coverslipped in glycerol-PB mixture (3 : 1).
3. Results 3.1. Light-induced Fos expression Retinas from dark-adapted untreated or salinetreated eyes displayed no or little detectable nuclear Fos-immunostaining as described previously [ 13,14,26]. Two days after crushing the optic nerve or 3 h after DAPI or 5,7-DHT injections, no Fos-immunostaining was observed in dark-adapted retinas. Flashing light induced a mosaic of Fos-IR cells in the ganglion cell layer and at the proximal border of the inner nuclear layer, as described previously [13,26] (Fig. 1). The num-
Fig. 1. Fos immunostaining Fos-immunoreactive nuclei Bar = 30 Wm.
Brain Research 29 (1995) 53-63
55
ber of Fos-IR cells appeared to be greater near the visual streak than in the periphery of the retina. In the inner nuclear layer there were always two or three times as many Fos-IR cells as in the ganglion cell layer; this difference was obvious throughtout the retina. Northern blots demonstrated no c-fos mRNA in dark-adapted retinas. After the onset of flashing light, the expression of c-fos mRNA peaked at 30 min, stayed high at 60 min and was back to the control level at 120 min (Fig. 2). 3.2. Effects of drug administration SCH 38393, kainic acid, NMDA and nicotine all induced nuclear Fos-staining in cells in the INL and GCL of dark-adapted retinas (Fig. 3). The density of immunostained neurons appeared to be greatest in nicotine-treated retinas and lowest in NMDA-treated
in a cross-section (A) and whole-mount (B) (focus are seen in both the inner nuclear layer and ganglion
on the inner nuclear layer) of a light-stimulated retina. cell layer. in, inner nuclear layer; on, outer nuclear layer.
J. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
56
c
0.5
1
retinas. Dopamine receptor antagonists (haloperidol and SCH 23390), nicotine receptor antagonists (mecamylamine) or glutamate receptor antagonists (MK-801 and kynuretic acid) did not block the light-induced Fos-expression, when administred 30 min prior to light exposure (Fig. 4). However, the receptor blockers markedly or completely prevented Fos expression induced by corresponding agonists (not shown).
2
3.3. Fos expression in different neuron populations
Fig 2. Northern blot of 5 pg per lane of total cellular RNA extracted fro) m a dark-adapted retina (C) and retinas exposed to flashing light for 0.5. 1 and 2 h. Arrow heads ooint to 18s and 2% bands. c-fos band is indicated with an arrow. _I
Ganglion cells of varying size displayed yellow-fluorescent cytoplasmic staining two days following intraorbital FluoroGold injection into the optic nerve. All FluoroGold-positive cells were located in the GCL. Only 3% of Fos-positive cells in the GCL were
Fig. 3. Fos-immunostained retinal flat mounts 2 h after administration of SCH 38393, a D, receptor agonist (A), kainate (B), nicotine (C) or NMDA CD). Focus is in the level of the inner nuclear layer. Immunoreactive nuclei were detected with a fluorescein-conjugated secondary antibody and are seen as bright dots. All the drugs induce Fos both in the ganglion and inner nuclear layers. The density of immunostained nuclei is greatest in the nicotine-treated retina. Bar = 35 km.
_I.Koistinaho, S.M. Sagar /Molecular
Brain Research 29 (1995) 53-63
Fig. 4. Fos-immunostained retinal flat mounts. Rabbits were dark-adapted and the retinas treated with haloperidol, a dopamine receptor antagonist (B), mecamylamine, a nicotinic receptor antagonist (C), MK-801, an NMDA-receptor antagonist CD), and kynuretic acid, a kainate receptor antagonist (E). The antagonists were administered 30 min prior light exposure. Light-induced Fos in saline-treated control retina is shown in A. Immunoreactive nuclei were detected with a fluorescein-conjugated secondary antibody. Bar = 35 pm.
