Journal of Chemical Neuroanatomy 12 (1997) 267–280

Enrichment of glutamate-like immunoreactivity in the retinotectalterminals of the viper Vipera aspis :

an electron microscope quantitative immunogold study

J. Reperant a,b,*, J.-P. Rio a, R. Ward b,c, M. Wasowicz b, D. Miceli c, M. Medina b, J. Pierre a,b

a INSERM U-106, Laboratoire de Neuromorphologie: De6eloppement, E6olution, Hopital de la Salpetriere, Batiment de Pediatrie,47 Bd de l’Hopital, 75651 Paris Cedex 13, France

b Laboratoire d’Anatomie Comparee, Museum National d’Histoire Naturelle, Paris, Francec Laboratoire de Neuropsychologie Experimentale, Uni6ersite du Quebec, Trois-Ri6ieres, Quebec, Canada

Received 30 September 1996; received in revised form 13 February 1997; accepted 16 March 1997

Abstract

A post-embedding immunogold study was carried out to estimate the immunoreactivity to glutamate in retinal terminals, Paxon terminals and dendrites containing synaptic vesicles in the superficial layers of the optic tectum of Vipera. Retinal terminals,identified following either intraocular injection of tritiated proline, horseradish peroxidase (HRP) or short-term survivals afterretinal ablation, were observed to be highly glutamate-immunoreactive. A detailed quantitative analysis showed that about 50%of glutamate immunoreactivity was localized over the synaptic vesicles, 35.8% over mitochondria and 14.2% over the axoplasmicmatrix. The close association of immunoreactivity with the synaptic vesicles could indicate that Vipera retino-tectal terminals mayuse glutamate as their neurotransmitter. P axon terminals and dendrites containing synaptic vesicles, strongly g-aminobutyric(GABA)-immunoreactive, were shown to be also moderately glutamate-immunoreactive, but two to three times less than retinalterminals. Moreover, in P axon terminals, the glutamate immunoreactivity was denser over mitochondria than over synapticvesicles, possibly reflecting the ‘metabolic’ pool of glutamate, which serves as a precursor in the formation of GABA. © 1997Elsevier Science B.V.

Keywords: Immunocytochemistry; Glutamate; Visual system; Reptile

1. Introduction

It is not altogether clear which neurotransmitters arereleased by the retinofugal fiber terminals in differentvertebrates species. A wide variety of neuroactive sub-stances have been identified in the somata of retinalganglion cells in a diversity of species; these includeL-glutamate (Raiguel and Marc, 1987; Ehinger et al.,1988; Kageyama and Meyer, 1989; Marc et al., 1990;Davanger et al., 1991; Crooks and Kolb, 1992; Sherry

and Ulshafer, 1992; Kalloniatis and Fletcher, 1993;Pow and Robinson, 1994), substance P (reviews inBrecha et al., 1987; Ehrlich et al., 1987; Cuenca andKolb, 1989; Karten et al., 1990), the acidic dipeptideN-acetylaspartylglutamate (NAAG) (Anderson et al.,1987; Tieman et al., 1987, 1988, 1991a,b; Williamson etal., 1991), L-aspartate (Ehinger, 1981; Yaqub and El-dred, 1991), catecholamines (Brunken et al., 1986;Britto et al., 1988), serotonin (Weiler and Ammer-muller, 1986), glycine (Eldred and Cheung, 1989) andg-aminobutyric acid (GABA) (Mosinger et al., 1986;Glasener et al., 1988; Yu et al., 1988; Caruso et al.,1989; Gayoso et al., 1989; Hurd and Eldred, 1989;

* Corresponding author. Tel.: +33 1 42162677; fax: +33 145709990.

0891-0618/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.PII S 0 891 -0618 (97 )00018 -5

J. Reperant et al. / Journal of Chemical Neuroanatomy 12 (1997) 267–280268

Koontz et al., 1989; Davanger et al., 1991; Gabriel etal., 1992; Watt et al., 1994; Gabriel and Straznicky,1995). In retinal terminals, on the other hand, gluta-mate exists in large quantities (Kageyama and Meyer,1989; Montero and Wenthold, 1989; Montero, 1990,1994; Morino et al., 1991; Nunes-Cardozo et al.,1991; Castel et al., 1993; de Vries et al., 1993; Ortegaet al., 1995; Mize and Butler, 1996) in normal speci-mens, but is no longer detectable after retinal ablation(Kageyama and Meyer, 1989; Morino et al., 1991;Ortega et al., 1995; Mize and Butler, 1996), whichsuggests that glutamate may be the neurotransmitterused by these structures. This hypothesis is supportedby studies in which electrical or natural visual stimulielicit responses through activation of NMDA andnon-NMDA (kainate, quisqualate) types of receptorswhich can be blocked by antagonists or reactivated byagonists such as L-glutamate (reviews in Funke et al.,1991; Binns and Salt, 1994).

This opinion is not, however, shared by all authors.Glutamate, aspartate and NAAG are released in pri-mary visual centers following optic nerve stimulation(Canzek et al., 1981; Sandberg and Jacobson, 1981;Liou et al., 1986; Tsai et al., 1990), and it has beensuggested that retinal ganglion cells may not use glu-tamate, but rather some endogenous agonist of gluta-mate receptors such as aspartate (Yaqub and Eldred,1991), NAAG (Anderson et al., 1987; Moffett et al.,1991; Tieman et al., 1987, 1991a; Molinar-Rode andPasik, 1992) or homocystic acid (Jones and Sillito,1992).

In this study, we have attempted to establish, byimmunocytochemical techniques, the pattern of gluta-mate-like immunoreactivity (GLU-ir) in the retino-tec-tal terminals of the snake Vipera aspis, a reptile inwhich the primary visual system has been particularlywell described (Reperant and Rio, 1976; Reperant etal., 1981, 1991, 1992). For this purpose, we have useda post-embedding immunogold procedure (Ottersenand Storm-Mathisen, 1985; Somogyi and Hodgson,1985; Somogyi et al., 1986; Ottersen, 1987, 1989a,b;Storm-Mathisen and Ottersen, 1990) that has enabledus to quantify the GLU-like immunoreactivity overthe retino-tectal terminals previously identified withaxonal tracing techniques. We have further attemptedto quantify the GLU-ir over different cellular com-partments (mitochondria, axoplasmic matrix andsynaptic vesicles) both in retinal terminals, GABA-im-munoreactive P axon terminals and dendrites contain-ing synaptic vesicles (Rio et al., 1995) with the aim todistinguish between ‘metabolic’ and ‘transmission’pools of glutamate (Somogyi et al., 1986; Monteroand Wenthold, 1989; Ji et al., 1991; Castel et al.,1993; Montero, 1994).

2. Materials and methods

A total of 33 adult, male or female, specimens ofVipera aspis were used (bred at Institut Pasteur,France). Of these four were normal, the remainingspecimens were subjected to one of the experimentalprocedures described below.

2.1. Surgical procedures

The intraocular injection of tracers and retinal abla-tion were carried out either under cold narcosis ornembutal anaesthesia (20–30 mg/kg, i.p.). The tracersemployed were either [3H]L-proline (specific activity 27Ci/mM, CEA, France) or horseradish peroxidase(HRP) (30% solution in 0.7% saline, Boehringer, Ger-many). Under direct visual control, 10 m l either oftritiated proline (20–25 mCi) or HRP solution wereinjected into the posterior chamber of the eye and thesnakes were subsequently maintained at 24–25°C.Each animal, together with the normal specimens, wasthen given an overdose of nembutal and perfusedthrough the single carotid artery with 300 ml of afixative composed of 1% paraformaldehyde and 1%glutaraldehyde in 0.12 M phosphate buffer at pH 7.3.

After perfusion, the skull was stored overnight infresh fixative at 4°C. The brain was then dissected outand the optic tecta separated. Except for specimens tobe used for HRP procedures (see below), the samplesof tectal tissue were postfixed for 3–4 h in 2%buffered osmium tetroxide, stained en bloc with 2.5%uranyl acetate, dehydrated in a graded series ofethanol and embedded in araldite.