58
.I. Koistinaho, S.M. Sagar / Molecular Brain Research 29 (1995) 53-63
Fig. 5. Fos-immunostained retinal flat mounts after light exposure. A: FluoroGold (FG)-labelled ganglion cells. B: Fos immunostaining in the same field as in A, arrows point to a cell double-labelled with FG and Fos. C: Fos-immunostained (fluorescein) retina reacted for NADPHd (dark precipitation); two NADPHd-reactive cells are double-labelled with Fos. Bar = 35 pm (A,B) and 25 pm (C).
double-labelled with FluoroGold after light exposure. The great majority of FluoroGold-positive cells did not display Fos-IR (Fig. 5A,B). NADPH diaphorase (NADPHd) stained two populations of amacrine cells near the inner border of the INL as has been described previously: large densely staining (Type I> and more numerous, smaller and lightly staining (Type II) cells [25] (Fig. 5C). 24% of Fos-IR cells in the INL were Type I NADPHd reactive cells and 7% were Type II NADPHd reactive cells. On the other hand, 65% of Type I NADPHd reactive cells showed Fos-IR. TH antibody strongly stained a sparse population of large amacrine cells with a dense network of neuronal processes. 11% of Fos-IR cells in the INL were TH-IR, and 100% of TH-IR cells showed Fos expression after light stimulation (Fig. 6A,B). Fos immunostaining in these cells was always relatively light. Cholinergic amacrine cells, which comprise to 12% of amacrine cells in the INL and 80% of amacrice cells in the GCL [37,39] were labelled either with DAPI or
ChAT antibody (Fig. 7). Double labelling with Fos immunostaining demonstrated that lo-15% of Fos-IR cells were DAPI-positive in the INL after light exposure. In the GCL 30-40% of Fos-IR stained with DAPI. Since DAPI is taken up by amacrine cells other than cholinergic amacrines, even though to a lesser extent, double-labelling with ChAT and Fos-antibodies was also performed. Twelve percent in the INL and 30% in the GCL of ChAT-immunoreactive amacrine cells was found to express Fos proteins. 5,7-DHT is taken up by five different populations of amacrice cells, two of which are located in the GCL and three in the INL [22,28]. 5,7-DHT demonstrated strongly yellow fluorescent populations of cells both in the INL and GCL 3 h after injection. Double-labelling with Fos antibody did not reveal any 5,7-DHT-positive cell with Fos-IR after light exposure (Fig. 6C,D). Somatostatin-IR cells were seen in the GCL and the vast majority of the cells were located in the inferior half of the retina. None of these cells expressed Fos proteins after light exposure. Amacrine cells expressing
J. Koistinaho, SM. Sagar/Molecular
another neuropeptide, VIP, were also devoid of Fosstaining after light exposure (Fig. 8).
4. Discussion Fos, a protein product of the c-fos immediate early gene, is rapidly induced by depolarization and agents that activate intracellular protein kinase C or elevate calcium and CAMP levels [1,20]. It forms a heterodimeric protein complex with a member of Jun family, binds then to the AP-1 binding site, and regu-
Brain Research 29 (1995) 53-63
59
lates transcription of genes containing this DNA element 11,201. It is therefore thought that Fos expression marks the activated neurons that undergo changes in gene expression after synaptic stimulation [10,20,27]. In vivo and in vitro experiments have shown that, in addition to Fos, several members of Jun family and protein products of other transcription factors than Fos and Jun families, are induced by depolarization [l]. Therefore, we cannot rule out the possibility that many Fos-negative retinal neurons ungergo changes in gene expression that exclusively require transcription factors other that Fos.
Fig. 6. Fos-immunostained retinal flat mounts after light exposure double-labelled for tyrosine hydroxylase (TH) (A,B) or serotonin (SER) (CD). In A, arrows point dark Fos-positive amacrine cells that show TH immunoreactivity when the same field is photographed under UV light (B). C shows fluorescent Fos-positive cells in a retina which was double-labelled with a fluorescent serotonin-derivative CD). None of the serotonin cells shows Fos expression. Bar = 25 pm.
hO
.I. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
Light induces Fos expression in dopaminergic, cholinergic and NOS-containing amacrines cells. Even though ON bipolar cells and serotoninergic amacrine cells depolarize in response to light [4,28,32,35,37,39], no Fos induction was seen in these cells. This indicates that light-stimulated synaptic input does not induce changes in Fos-regulated gene expression in all depolarized retinal neurons. Retinal neurons vary in the extent they synthesize Fos, possibly depending on their degree of depolarization. A detectable Fos immunos-
taining would be expected to be seen only in those neurons that respond to flashing light by a sustained depolarization. It is also possible that the identity of intracellular signal tranduction systems that are activated determines the degree of c-fos expression. Flashing light may trigger the second messengers that activate c-fos only in a proportion of retinal neurons. Administration of nicotine, glutamate receptor and dopamine receptor agonists mimicked the overall pattern of light-evoked Fos immunostaining, but their
double-labelled with either Fos-antiserum Fig. 7. A retinal flat mount double-labelled with Fos-antiserum (A) and DAPI (B), and cross-sections (Cl and DAPI (D) or Fos-antiserum (El and ChAT-antibody (Fl. Fos-immunostained nuclei in A and C and the corresponding DAPI-labelled nuclei in B and D are shown with white arrows. In E, black arrows point to faintly and strongly labelled Fos-positive nucleus in the ganglion cell layer. F shows the same corresponding ChAT-immunolabelled cells (black arrows) in the same field. IN, inner nuclear layer; ON, outer nuclear layer. Bar = 25 pm.
.I. Koistinaho, SM. Sagar / Molecular Brain Research 29 (1995) 53-63
antagonists were unable to block the light-evoked Fos immunostaining. Previous studies have indicated changes in the release of acetylcholine [15,21], glutamate [16] and dopamine [5,9,11,121 in light-stimulated mammalian retina. All these neurotransmitters may be involved in retinal Fos induction, even though none of them alone is required for light-stimulated Fos induction in rabbit amacrine cells. Virtually all dopaminergic neurons in the retina displayed Fos staining in response to flashing light. Since tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis, has an AP-1 binding site in the promoter region of its gene [34], it is likely to be among the genes that are regulated by light in these cells. A long-lasting activation of TH by light requires an increased protein synthesis and gene expression to provide sufficient enzyme to synthesize dopamine, which is secreted in the light. Genes encoding other neurotransmitter, second messenger and metabolic enzymes in dopaminergic amacrine cells may also be regulated by Fos. In the rabbit retina there are two populations of cholinergic neurons: a population that releases acetylcholine in response to lights OFF is located in the INL, and a population that releases acetylcholine in response to lights ON is located in the GCL [6,21,39]. In the present study, a proportion of both populations of the cells displayed Fos staining in response to flashing
Fig. 8. Fos-immunostained retinal flat mounts fluorescein-conjugated secondary antibodies. Fos-induction in VIP- or somatostatin-positive
61
light, as can be expected. Even though alternative splicing can produce several types of mRNA for choline acetyltrasferase, the rate-limiting enzyme of acetylcholine synthesis, the enzyme has an AP-1 binding site in the promoter region of the gene [19]. It is therefore likely that Fos is among the transcription factors that regulate acetylcholine synthesis, but that factors other than Fos are required to determine the mRNA level of choline acetyltransferase in different populations of cholinergic amacrine cells. The vast majority of Type I NADPHd reactive amacrine cells were Fos-immunoreactive after light exposure. NADPHd histochemistry stains the neuronal type of nitric oxide synthase (NOS) [3,14]. Because neuronal NOS is a Ca2+-sensitive enzyme [3], and Fos expression was induced by light-evoked synaptic input which presumably involves Ca2+ influx, it is possible that light stimulates NOS in these amacrine cells causing NO release in the retina. NO is well known to increase cGMP levels in its target cells by stimulating soluble guanylyl cyclase [3,141. We have previously shown that cGMP levels are increased in On cone bipolar cells after administration of nitric oxide donors to the retina [14]. In addition, others have shown that light increases cGMP levels in ON bipolar cells in a NO-dependent manner [32]. The present results taken together with previous studies suggest that NOS is stimulated by light in NADPHd reactive amacrine cells
after light exposure double-labelled with VIP (A) or somatostatin Fos-positive nuclei are seen as dark shadows (pointed with cells. Bar = 25 pm.