2.2. Normal material

The polymerized blocks were oriented in such away that they would be cut in the coronal plane.Semi-thin (1 mm) sections were cut on a Reichert OMU2 microtome, stained with toluidine blue and pho-tographed under a Leitz photomicroscope. Thin (70–80 nm) sections were then cut, mounted on neckedcopper grids, double stained with uranyl acetate andlead citrate and examined under a Philips 400 electronmicroscope.

2.3. Degeneration experiments

After either eye removal or retinal ablation, 21snakes were allowed to survive for periods varyingfrom 5–113 days. Thin sections of the optic tectum,contralateral to the lesion, were mounted onto un-coated nickel grids. In order to analyze the sequentialevents of degeneration of optic terminals, some sec-tions were double stained with uranyl acetate andlead citrate, and others were prepared for post-embed-ding immunocytochemistry (see below).

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2.4. Radioautography experiments

A total of five snakes received a unilateral intraocularinjection of tritiated proline and were sacrificed 1–2days later. Thin sections of the optic tectum contralat-eral to the injection were prepared for high-resolutionradioautography (Larra and Droz, 1970). They weremounted on formvar-coated grids, dipped in Ilford L4emulsion, and stored at 4°C for 5–9 months prior todevelopment either in microdol or phenidon. The back-ground level was assessed from grain counts made oversquares containing no tissue or tissue devoid of retinalprojections. Further analysis followed the methods de-scribed by Bachman and Salpeter (1965) and bySalpeter and McHenry (1973).

2.5. HRP experiments

A total of three vipers received an intraocular injec-tion of HRP and were allowed to survive for 7–10days. The optic tectum contralateral to the injectionwas cut on a vibratome into 100 mm slices which werethen treated either by the cobalt-nickel intensified3,3%diaminobenzidine (DAB) method (Adams, 1981) orby Mesulam’s (Mesulam, 1982) tetramethylbenzidine(TMB) procedure and prepared for conventional elec-tron microscopy. Thin sections were cut and eitherdouble stained with uranyl acetate and lead citrate, orprepared for post-embedding immunocytochemistry(see below).

2.6. Immunocytochemical procedures

The procedures were carried out both in normal andexperimental (HRP and degeneration) material. Singleor alternate serial thin sections were mounted on nickelgrids and immersed in the following solutions: (1) 1%aqueous periodic acid (7 min); (2) rinse in distilledwater; (3) 1% sodium metaperiodate (7 min); (4) rinsein distilled water; (5) 5% bovine serum albumin in 0.05M Tris-buffered saline (TBS) pH 7.6 (30 min); and (6)rinse in TBS. Sections were then incubated overnight at4°C in the primary antibody (either anti-GLU, Chemi-con, USA, at a dilution of 1/300, or anti-GABA,Immunotech, France, at a dilution of 1/2000) diluted inTBS pH 7.6. After several rinses in TBS, sections werereacted with a goat anti-rabbit immunoglobulin cou-pled to colloidal (15 nm) gold particles (Janssen, Bel-gium), at a dilution of 1/75 in TBS pH 8.2 for 1 h.Sections were then rinsed in TBS and distilled waterand double counterstained with uranyl acetate and leadcitrate prior to examination under the electron micro-scope. Some sections were also processed for doubleimmunolabeling. After incubation in the first anti-GLUantibody and visualization with 20 nm gold particles,sections were exposed to vapours of 4% paraformalde-

hyde at 80°C for 1 h (Wang and Larsson, 1985),thoroughly rinsed in PBS and subsequently incubatedin the second anti-GABA antibody, rinsed and reactedwith 10 nm gold particles.

2.7. Control procedures

The two antibodies have been already characterizedin previous studies (anti-GLU, Chemicon: Sherry andUlshafer, 1992; Kalloniatis and Fletcher, 1993; anti-GABA, Immunotech; Seguela et al., 1984). Each anti-body shows a high specificity for the particular haptene(glutaraldehyde-fixed glutamate or glutaraldehyde-fixedGABA) and negligible cross-reactivity, either with theother haptene of the pair or with other amino acids, inparticular aspartate. In the present study, in each im-munolabeling session, control grids were tested for anycross-reactivity with glutamate, GABA and aspartate.Viper brain macromolecules were treated with glu-taraldehyde either alone or in the presence of one ofthese amino acids. The conjugates thus formed wereembedded and processed according to Ottersen’s tech-nique (Ottersen, 1987) and these conjugate sectionsthen underwent the same immunogold procedure assections of the SGFS. Electron micrographs were takenat the same magnification as that used for tissue sec-tions and the density of gold particles determined.Additional controls were made either by omitting theprimary antibody, replacing it with non-immune nor-mal serum, or immunoadsorption of free glutamate (forthe anti-GLU antibody) or free GABA (for the anti-GABA antibody) on polyacrylamide beads with glu-taraldehyde as a linking agent. Under none of theseconditions was any labeling ever observed.

2.8. Quantitati6e methods

The cross-sectional areas of profiles containingsynaptic vesicles (retinal and P axon terminals, den-drites containing synaptic vesicles) were measured bymeans of an automated image-analysis system(SAMBA, Alcatel, France) in electron micrographsprinted at a final magnification of 28 000× . The cross-sectional areas and perimeters of synaptic vesicles weremeasured by means of the same system, in electronmicrographs printed at a final magnification of112 000× . From these two parameters, a dimensionlessindex of roundness was derived as R=4pA/P2 for eachvesicle, in which A represents the synaptic vesicle cross-sectional area and P the perimeter, varying from 0 forflattened vesicles to 1 for round vesicles.

The density of immunogold particles over randomlyselected retinal (Rt), P axon terminals and dendritescontaining synaptic vesicles (DCSVs) was estimated inmicrographs printed at a final magnification of56 000× and all the gold particles lying over these

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terminals were manually counted on areas of 1 mm2.Subsequent analyses of the density of gold particleslying over the different subcellular compartments (mito-chondria, synaptic vesicles and axoplasmic matrix) werealso made. To do this, the surfaces occupied by mito-chondria and vesicles were determined, for each profile,by the image-analysis system and the surface occupiedby the axoplasmic matrix was calculated by subtractingthese surfaces from the total area of the terminal. Asbefore, the number of gold particles lying over each ofthe axoplasmic compartments was counted manually.

The following criteria were used to assign gold parti-cles to each compartment: particles over synaptic vesi-cles or the vesicular membrane were included in thevesicular count, those lying over mitochondria or theexternal mitochondrial membrane were included in themitochondrial count; and all particles not includedeither in the vesicular or mitochondrial count (includingparticles lying over dense-core vesicles) were assigned tothe cytoplasmic count.

The background was estimated from counts of goldparticles lying over the lumen of blood vessels or emptyresin, and expressed as n particles per square microme-tre. All data are presented as mean values together withthe standard error of the mean.

3. Results

3.1. Selecti6ity of the antisera

The results obtained with the conjugate materialrevealed a high degree of selectivity for each antiserum.Thus, for the sections of glutamate-conjugate, 32859295 (n=10) particles/mm2 were observed when thesewere treated with the anti-GLU and only 11299 (n=12) particles/mm2 with anti-GABA. Conversely, the im-munoprocessing of sections of GABA-conjugate withanti-GABA led to the deposition of 28859305 (n=10)particles/mm2 but of only 3.291.5 (n=10) particles/mm2 when anti-GLU was used. Finally, the sections ofaspartate-conjugate showed 62917 (n=8) particles/mm2 when treated with anti-GABA and 109912(n=8) particles/mm2 when treated with anti-GLU.

3.2. Identification and cytological characterization ofretino-tectal terminals and their targets

The retino-recipient layer of the viper optic tectumcomprises three distinct strata: the stratum zonale (SZ),the stratum opticum (SOP), and the stratum griseum etfibrosum superficiale (SGFS) (Reperant et al., 1991).The SZ is a thin plexiform layer 25–30 mm thick. On itsinternal surface, a layer of optic tract fibers occupiesthe SOP, a layer some 60 mm thick in which themyelinated optic fibers are organized into some 200–

250 small densely packed fascicles. Below the SOP liesthe SGFS, which is 350–420 mm thick (Fig. 1). Thislatter layer is a relatively homogeneous structure com-posed of cell bodies dispersed within a neuropil. TheSGFS is the principal layer in which the optic fibersarborize (Reperant et al., 1991), and our observationsconcern this layer.