(B). Peptides were stained with an arrow in B). There are no
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J. Koistinaho, S. M. Sagar / Molecular Brain Research 29 (1995) 53-63
in the rabbit retina. However, there is no evidence suggesting that Fos is a transcription factor for the NOS gene in neurons. A,, amacrine cells are glycinergic narrow-field neurons that receive their major input from rod bipolar cells [5,32,37-391. Rabbit An cells respond to light with a triphasic potential, characterized by a depolarizing transient at light ON, followed by a sustained plateau phase, and finally by a hyperpolarizing transient at light OFF [4,5,6,35,37-391. These cells can be labelled in vivo with nuclear yellow, which stains the nuclei of A,, cells yellow [37]. We were unable to combine this staining method with Fos immunostaining, since the yellow fluorescence did not last through immunocytochemical procedure. Another way of demonstrating these cells is glycine immunocytochemistry, which requires inclusion of glutaraldehyde into fixation solution. Because the Fos antibody used in the study is sensitive to aldehydes, we could not use this approach either, and the relationship between An amacrine cells and Fos induction after light exposure remains unsolved. Considering that A,, amacrine cells constitute up to 11% of amacrine cells in the INL and that they depolarize at light ON [4,5,35,37-391, it is highly possible that some A,, amacrine cells are among the 50% of Fos-positive cells that were not characterized in the present study. VIP [24] and somatostatin [23] are neuropeptides present in the specific populations of amacrine cells in the rabbit retina. Both VIP gene and preprosomatostatin gene have an AP-1 binding site in their promoter regions [7,33], indicating their protein synthesis as potentially regulated by Fos transcription factor. VIP-positive cells have their cell bodies in the INL and are more numerous in the central than in the peripheral retina [24]. Even though the distribution and morphology of VIP-positive cells have been described in the mammalian retina, the function and target cells of the peptide have remained obscure. VIP stimulates adenylate cyclase and glycogenolysis in the rabbit retina and VIP has been suggested to act on Miiller cells rather than to have a role in retinal neurotransmission [30,31]. Flashing light did not induce Fos in VIP-positive cells in the rabbit retina, suggesting that VIP-positive cells are not strongly stimulated by light in the rabbit retina. Somatostatin-immunoreactive cells in the rabbit retina are displaced amacrine cells in the GCL, but extend long axon-like processes in the IPL [271. Their cell bodies are almost exclusively localized in the inferior half of the retina 1271. No Fos-expressing somatostatin-immunoreactive cells were seen after exposure to flashing light. The function of somatostatin-immunoreactive cells in the retina is still unclear, but a role in modulation of global retinal visual function, such as light sensitivity, or metabolic or trophic func-
tion, and generation of direction selectivity, has been suggested previously 123,411. The few ganglion cells that expressed Fos after light exposure had a large cell body and were presumably cx ganglion cells. These cells belong to ON ganglion cells and depolarize in response to light [6,39]. Since the density of Fos-stained ganglion cells was very low, only a small proportion of ON ganglion cells respond to light stimulation by Fos gene induction. Fos immunocytochemistry is only an indirect indication of physiological activation of neurons. Although no statement can be rigorously made about neurons that fail to express c-fos in response to an experimental stimulus, the preponderance of the evidence is that neurons that do express the gene have undergone potent synaptic excitation [1,10,34,40]. The neuronal localization of Fos permits the detailed anatomic dissection of physiologic pathways through the visual system. The experiments reported here employed the most general and potent visual stimulus, bright flashing light. Whether the same approach can be applied to more refined visual stimuli, such as moving targets or color remains to be demonstrated. Our finding in vivo support previous evidence from neurotransmitter release studies in vitro that light exposure excites dopaminergic and cholinergic amacrine cells [5,6,9,11,12,15,21,39]. Our most novel finding is that presumptive NOS amacrine cells are excited by light. The morphology of these cells, especially the Type I NADPHd amacrine cells that have multiple very long axon-like processes in the rabbit IPL [14,25,36], suggests that light induces a diffuse release of NO in the IPL. The presumed target of this NO is a soluble guanylyl cyclase in ON cone bipolar cells, which contain cGMP-gated cation channel [ 14,321. Illumination would thereby stimulate cGMP synthesis diffusely in ON cone bipolar cells. Focal light exposure would then act by a G protein-coupled stimulation of cGMP hydrolysis to provide contrast detection. This hypothesis is testable electrophysiologically, as selective NOS inhibitors are readily available.
Acknowledgements This study was supported by the National Institute of Neurology Disease and Stroke (NS27488) and Department of Veterans Affairs Merit Review Program.
References [l] Bartel, D.P., Sheng, M., Lau, L.F. and Greenberg, M.E., Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction, Genes Del,.. 3 (1989) 304-313.
J. Koistinaho,
S.M. Sagar/Molecular
[2] Bhat, R.V., Worley, P.F., Cole, A.J. and Baraban, J.M., Activation of the zinc finger encoding gene krox-20 in adult rat brain: comparison with zif268, Mol. Brain Rex, 13 (1989) 263-266. [3] Bredt. D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snider, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615-624. [4] Dacheux, R.F. and Raviola, E., The rod pathway in the rabbit retina: depolarizing bipolar and amacrine cells, J. Neurosci., 6 (1986) 331-345. [5] Dowling, J.E. and Ehringer, B., Synaptic organization of the dopaminergic neurons in the rabbit retina, J. Camp. Neural., 180 (1978) 203-220. [6] Dowling, J.E., The Retina, Harvard University Press, Cambridge, MA, 1987. [7] Fink, J.S., Verhave, M., Walton, K., Mandel, G. and Goodman, R.H., Cyclic AMP- and phorbol ester-induced transcriptional activation are mediated by the same enhancer element in the human vasoactive intestinal peptide gene, J. Biol. Chem., 266 (1991) 3882-3887. [B] Hadjiconstantinou, M., Rossetti, Z.L., Krajnc, D. and Neff, N.H., Aromatic L-/amino acid decarboxylase activity of the rat retina in modulated in vivo by environmental light, J. Neurochem., 51 (1988) 1560-1564. [9] Hokoc, J.N. and Mariani, A.P., Synapses from bipolar cells onto dopaminergic amacrine cells in cat and rabbit retinas, Brain Res., 461 (1988) 17-26. [lo] Hunt, S.P., Pini, A. and Evan, G., Induction of c-fos like protein in spinal cord neurones following sensory stimulation, Nature, 328 (1987) 632-634. [ll] Jensen, R.J., Mechanism and site of action of a dopamine Dl antagonist in the rabbit retina, vis. Neurosci., 3 (1989) 573-585. [ 121 Jensen, R.J. and Daw, N.W., Effect of dopamine antagonists on receptive fields of brisk cells and directionally selective cells in the rabbit retina, J. Neurosci., 4 (1984) 2972-2985. [13] Koistinaho, J., Hicks, K. and Sagar, S.M., Tetrodotoxin enhances light-induced c-fos expression in the rabbit retina, Mol. Brain Res., 17 (1993) 179-183. [14] Koistinaho, J., Swanson, R.A., de Vente, S. and Sagar, S.M., NAPDH-diaphorase (nitric oxide synthase) reactive amacrine cells of rabbit retina: putative target cells and stimulation by light, Neuroscience, 57 (1993) 587-597. [15] Masland, R.H. and Livingston, C.J., Effect of stimulation with light on synthesis and release of acetylcholine by an isolated mammalian retina, J. Neurophysiol., 39 (1976) 1210-1219. [16] Massey, S.C., Cell types using glutamate as a neurotransmitter in vertebrate retina. In N. Osborne and J. Chader (Eds.), Progress in Retinal Research, Vol. 9, Pergamon, Oxford, 1990, pp. 399-425. [17] McLean, I.W. and Nagane, P.K., Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. [18] Millbrandt, J., A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor, Science, 238 (1987) 797-799. 1191 Misawa, H., Ishii, K. and Deguchi, T., Gene expression of mouse choline acetyltransferase, J. Biol. Chem., 267 (1992) 20392-20399. [20] Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-197.