The retino-tectal terminals have been clearly iden-tified with the use of anterograde degeneration, tritiatedamino acid (Fig. 2A, B) and HRP tracing techniques(Fig. 2C). These terminals (Fig. 2) are of varying size(0.91–3.36 mm2) and are often clustered. They show anirregular, sometimes scalloped contour (Fig. 2); theaxoplasm is electron lucent and contains denselypacked agranular synaptic vesicles of an ovoid shape

Fig. 1. Transverse semi-thin section of the optic tectum of the viper,stained with toluidine blue. SZ, stratum zonale ; SOP, stratum op-ticum ; SGFS, stratum griseum et fibrosum superficiale ; SGC, stratumgriseum centrale ; SAC, stratum album centrale. Scale bar: 84 mm.

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Fig. 2. Electron micrographs of the SGFS. A and B: high-resolution autoradiographs showing labeled retinal terminals (triangles) 24 h afterinjection of 3H-proline into the contralateral eye. A, developed in microdol; B, developed in phenidon; C, examples of retinal terminals (triangles)containing TMB reaction product (8 days after contralateral eye injection of HRP). Scale bars: A: 0.50 mm; B–C: 0.47 mm.

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(mean diameter, 46.596.0 nm, roundness index, 0.7890.11). The active zone is small (0.2190.03 mm). Theycontain from one to four small, round, dark mitochondriaand occasionally microtubules, neurofilaments, vacuolesand irregular aggregations of smooth endoplasmic reticu-lum, as well as one to three dense core vesicles. Theseterminals only establish asymmetrical synaptic contacts(Fig. 2A) with a variety of postsynaptic elements: dendriticspines and small (B1 mm) dendritic profiles, large (\1mm) dendritic profiles devoid of synaptic vesicles and largedendrites containing synaptic vesicles (DCSVs, Fig. 2A),belonging to interneurons (Rio et al., 1995).

3.3. Immunocytochemical data

3.3.1. Intact retinal terminals (labeled with HRP ornot)

Our use of the two antibodies has shown that the Rtterminals are never GABA-immunoreactive (Rio et al.,1995; present results; Fig. 4B, D), their labeling being veryclose to the background level (0.9590.15 particles/mm2).On the other hand, these terminals are strongly GLU-im-munoreactive (42.7192.14 particles/mm2, n=71), uncor-rectedforbackground(1.4090.56particles/mm2,Fig.4A,Fig. 5). A more detailed analysis has shown that about 50%of these particles are located over synaptic vesicles, 35.8%over mitochondria and 14.2% over the axoplasmic matrix(Fig. 3A, Fig. 4B, C). The labeling of synaptic vesicles ismore intense (t(100)=6.55, PB0.001) than that of themitochondria, which in turn is more intense (t(100)=6.93,PB0.001) than that of the axoplasmic matrix. Theaxoplasmicmatrixis,however,moreintenselylabeledthanthe background (t(100)=8.00, PB0.001, Fig. 6).

3.3.2. Degenerating retinal terminalsAs we have already shown (Reperant et al., 1991), the

degeneration of retino-tectal terminals in the viper is slow,occurs at widely varying rates and can be classified in threestages: (1) degeneration with swelling of synaptic vesicles(Fig. 3B); (2) degeneration with darkening of the axoplasmaccompanied or not by a transitory hypertrophy of theneurofilaments (Fig. 3C); and (3) late degenerationcharacterized by a more pronounced darkening of theaxoplasm, a progressive disappearance of organelles andthe engulfment by reactive glial cells (Fig. 3D).

The immunocytochemical studies of this material withthe anti-GLU antibody has revealed the following results.During stage one degeneration, the glutamate immunore-activity isessentiallysimilar tothatestimated inthenormalmaterial (41.1292.70 particles/mm2, n=40, t(109)=0.94,P\0.05, Fig. 3B). In stage two degeneration, the Rtterminals are considerably less immunoreactive (24.8694.01 particles/mm2, n=38, Fig. 3C). In the initial stagesof stage three degeneration, the immunoreactivity fallseven further (16.8392.95 particles/mm2, n=42), andwhen the degenerating retinal terminal is engulfed in thereactive glia, the density of gold particles is not distinguish-

able fromthebackgroundlevel (2.8990.92particles/mm2,t(67)=1.38, P\0.05, Fig. 3D).

3.3.3. GABA-immunoreacti6e P axon terminals andDCSVs

Inapreviouselectronmicroscopicstudyoftheretino-re-cipient layers of the viper optic tectum (Rio et al., 1995),we have described five types of profiles containing synapticvesicles (PCSVs) following different ultrastructural crite-ria (profile size, shape, size and density of synaptic vesicles,type of synaptic contact). The first four types correspondto axon terminals (P1, P2, P3 and P4). The last type repre-sents dendrites containing synaptic vesicles (DCSVs),belonging to interneurons. These profiles have been shownto be strongly GABA-immunoreactive (Rio et al., 1995present results,Fig.4B).Themeandensityofgoldparticlesper square micrometre over P axon terminals and DCSVswasrespectivelyof43.9391.25and34.391.24(Rioetal.,1995).Theanalysisofdoubleimmunolabeledpreparations(anti-GLU and anti-GABA, Fig. 4B), or alternate serialsections (anti-GLU or anti-GABA, Fig. 4C, D), and thequantitative analysis of glutamate immunoreactivity onthese profiles simply identified on ultrastructural criteria(Fig. 4A) has revealed that they are also moderatelyGLU-immunoreactive (17.1890.34 particles/mm2, n=82forPaxonterminals;17.0190.41particles/mm2,n=80for DCSVs, Fig. 4B, Figs. 5 and 6), but less than the retinalterminals (t(79)=4.68, PB0.001). A more detailed analy-sis has shown that the latter particles are generally situatedover mitochondria (38.993.71 particles/mm2), less overthe synaptic vesicles (8.5390.81 particles/mm2, t(62)=3.65; PB0.001), and less than over the axoplasmic matrix(8.1390.74 particles/mm2, t(62)=1.51, P\0.05, Figs. 5and 6). We also point out that these P axon terminals showabout 2.3 times less glutamate reactivity than the retinalterminals,andtheparticledensityoverthesynapticvesiclesof Rt terminals is more than 7.5 times greater than overthe GABA-immunoreactive P axon terminals (Table 1).

4. Discussion

Previous ultrastructural studies of the degeneration ofretino-tectal terminals after enucleation (Reperant et al.,1991) have enabled us to describe some morphologicalcharacteristicsof these terminals.Theuseofaxonal tracingtechniques in the present investigation confirm andcomplete these previous data.

4.1. Glutamate as a possible neurotransmitter of opticterminals; an hypothesis based on the present resultsand pre6ious findings

4.1.1. Immunocytochemical data in fa6our of thishypothesis

Recent methodological improvements, in particularthe immunogold technique combined with electron mi-

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Fig. 3. A: High-magnification micrograph showing the glutamate-immunoreactive labeling of synaptic vesicles in a retinal terminal in the SGFS(triangle, HRP reaction product, DAB method). B–D: Glutamate-immunoreactive labeling of retino-tectal terminals at various stages ofdegeneration. B: Degeneration with swelling of synaptic vesicles (stage one) showing a high density of immunogold particles. C: Degeneration withdarkening of the axoplasm and neurofibrillar hypertrophy (nf). Note that the density of immunogold particles is lower than in (A). D: Latedegeneration with more pronounced darkening of the axoplasm associated with a loss of organelles and the distortion of mitochondria. Thedensity of gold particles is very low. The reactive glial profiles (rg) which engulf the degenerating retinal terminal are not glutamate-immunoreac-tive. Scale bars: A: 0.11 mm; B, D: 0.50 mm; C: 0.40 mm.