Brain Research
29 (1995) 53-63
63
[21] O’Malley, D.M., Sandell, J.H. and Masland, R.H., Co-release of acetylcholine and GABA by the starburst amacrine cells, J. Neurosci., 12 (1992) 1394-1408. [22] Osborne, N.N. and Beaton, D.W., Direct histochemical localization of 5,7_dihydroxytryptamine and the uptake of serotonin by a subpopulation of GABA neurones in the rabbit retina, Brain Res., 382 (1986) 158-162. [23] Sagar, S.M., Somatostatin-like immunoreactive material in the rabbit retina: Immunohistochemical staining using monoclonal J. Camp. Neural., 266 (1987) 291-299. antibodies, [24] Sagar, S.M., Vasoactive intestinal polypeptide (VIP) immunohistochemistry in the rabbit retina, Brain Res., 426 (1987) 1577163. [25] Sagar, S.M., NADPH-diaphorase reactive neurons of the rabbit retina: differential sensitivity to excitotoxins and unusual morphologic features, J. Camp. Neural., 300 (1990) 309-319. [26] Sagar, SM. and Sharp, F.R., Light induces a Fos-like nuclear antigen in retinal neurons, Mol. Brain Rex, 7 (1990) 17-21. [27] Sagar, S.M., Sharp, F.R. and Curran, T., Expression of the c-fos protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. [28] Sandell, J.H., Masland, R.H., Raviola, E. and Dacheux, R.F., Connections of indolamine-accumulating cells in the rabbit retina, J. Neurosci., 6 (1989) 3331-3347. [29] Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229-234. [30] Schoderet, M., Sovilla, J.-Y. and Magisstretti, P.J., The effects of VIP on cyclic AMP and glycogen levels in vertebrate retina, Peptides, 5 (1984) 295-298. [31] Schoderet, M., Sovilla, J.-Y. and Magisstretti, P.J., VIP and glucagon-induced formation of cyclic AMP in intact retinae in vitro, Eur. J. Pharmacol., 71 (1981) 131-133. [32] Shieldls, R. and Falk, G., Retinal on bipolar cells contain a nitric oxide sensitive guanylate cyclase, NeuroReport, 3 (1992) 845-848. [33] Smith, S.E., Papavassilou, A.G. and Bohmann, D., Differential TRE-related elements are distinguished by sets of DNA-binding proteins with ovelapping sequence specificity, Nucl. Acids Res., 21 (1993) 1581-1985. [34] Sonnenberg, J.L., Rauscher, III F.J., Morgan, J.I. and Curran, T., Regulation of proenkephalin by Fos and Jun, Science, 246 (1989) 1622-1625. [35] Strettoi, E., Dacheux, R.F. and Raviola, E., Synaptic connections of rod bipolar cells in the inner plexiform layer of the rabbit retina, J. Camp. Neurol., 295 (1990) 449-466. [36] Vaney, D.I. and Young, H.M., GABA-like immunoreactivity in NADPH-diaphorase amacrine cells of the rabbit retina, Brain Res., 240 (1988) 380-385. [37] Vaney, D.I., Young, H.M. and Gynther, I.C., The rod circuit in the rabbit retina, I/is. Neurosci., 7 (1991) 141-154. [38] Voigt, T. and WBssle, H., Dopaminergic innervation of AI1 amacrine cells in mammalian retina, J. Neurosci., 7 (1987) 41154128. [39] Wgssle, H. and Boycott, B.B., Functional architecture of the mammalian retina, Physiol. Reu., 71 (1991)141-154. [40] Wisden, W., Errington, M.L., Williams, S., Dunnet, S.B., Waters, C., Hitchcock, D., Evan, G., Bliss, T.V.P. and Hunt, S.P., Differential expression of immediate early genes in the hippocampus and spinal cord, Neuron, 4 (1990) 603-614. 1411 Zalutsky, R.A. and Miller, A., The physiology of somatostatin in the rabbit retina, J. Neurosci., 10 (1990) 383-393.