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Fig. 4. Glutamate and/or GABA immunoreactivity in the SGFS. A: Two glutamate-immunoreactive retinal terminals (Rt) contact (arrows) adendrite containing synaptic vesicles (DCSV) which makes a symmetrical synaptic contact (arrowhead) on a small dendrite. The density of goldparticles is lower in the DCSV than in Rt terminals. B: Double immunolabeled section of a P1 axon terminal containing a denser amount of smallgold particles (10 nm, anti-GABA) than large particles (20 nm, anti-GLU). On the contrary, the two retinal terminals (Rt) only contain large goldparticles. C, D: Alternate serial sections either processed with anti-GLU (C) or anti-GABA (D) antibody. The retinal terminals (Rt) are denselyglutamate-immunoreactive (C), whereas a P3 axon terminal is strongly GABA-immunoreactive (D). Low levels of glutamate- and GABAimmunoreactivity are respectively observed in P3 (C) and Rt (D) axon terminals. Scale bars: 0.25 mm.

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Fig. 5. Histogram showing the distribution of glutamate immunoreac-tivity in retinal (Rt), P axon terminals and dendrites containing synapticvesicles (DCSV) in Vipera retino-recipient tectal layers. B: Background.

Table 1Distribution of glutamate immunoreactivity in GABA negative (+,vertical hatchings) and GABA-ir (−, horizontal hatchings)

B, background; D, dendrites; DCSV, dendrites containing synapticvesicles; retino-tectal (Rt), P and S1 terminals; So, somata.

croscopy, have made it possible to evaluate this hypoth-esis by the precise location of glutamate within neuronsat an unrivalled level of morphological definition (Som-ogyi et al., 1986; Ottersen, 1987, 1989a,b; Ottersen andStorm-Mathisen, 1987; Storm-Mathisen and Ottersen,1990), and this technique has been used in the presentstudy. In contrast to the immunocytochemical identifi-cation of other neuroactive substances (such as GABA,for which a positive immunoreactivity appears to be aspecific marker of GABAergic neurons) the glutamateimmunoreactivity is more difficult to interpret. Thisamino acid is present in most, if not all, cell compart-ments and is involved in a number of metabolic path-ways within the central nervous system and someauthors have suggested that the glutamate immunoreac-tivity reveals both the ‘metabolic’ and ‘transmitter’ poolin neural processes (Somogyi et al., 1986; Ottersen andStorm-Mathisen, 1987; Ottersen, 1989a,b).

Our results have demonstrated a high level of gluta-mate immunoreactivity in the retino-tectal terminals ofthe viper SGFS, whereas a lower level of glutamateimmunoreactivity has been estimated in GABA-im-munopositive P axon terminals. This result has led usto quantify the level of glutamate immunoreactivity in

different cellular compartments (synaptic vesicles, mito-chondria and axoplasmic matrix). Within the retinalterminals, the gold particle density over the synapticvesicles is about 1.5 times as high as over mitochondria,and about 3.5 times higher than over the axoplasmicmatrix. Several of the enzymes involved in themetabolism of glutamate and glutamine (the principalsource of glutamate as a neurotransmitter), such as thephosphate-activated glutaminase, glutamate dehydroge-nase and aspartate amino-transferase, have been iden-tified in mitochondria (Palaiologos et al., 1989;Erecinska and Silver, 1990; Kvamme et al., 1991), andthe mitochondrial glutamate immunoreactivity mayreflect the presence of these enzymes. On the otherhand, the accumulation of glutamate in the synapticvesicles of retinal terminals strongly suggests that gluta-mate is likely to be the neurotransmitter.

In other respects, the GABA-immunoreactive P axonterminals are 2.3 times less glutamate-immunoreactivethan are retinal terminals. However, with the anti-GLUantibody, the gold particle density over mitochondria isproportionally higher in GABA-immunoreactive Paxon terminals than in the retinal terminals. The gluta-mate contained in P axon terminals may thus wellcorrespond to the ‘metabolic’ pool of glutamate whichserves as a precursor in the formation of GABA by theaction of glutamate decarboxylase (Ottersen andStorm-Mathisen, 1987 for review).

Comparable immunocytochemical data have beenobtained in the primary visual centers of other species.Thus, a high level of glutamate immunoreactivity hasbeen described in the retino-geniculate terminals of thecat (Montero, 1990, 1994) and macaque (Montero andWenthold, 1989), in the retino-tectal terminals of thepigeon (Morino et al., 1991) and goldfish (Kageyamaand Meyer, 1989), in the retino-collicular terminals ofthe rat (Ortega et al., 1995) and cat (Mize and Butler,1996), in the retino-suprachiasmatic terminals of the

Fig. 6. Quantitative estimation of glutamate gold particle density overdifferent subcellular compartments in retinal (Rt) and P axon terminals.sv, synaptic vesicles; m, mitochondria; am, axoplasmic matrix.

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mouse (Castel et al., 1993), rat (de Vries et al., 1993)and cat (Chen and Pourcho, 1995) and in the retinalterminals in the nucleus of the optic tract (Nunes-Car-dozo et al., 1991). These results have led these investi-gators to conclude that glutamate immunoreactivityover the retinal terminals is strong evidence in supportof the hypothesis of a glutamatergic neurotransmission.

4.1.2. Other data in support of this hypothesisIn a variety of primary visual centers (optic tectum,

dorsal lateral geniculate nucleus, nucleus of the optictract, suprachiasmatic nucleus) of a wide variety ofspecies (cat, rabbit, mouse, rat, pigeon, frog andgoldfish), extra- and intracellular recordings in vivo orin vitro have shown that antagonists of EAAs block theexcitatory postsynaptic potentials induced by stimula-tion of the optic nerve or photic stimulation of theretina. The iontophoretic administration of EAAs, suchas glutamate, produces an excitatory response of visu-ally sensitive neurons (Felix and Frangi, 1977; Langdonand Freeman, 1986, 1987; Shibata et al., 1986; Debskiet al., 1987; Fox and Fraser, 1987; van Deusen andMeyer, 1988; Cahill and Menaker, 1989a,b; Murphy etal., 1989; Hartveit and Heggelund, 1990; Heggelundand Hartveit, 1990; Sillito et al., 1990a,b; Funke et al.,1991; Kim and Dudek, 1991; Kwon et al., 1991;Roberts et al., 1991; Schmidt, 1991; Hestrin, 1992;Binns and Salt, 1994).

In the pigeon optic tectum (Canzek et al., 1981) andrabbit superior colliculus (Sandberg and Jacobson,1981), the electrical stimulation of the optic nerve leadsto a release of endogenous glutamate and aspartate invivo. In vitro, a calcium-dependent, potassium-inducedrelease of glutamate has been demonstrated in pigeontectal slices (Reubi, 1980; Toggenburger et al., 1982),while in rat brain slices of the suprachiasmatic nucleus,the stimulation of the optic nerve induces the release ofglutamate and aspartate (Liou et al., 1986).

Several previous studies have examined the effects ofenucleation or retinal ablation. In the retino-recipientlayers of the avian optic tectum, the high-affinity up-take of glutamate is drastically reduced in tectal synap-tosomes prepared from enucleated specimens (pigeon:Henke et al., 1976; chicken: Bondy and Purdy, 1977).Retinal ablation also leads to a significant reduction ofglutamate and aspartate levels in the optic nerve andretino-recipient tectal layers of the pigeon (Fonnumand Henke, 1982).

4.2. Other putati6e neurotransmitters of optic ner6eterminals

The notion that glutamate is the neurotransmitter ofretinal terminals is not, however, universally accepted.Several other molecules have been proposed for thisfunction (see Section 1), which we discuss below in thecontext of our results.

4.2.1. AspartateWhile our results show a relatively high level of

glutamate immunoreactivity in the retino-tectal termi-nals of the viper, suggesting that this substance is theneurotransmitter of these terminals, the possibility re-mains that it may be aspartate which serves this func-tion. These two amino acids act on the same type ofEAA receptor, although with different affinities (Fon-num, 1984; Watkins and Oliverman, 1987; Villanuevaand Orrego, 1988; Collingridge and Lester, 1989), andare taken up by similar or identical carriers (Logan andSnyder, 1972; Davies and Johnston, 1976). Further-more, it has been shown that the stimulation of theoptic nerve leads not only to the release of glutamate,but also of aspartate (Canzek et al., 1981; Sandbergand Jacobson, 1981; Liou et al., 1986). In light of theseresults, we are inclined to consider the possibility thatour glutamate antibody may cross-react with aspartatein fixed tissue. However, previous studies (Sherry andUlshafer, 1992; Kalloniatis and Fletcher, 1993; and ourcontrol procedure) have shown that this antibody dis-criminates well between these two amino acids andsome preliminary data with an anti-aspartate antibodyappear to indicate that, in our material, the level ofimmunoreactivity in the retino-tectal terminals is barelydistinguishable from the background (Reperant et al.,unpublished observations). These results are in agree-ment with those of Montero (1994), who has demon-strated high levels of glutamate immunoreactivity, butnot of aspartate, in the GLd of the cat, and hasconcluded that glutamate is likely the neurotransmitterof the retinofugal pathway in this species.

4.2.2. Substance P and NAAGThe presence of a small number of dense-core vesicles

in almost every terminal that we have observed stronglysuggests the coexistence of other neuroactive peptidesor amines in these terminals. Some preliminary investi-gations (Reperant et al., unpublished observations)have allowed us to demonstrate an immunoreactivity tosubstance P and NAAG in the retino-recipient layers ofthe optic tectum of the viper, at the light microscopiclevel, but we are not at present able to identify theprecise intracellular localization of this substance. Itscoexistence in retinal terminals remains thus an openempirical question. It has been shown that NAAG maypossibly coexist in retinal terminals in mammals andbirds (Anderson et al., 1987; Tieman et al., 1987, 1991a;Moffett et al., 1991; Williamson et al., 1991; Molinar-Rode and Pasik, 1992). These authors have suggestedthat NAAG could be the neurotransmitter of retinalterminals on the basis of the following results. Itspresence may be demonstrated immunochemically inthe somata, fibers and axon terminals of retinal gan-glion cells and the level of NAAG immunoreactivitywithin the retinal targets decreases after eye removal

J. Reperant et al. / Journal of Chemical Neuroanatomy 12 (1997) 267–280 277

(Moffett et al., 1991; Tieman et al., 1991b; Molinar-Rode and Pasik, 1992). Finally, the calcium-dependentrelease of NAAG within the rat superior colliculus andchick optic tectum is achieved by stimulation of theoptic tract (Tsai et al., 1990; Williamson et al., 1991).Nevertheless, other results suggest that NAAG mightnot subserve the function of a ‘classical’ neurotransmit-ter. The iontophoretic injection of NAAG into the GLdof the cat leads to a weak and highly variable effect(Jones and Sillito, 1992). Its effects on the two principaltypes of excitatory receptors of the primary visualcenters are extremely different; whatsoever, it has noeffect on the non-NMDA receptors, whereas its effectson the NMDA receptors is in the magnitude of 100times less than that of glutamate (Sekiguchi et al.,1992). This substance has no effect on the activity ofthalamic somatosensory neurons in the cat (Hendersonand Salt, 1988) and it has been shown to coexist inneurons that are demonstrably cholinergic, noradrener-gic or serotoninergic (Forloni et al., 1987), in additionto GABAergic neurons (Henderson and Salt, 1988).

4.2.3. MonoaminesImmunohistochemical methods have demonstrated

the existence of a small population of retinal ganglioncells that are immunoreactive to the enzyme of synthe-sis of catecholamines (tyrosine hydroxylase, TH) in theretina of elasmobranchs (Brunken et al., 1986), turtles(Weiler and Ammermuller, 1986) and birds (Britto etal., 1988). We have not examined, under the electronmicroscope, the retinal targets of the viper after TH- or5-HT-immunocytochemistry; however, at the ultra-structural level, both the optic chiasma and optic tractof the viper are totally devoid of TH- or 5-HT-im-munoreactivity (Challet et al., 1991; Reperant et al.,unpublished observations).

4.2.4. GABAImmunocytochemical studies have demonstrated the

presence of GABA in some retinal ganglion cells ofurodeles (Glasener et al., 1988; Watt et al., 1994),anurans (Gabriel et al., 1992; Gabriel and Straznicky,1995), chelonians (Hurd and Eldred, 1989) and a vari-ety of mammalian species including man (Mosinger etal., 1986; Yu et al., 1988; Caruso et al., 1989; Gayoso etal., 1989; Koontz et al., 1989; Davanger et al., 1991). Ina double-labeling study, the combination of a fluores-cent tracer injection into the superior colliculus withGABA immunohistochemistry has led Caruso et al.(1989) to estimate that approximately 5–6% of theretinal ganglion cells that project to the rat superiorcolliculus are GABA-immunoreactive. GABA im-munoreactivity has been also reported in optic nervesof some mammalian species (Rogers and Pow, 1995;Wilson et al., 1996). In addition, a strong GABAimmunoreactivity has been also demonstrated in some

retinal terminals of the superficial layers of the toadoptic tectum (Gabriel and Straznicky, 1995) andmacaque superior colliculus (Kisvarday et al., 1991).On the other hand, neither in the present study, nor inprevious investigations (Rio et al., 1995) did we observeany retinal terminals of Vipera that are GABA-im-munopositive. Indeed, the GABA-immunoreactive Paxon terminals in the optic tectum of the viper neverdegenerate after retinal ablation (Reperant et al., 1991).

5. Conclusions

The present results strongly suggest that the retino-tectal terminals in the viper may use glutamate as theirneurotransmitter. Furthermore, both our results andthe literature data support the hypothesis that gluta-mate may be the endogenous agonist for EAA recep-tors throughout the primary visual system of a widevariety of vertebrate species, implying that glutamateneurotransmission by retinal ganglion cells is phyloge-netically conserved.

Acknowledgements

This article is dedicated to Professor P.P. Gambarianof the Zoological Institute of St. Petersburg (Russia),on the occasion of his birthday. Financial support forthis research was provided by INSERM, MNHN,FCAR and NSERC/CRSNG. The authors thank F.Roger and S. Arnold for their excellent technical assis-tance, and D. LeCren for his habitually skilled photo-graphic work.

References

Adams, J.C., 1981. Heavy metal intensification of DAB-based HRPreaction product. J. Histochem. Cytochem. 29, 775.

Anderson, K.J., Borja, M.A., Cotman, C.W., Moffett, J.R., Nam-boodiri, M.A.A., Neale, J.H., 1987. N-acetylaspartylglutamateidentified in the rat ganglion cells and their projections in thebrain. Brain Res. 411, 172–177.

Bachman, L., Salpeter, M.M., 1965. Autoradiography with electronmicroscope: A quantitative evaluation. Lab. Invest. 14, 1041–1053.

Binns, K.E., Salt, T.E., 1994. Excitatory amino acid receptors partic-ipate in synaptic transmission of visual responses in the superficiallayers of the cat superior colliculus. Eur. J. Neurosci. 6, 161–169.

Bondy, S.C., Purdy, J.L., 1977. Putative neurotransmitters of theavian visual pathway. Brain Res. 119, 417–426.

Brecha, N., Johnson, D., Bolz, J., Sharma, S., Parnavelas, J.G.,Lieberman, A.R., 1987. Substance-P immunoreactive retinal gan-glion cells and their central axons in the rabbit. Nature 327,155–158.

Britto, R.G., Keyser, K.T., Hamassaki, D.E., Karten, H.J., 1988.Catecholaminergic subpopulation of retinal displaced ganglioncells project to the accessory optic nucleus in the pigeon (Columbali6ia). J. Comp. Neurol. 269, 109–117.

J. Reperant et al. / Journal of Chemical Neuroanatomy 12 (1997) 267–280278

Brunken, W.J.P., Witkowsky, P., Karten, H.J., 1986. Retinal neuro-chemistry of three elasmobranch species: An immunohistochemi-cal approach. J. Comp. Neurol. 243, 1–12.

Cahill, G.M., Menaker, M., 1989a. Responses of the suprachiasmaticnucleus to retinohypothalamic tract volleys in a slice preparationof the mouse hypothalamus. Brain Res. 469, 65–75.

Cahill, G.M., Menaker, M., 1989b. Effects of excitatory amino acidreceptor antagonists and agonists on suprachiasmatic nucleus toretinohypothalamic tract volleys. Brain Res. 479, 76–82.

Canzek, V., Wolfensberger, M., Amsler, V., Cuenod, M., 1981. Invivo release of glutamate and aspartate following optic nervestimulation. Nature 293, 572–574.

Caruso, D.M., Owczarzak, M.T., Goebel, D.J., Hazlett, J.C., Pour-cho, R.G., 1989. GABA-immunoreactivity in ganglion cells of therat retina. Brain Res. 476, 129–134.

Castel, M., Belenky, M., Cohen, S., Ottersen, O.P., Storm-Mathisen,J., 1993. Glutamate-like immunoreactivity in retinal terminals ofthe mouse suprachiasmatic nucleus. Eur. J. Neurosci. 5, 368–381.

Challet, E., Reperant, J., Pierre, J., Ward, R., Miceli, D., 1991. Theserotoninergic system of the brain of the viper Vipera aspis. Animmunohistochemical study. J. Chem. Neuroanat. 4, 233–248.

Chen, B., Pourcho, R.A., 1995. Morphological diversity and gluta-mate immunoreactivity of retinal terminals in the suprachiasmaticnucleus of the cat. J. Comp. Neurol. 361, 108–118.

Collingridge, G.L., Lester, R.A.J., 1989. Excitatory aminoacids andreceptors in the vertebrate central nervous system. Pharmacol.Rev. 40, 143–210.

Crooks, J., Kolb, H., 1992. Localisation of GABA, glycine, gluta-mate and tyrosine hydroxylase in the human retina. J. Comp.Neurol. 315, 287–302.

Cuenca, N., Kolb, H., 1989. Morphology and distribution of neuronsimmunoreactive for substance P in the turtle retina. J. Comp.Neurol. 290, 391–411.

Davanger, S., Ottersen, O.P., Storm-Mathisen, J., 1991. Glutamate,GABA and glycine in the human retina: An immunocytochemicalinvestigation. J. Comp. Neurol. 311, 483–494.

Davies, L.P., Johnston, G.A.R., 1976. Uptake and release of D- andL-aspartate by rat brain slices. J. Neurochem. 26, 1007–1014.

Debski, E.A., Cline, H.T., Constantine-Paton, M., 1987. Kynurenicacid blocks retinal-tectal transmission in Rana pipiens. Soc. Neu-rosci. Abstr. 13, 1691.

de Vries, M.J., Nunes Cardozo, B., van der Want, J., de Wolf, A.,Meijer, J.H., 1993. Glutamate immunoreactivity in terminals ofthe retinohypothalamic tract of the brown norvegian rat. BrainRes. 612, 231–237.

Ehinger, B., 1981. [3H]DAspartate accumulation in the retina ofpigeon, guinea-pig and rabbit. Exp. Eye Res. 33, 381–391.

Ehinger, B., Ottersen, O.P., Storm-Mathisen, J., Dowling, J.E., 1988.Bipolar cells in the turtle retina are strongly immunoreactive forglutamate. Proc. Natl. Acad. Sci. USA 85, 8321–8325.

Ehrlich, D., Keyser, K.T., Karten, H.J., 1987. The distribution ofsubstance P-like immunoreactive retinal ganglion cells and theirpattern of termination in the optic tectum of chick (Gallus gallus).J. Comp. Neurol. 266, 220–233.

Eldred, W.D., Cheung, K., 1989. Immunocytochemical localisation ofglycine in the retina of the turtle (Pseudemys scripta). Vis. Neu-rosci. 2, 331–338.

Erecinska, M., Silver, I.A., 1990. Metabolism and role of glutamatein mammalian brain. Prog. Neurobiol. 35, 245–296.

Felix, D., Frangi, V., 1977. Dimethoxyaporphine as an antagonist ofchemical excitation in the pigeon optic tectum. Neurosci. Lett. 4,347–350.

Fonnum, F., 1984. Glutamate: A neurotransmitter in mammalianbrain. J. Neurochem. 42, 1–11.

Fonnum, F., Henke, H., 1982. The topographical distribution ofalanine, aspartate, gamma-aminobutyric acid, glutamate, glu-tamine and glycine in the pigeon optic tectum and the effect ofretinal ablation. J. Neurochem. 38, 1130–1134.

Forloni, G., Grzanna, R., Blakely, R.D., Coyle, J.T., 1987. Colocali-sation of N-acetylaspartylglutamate in central cholinergic, nora-drenergic and serotoninergic neuron. Synapse 1, 455–460.

Fox, B.E.S., Fraser, S.E., 1987. Excitatory amino acids in the retino-tectal system of Xenopus lae6is. Soc. Neurosci. Abstr. 13, 766.

Funke, K., Eysel, U.T., Fitzgibbon, T., 1991. Retinogeniculate trans-mission by NMDA and non-NMDA receptors in the cat. BrainRes. 547, 229–238.

Gabriel, R., Straznicky, C., Wye-Dvorak, J., 1992. GABA-like im-munoreactive neurons in the retina of Bufo marinus : Evidence forthe presence of GABA-containing ganglion cells. Brain Res. 571,175–179.

Gabriel, R., Straznicky, C., 1995. Synapses of optic axons withGABA- and glutamate-containing elements in the optic tectum ofBufo marinus. J. Brain Res. 36, 329–340.

Gayoso, M.J., Bullon, M.M., Abia, J., Al-Majdalawi, A., 1989.GABA-immunoreactivity in displaced amacrine and ganglion cellsin the cat retina. Eur. Neurosci. Abstr. 78.

Glasener, G., Himstedt, W., Weiler, R., Matute, C., 1988. Putativeneurotransmitters in the retinae of three urodele species (Triturusalpestris, Salamandra salamandra, Pleurodeles waltli ). Cell TissueRes. 252, 317–328.

Hartveit, E., Heggelund, P., 1990. Neurotransmitter receptors mediat-ing excitatory input to cells in the cat lateral geniculate nucleus II.Nonlagged cells. J. Neurophysiol. 63, 1361–1372.

Heggelund, P., Hartveit, E., 1990. Neurotransmitter receptors mediat-ing excitatory input to cells in the cat lateral geniculate nucleus I.Lagged cells. J. Neurophysiol. 63, 1347–1360.

Henderson, Z., Salt, T.E., 1988. The effects of N-acetylaspartylgluta-mate like immunoreactivity in the rat somatosensory thalamus.Neuroscience 25, 899–906.

Henke, H., Schenker, T.M., Cuenod, M., 1976. Effects of retinalablation on uptake of glutamate, glycine, GABA, proline andcholine in pigeon tectum. J. Neurochem. 26, 131–134.

Hestrin, S., 1992. Developmental regulation of NMDA receptormediated synaptic currents at a central synapse. Nature 357,686–689.

Hurd, L.B., Eldred, W.D., 1989. Localisation of GABA- and GAD-like immunoreactivity in the turtle retina. Vis. Neurosci. 3, 9–20.

Ji, Z., Aas, J.-E., Laake, J., Walberg, F., Ottersen, O.P., 1991. Anelectron microscopic, immunogold analysis of glutamate and glu-tamine in terminals of rat spinocerebellar fibers. J. Comp. Neurol.307, 296–310.

Jones, H.E., Sillito, A.M., 1992. The action of the putative neuro-transmitters N-acetylaspartylglutamate and L-homocysteate in catdorsal lateral geniculate nucleus. J. Neurophysiol. 68, 663–672.

Kageyama, G.H., Meyer, R.L., 1989. Glutamate-immunoreactivity inthe retina and optic tectum of goldfish. Brain Res. 503, 118–127.

Kalloniatis, M., Fletcher, E.L., 1993. Immunocytochemical localiza-tion of the amino-acid neurotransmitters in the chicken retina. J.Comp. Neurol. 336, 174–193.

Karten, H.J., Keyser, K.T., Brecha, N.C., 1990. Biochemical andmorphological heterogeneity of retinal ganglion cells. In: Cohen,B., Bodis-Wollner, I. (Eds.), Vision and the Brain. New York:Raven Press, pp. 19–33.

Kim, Y.I., Dudek, F.E., 1991. Intracellular electrophysiological studyof the suprachiasmaticus nucleus neurons in rodents: Excitatorysynaptic mechanisms. J. Physiol. 444, 269–287.

Kisvarday, Z.F., Cowey, A., Stoerig, P., Somogyi, P., 1991. Directand indirect retinal input into degenerated dorsal lateral genicu-late nucleus after striate cortical removal in monkey: Implicationsfor residual vision. Exp. Brain Res. 86, 271–292.

Koontz, M.A., Hendrickson, A.E., Ryan, M.K., 1989. GABA-im-munoreactive synaptic plexus in the nerve fiber layer of primateretina. Vis. Neurosci. 5, 17–28.

Kvamme, E., Torgner, I.A., Roberg, B., 1991. Evidence indicatingthat pig renal phosphate-activated glutaminase has a functionally

J. Reperant et al. / Journal of Chemical Neuroanatomy 12 (1997) 267–280 279

predominant external localization in the inner mitochondrialmembrane. J. Biol. Chem. 266, 13185–13192.

Kwon, Y.H., Esguerra, M., Sur, M., 1991. NMDA and non-NMDAreceptors mediate visual geniculate nucleus. J. Neurophysiol. 66,414–428.

Langdon, R.B., Freeman, J.A., 1986. Antagonists of glutaminergicneurotransmission block retinotectal transmission in goldfish.Brain Res. 398, 169–174.

Langdon, R.B., Freeman, J.A., 1987. Pharmacology of retino-tectaltransmission in the goldfish: Effects of nicotinic ligands strychnineand kynurenic acid. J. Neurosci. 7, 760–773.

Larra, F., Droz, B., 1970. Techniques radioautographiques et leurapplication au renouvellement des constituants cellulaires. J. Mi-croscop. 9, 845–880.

Liou, S.Y., Shibata, S., Iwasaki, K., Ueki, S., 1986. Optic nervestimulation induced increase of release of [3H]glutamate and[3H]aspartate but not [3H]GABA from the suprachiasmatic nu-cleus in slices of rat hypothalamus. Brain Res. Bull. 16, 527–531.

Logan, W.J., Snyder, S.H., 1972. High-affinity uptake systems forglycine, glutamate and aspartate in synaptosomes of rat centraltissues. Brain Res., 413–431.

Marc, R.E., Liu, W.L.S., Kalloniatis, M., Raiguel, S.F., vanHaesendonck, E., 1990. Patterns of glutamate immunoreactivityin the goldfish retina. J. Neurosci. 10, 4006–4034.

Mesulam, M.M., 1982. Principles of horseradish peroxidase neurohis-tochemistry and their applications for tracing pathways-axonaltransport, enzyme histochemistry and light microscopic analysis.In: Mesulam, M.M. (ed.), Tracing Neural Connections withHorseradish Peroxidase. New York: Wiley, pp. 1–151.

Mize, R.R., Butler, G.D., 1996. Postembedding immunocytochem-istry demonstrates directly that both retinal and cortical terminalsin the cat superior colliculus are glutamate immunoreactive. J.Comp. Neurol. 371, 633–648.

Moffett, J.R., Williamson, L.C., Neale, J.H., Palkovits, M., Nam-boodiri, M.A.A., 1991. Effect of optic nerve transection on N-acetylaspartylglutamate immunoreactivity in the primary andaccessory optic projection systems in the rat. Brain Res. 538,86–94.

Molinar-Rode, R., Pasik, P., 1992. Amino acids and N-acetyl-as-partylglutamate as neurotransmitter candidates in the monkeyretinogeniculate pathways. Exp. Brain Res. 89, 40–48.

Montero, V.M., 1990. Quantitative immunogold analysis reveals highglutamate levels in synaptic terminals of retino-geniculate, cortico-geniculate and geniculo-cortical axons in the cat. Vis. Neurosci. 4,437–443.

Montero, V.M., 1994. Quantitative immunogold evidence for enrich-ment of glutamate but not aspartate in synaptic terminals ofretino-geniculate, geniculo-cortical and cortico-geniculate axons inthe cat. Vis. Neurosci. 11, 675–681.

Montero, V.M., Wenthold, R.J., 1989. Quantitative immunogoldanalysis reveals high glutamate levels in retinal and cortical synap-tic terminals in the lateral geniculate nucleus of the macaque.Neuroscience 31, 639–647.

Morino, P., Bahro, M., Cuenod, M., Streit, P., 1991. Glutamate-likeimmunoreactivity in the pigeon optic tectum and effects of retinalablation. Eur. J. Neurosci. 3, 366–378.

Mosinger, J.L., Yazulla, S., Studholme, K.M., 1986. GABA-likeimmunoreactivity in the vertebrate retina: A species comparison.Exp. Eye Res. 42, 631–644.

Murphy, P.C., Salt, T.E., Sillito, A.M., 1989. Contribution of bothNMDA and non-NMDA receptors to transmission of the visualinput to X and Y cells in the anesthetized feline dorsal lateralgeniculate nucleus. Proc. Physiol. Soc. J. Physiol. 415, 46.

Nunes-Cardozo, B., Buijs, R., van der Want, J., 1991. Glutamate-likeimmunoreactivity in retinal terminals in the nucleus of the optictract in rabbit. J. Comp. Neurol. 309, 261–270.

Ortega, F., Hennequet, L., Sarria, R., Streit, P., Grandes, P., 1995.Changes in the pattern of glutamate-like immunoreactivity in therat superior colliculus following retinal and visual cortical lesions.Neuroscience 67, 125–134.

Ottersen, O.P., 1987. Postembedding light- and electron microscopicimmunocytochemistry of amino acids: Description of a newmodel system allowing identical conditions for specificity testingand tissue processing. Exp. Brain Res. 69, 167–174.

Ottersen, O.P., 1989a. Quantitative electron microscopic immunocy-tochemistry of neuroactive amino acids. Anat. Embryol. 180,1–15.

Ottersen, O.P., 1989b. Postembedding immunogold labelling of fixedglutamate: An electron microscopic analysis of the relationshipbetween gold particle density and antigen concentration. J. Chem.Neuroanat. 2, 57–66.

Ottersen, O.P., Storm-Mathisen, J., 1985. Different neuronal localiza-tion of aspartate-like and glutamate-like immunoreactivities in thehippocampus of rat, Guinea-pig and Senegalese-baboon (Papiopapio) with a note on the distribution of g-aminobutyrate. Neuro-science 16, 589–606.

Ottersen, O.P., Storm-Mathisen, J., 1987. Immunocytochemistry visu-alization of glutamate and aspartate. In: Hicks, T.P., Lodge, D.,McLean, H. (Eds.), Excitatory Amino-acid Transmission. Neuro-logical Neurobiology. New York: Alan R. Liss, Vol. 24, pp.131–138.

Palaiologos, G., Hertz, L., Schousboe, A., 1989. Role of aspartateaminotransferase and mitochondrial dicarboxylate transport forrelease of endogenously and exogenously supplied neurotransmit-ter in glutamatergic neurons. Neurochem. Res. 14, 359–366.

Pow, D.V., Robinson, S.R., 1994. Glutamate in some retinal neuronsis derived solely from glia. Neuroscience 60, 355–366.

Raiguel, S.F., Marc, R.E., 1987. Evidence that glutamate is a neuro-transmitter in avian retinal ganglion cells. Soc. Neurosci. Abstr.USA 13, 180.

Reperant, J., Rio, J.-P., 1976. Retinal projections in Vipera aspis. Areinvestigation using light radioautographic and electron micro-scopic degeneration techniques. Brain Res. 107, 603–609.

Reperant, J., Peyrichoux, J., Rio, J.-P., 1981. Fine structure of thesuperficial layers of the viper optic tectum. A Golgi and electronmicroscopic study. J. Comp. Neurol. 199, 393–417.

Reperant, J., Rio, J.-P., Ward, R., Miceli, D., Vesselkin, N.P.,Hergueta, S., Lemire, M., 1991. Sequential events of degenerationand synaptic remodelling in the viper optic tectum followingretinal ablation. A degeneration, radioautographic and immuno-cytochemical study. J. Chem. Neuroanat. 4, 397–419.

Reperant, J., Rio, J.-P., Ward, R., Hergueta, S., Miceli, D., Lemire,M., 1992. Comparative analysis of the primary visual system ofreptiles. In: Gans, C., Ulinski, P.S., (Eds.), Biology of the Rep-tilia. Sensorimotor Integration. Chicago: University of ChicagoPress, Vol. 17, pp. 175–240.

Reubi, J.C., 1980. Comparative study of the release of glutamate andGABA newly synthesized from glutamate in various regions ofthe central nervous system. Neuroscience 5, 2145–2150.

Rio, J.-P., Reperant, J., Herbin, M., Miceli, D., 1995. Distribution ofGABA immunoreactivity in the retino-recipient layer of the viperoptic tectum. A light and electron microscope quantitative study.Anat. Embryol. 191, 251–265.

Roberts, W.A., Eaton, S.A., Salt, T.E., 1991. Excitatory amino acidreceptors mediate synaptic responses to visual stimuli in superiorcolliculus neurons of the rat. Neurosci. Lett. 129, 161–164.

Rogers, P.C., Pow, D.V., 1995. Immunocytochemical evidence for anaxonal localization of GABA in the optic nerves of rabbits, ratsand cats. Vis. Neurosci. 12, 1143–1159.

Salpeter, M.M., McHenry, F.A., 1973. Electron microscopic autora-diography. Analysis of autoradiograms. In: Koehler, J.K., (Ed.),Advanced Techniques in Biological Electron Microscopy. NewYork: Springer-Verlag, pp. 113–152.

J. Reperant et al. / Journal of Chemical Neuroanatomy 12 (1997) 267–280280

Sandberg, M., Jacobson, I., 1981. b-alanine, a possible neurotrans-mitter in the visual system. J. Neurochem. 37, 1353–1356.

Schmidt, M., 1991. Mediation of visual responses in the nucleus ofthe optic tract in cats and rats by excitatory amino acid receptors.Neurosci. Res. 12, 111–121.

Seguela, P., Geffard, M., Buijs, R.M., Le Moal, M., 1984. Antibodiesagainst g-aminobutyric acid: Specificity studies and immunocyto-chemical results. Proc. Natl. Acad. Sci. USA 81, 3888–3892.

Sekiguchi, M., Wada, K., Wenthold, R.J., 1992. N-acetylaspartylglu-tamate acts as an agonist upon homomeric NMDA receptor(NMDAR1) expressed in Xenopus oocytes. Fed. Eur. Biochem.Soc. 311, 285–289.

Sherry, D.M., Ulshafer, R.J., 1992. Neurotransmitter-specific identifi-cation and characterization of neurons in the all-cone retina ofAnolis carolinensis II: glutamate and aspartate. Vis. Neurosci. 9,313–323.

Shibata, S., Liou, S.Y., Ueki, S., 1986. Influence of excitatory aminoacid receptor antagonists and baclofen on synaptic transmissionin the optic nerve of the suprachiasmatic nucleus in slices of rathypothalamus. Neuropharmacology 25, 403–409.

Sillito, A.M., Murphy, P.C., Salt, T.E., 1990a. The contribution ofthe non-NMDA group of excitatory amino acid receptors toretinogeniculate transmission in the cat. Neuroscience 34, 273–280.

Sillito, A.M., Murphy, P.C., Salt, T.E., Moody, C.I., 1990b. Thedependence of retinogeniculate transmission in the cat on NMDAreceptors. J. Neurophysiol. 63, 347–355.

Somogyi, P., Hodgson, A.J., 1985. Antiserum to g-aminobutyric acid.III. Demonstration of GABA in Golgi-impregnated neurons andin conventional electron microscopic sections of cat striate cortex.J. Histochem. Cytochem. 33, 249–257.

Somogyi, P., Halasz, K., Somogyi, J., Storm-Mathisen, J., Ottersen,O.P., 1986. Quantification of immunogold labelling reveals enrich-ment of glutamate in mossy and parallel fiber terminals in catcerebellum. Neuroscience 19, 1045–1050.

Storm-Mathisen, J., Ottersen, O.P., 1990. Immunocytochemistry ofglutamate at the synaptic level. J. Histochem. Cytochem. 38,1733–1743.

Tieman, S.B., Cangro, C.B., Neale, J.H., 1987. N-Acetylaspartylglu-tamate immunoreactivity in neurons of the cat’s visual system.Brain Res. 420, 188–193.

Tieman, S.B., Butler, K., Neale, J.H., 1988. N-acetylaspartylgluta-mate: A neuropeptide in human visual system. J. Am. Med.Assoc. 259, 2020.

Tieman, S.B., Neale, J.H., Tieman, D.G., 1991a. N-acetylaspartylglu-tamate immunoreactivity in neurons of the monkey’s visual path-way. J. Comp. Neurol. 313, 45–64.

Tieman, S.B., Moffett, J.R., Irtenkauf, S.M., 1991b. Effect of eyeremoval on N-acetylaspartylglutamate immunoreactivity in retinaltargets of the cat. Brain Res. 562, 318–322.

Toggenburger, G., Felix, D., Cuenod, M., Henke, H., 1982. In vivorelease of endogenous b-alanine, GABA and glutamate and elec-trophysiological effect of b-alanine in pigeon optic tectum. J.Neurochem. 39, 176–183.

Tsai, G., Stauch, B.L., Vornov, J.J., Deshpande, J.K., Coyle, J.T.,1990. In vivo release of N-acetylaspartylglutamate following opticnerve stimulation. Soc. Neurosci. Abstr. USA 15, 840.

van Deusen, E.B., Meyer, R.L., 1988. Differential effects of KA/QA,NMDA and APB receptor antagonists on optic and toral afferentstimulated activity of goldfish tectal neurons in vitro. Soc. Neu-rosci. Abstr. USA 14, 96.

Villanueva, S., Orrego, F., 1988. Endogenous ligands for thequisqualate receptor: Presence in rat brain cortex synaptic vesi-cles. Brain Res. 440, 363–365.

Wang, B.-L., Larsson, L.-I., 1985. Simultaneous demonstration ofmultiple antigens by indirect fluorescence or immunogold stain-ing: Novel light and electron microscopical double and triplestaining method employing primary antibodies from the samespecies. Histochemistry 83, 47–56.

Watkins, J.C., Oliverman, H.J., 1987. Agonists and antagonists forexcitatory amino acid receptors. Trends Neurosci. 10, 265–272.

Watt, C.B., Glazebrook, P.A., Florack, V.J., 1994. Localisation ofsubstance P and GABA in retinotectal ganglion cells of the larvaltiger salamander. Vis. Neurosci. 11, 355–362.

Weiler, R., Ammermuller, J., 1986. Immunocytological localization ofserotonine in intracellularly analyzed and dye-injected ganglioncells of the turtle retina. Neurosci. Lett. 72, 147–152.

Williamson, L.C., Eagles, D.A., Brady, M.J., Moffett, J.R., AryanNamboodiri, M.A., Neale, J.H., 1991. Localisation and synapticrelease of N-acetylaspartylglutamate in the chick retina and optictectum. Eur. J. Neurosci. 3, 441–451.

Wilson, J.R., Cowey, A., Somogyi, P., 1996. GABA immunopositiveaxons in the optic nerve and optic tract of macaque monkeys. Vis.Res. 36, 1357–1363.

Yaqub, A., Eldred, W.D., 1991. Localization of aspartate-like im-munoreactivity in the retina of the turtle (Pseudemys scripta). J.Comp. Neurol. 312, 584–598.

Yu, B.C.Y., Watt, C.B., Lam, D.M.K., Fry, K.R., 1988. GABAergicganglion cells in the rabbit retina. Brain Res. 439, 376–382.

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