Identification of Putative Neurotransmitters in the Lizard Parietal Eye

Gusrav A. Engbrerson* and Barbara A.

The retina of the parietal eye of lizards is a simple vertebrate retina that may be useful in studies of basic principles of information processing in visual systems. The chemical substances which mediate the cell-to-cell communication of this eye are not known. As a step in attempting to understand the cellular basis for visual information processing the authors have studied the ability of the parietal eye to synthesize conventional neurotransmitter substances. The eyes were incubated in radiolabeled precursors, then an extract of the tissue was subjected to high-voltage paper electrophoresis. The results of this study indicate that 7-amino butyric acid (GABA) was synthesized from glutamic acid, acetylcholine (ACh) was synthesized from choline and serotonin (5-HT) was synthesized from tryptophan. Endogenous 5-HT was localized at sites outside the sensory epithelium by immunohis- tochemical means. Autoradiography following an incubation with 3H-GABA revealed uptake by lens cells and probably both glia and neurons. The authors conclude that GABA and ACh may be involved in the processing of visual information within the parietal eye but that 5-HT probably is not. Invest Ophthalmol Vis Sci 26:670-678, 1985

The parietal eye is a highly organized photoreceptive principles of information processing by vertebrate organ found on the dorsal midline in many lizard visual systems. species. The neural circuitry of this retina is simple The parietal eye appears to extract and send to the compared to that of other vertebrate retinas. Parietal brain information concerning the wavelength and eye photoreceptors send basal processes into a single intensity of light which reaches its retina.8"10 The plexiform layer where they make synaptic contact specific cellular connectivity which constitutes the with other photoreceptors, with the dendritic arbors framework for generating this neural output is not and somata of ganglion cells,1 and perhaps with a known. Nor do we know what biochemical substances population of interneurons2"4 (Fig. 1). This is in might be utilized to carry messages within that frame- contrast to the two plexiform layers and the five work. How the parietal eye processes visual infor- principle cell types of lateral eye retinas. mation will be more easily understood if the sub- Despite the relative simplicity of its structure, the stances that subserve neurotransmission can be deter- parietal eye is similar functionally to other vertebrate mined. If we know what substances the neurons in retinas in that it processes light information into this eye use to communicate with one another we nerve impulses that course to the brain.5"7 The struc- can better predict specific interactions of the neural tural simplicity of the parietal eye may, therefore, circuitry. Also, with such knowledge we can formulate provide a suitable model in which to study general experiments utilizing pharmacologic agents to sys- tematically probe elements of that circuitry and reveal a cellular basis for visual information processing. From the Institute for Sensory Research,* Syracuse University, The purpose of this study is to identify possible Syracuse, New York, and the Laboratory of Vision Research,! neurotransmitter substances used by cells in the pa- National Eye Institute, National Institutes of Health, Bethesda, rietal eye. Little information is available on this Maryland. 11 Presented in part at the ARVO Meeting, Sarasota, Florida, May subject. Quay detected in the parietal eye the activity 1983. of hydroxyindole-O-methyltransferase (HIOMT), the Supported in part by NIH grant EY-03359. enzyme that converts N-acetylserotonin to melatonin. t Present address: C.V. Whitney Laboratory, University of Florida, Results of other studies on the identification of possible Rt. 1, Box 121, St. Augustine, FL 32086. transmitter substances in this retina are all negative Submitted for publication: June 22, 1984. and include the following: the failure to detect HIOMT Reprint requests: Gustav A. Engbretson, PhD, Institute for 12 Sensory Research, Syracuse University, Syracuse, NY 13210. activity ; the inability to take up 5-hydroxytrypto-

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phan, the immediate precursor of serotonin (5- HT)1314; the absence of formaldehyde-induced histofluorescence indicative of 5-HT or norepinephrine (NE)13; and the lack of acetylcholinesterase (AChE) staining.15 Additionally, when 5-HT or NE was added to the medium bathing a parietal eye, no action potentials were elicited in afferent fibers of the parietal 5 16 nerve. Finally, Engbretson et al detected substance PrL P-like immunoreactivity in processes coursing in the plexiform layer, but these appeared to be efferent fibers as no labeled somata were found, and lesions of the parietal nerve eliminated the immunoreactivity. We undertook to determine if mechanisms for the synthesis of several conventional neurotransmitter substances are present in the parietal eye. Specifically, PL we examined this simple visual organ for its ability to synthesize catecholeamines, 5-HT, 7-aminobutyric acid (GABA), acetylcholine (ACh), and octopamine from radiolabeled precursors. GCL

Materials and Methods Fig. 1. A diagram of the retinal structure of the lizard parietal Synthesis of Neurotransmitter Candidates eye. Light enters the eye from above. The general morphology of The treatment and use of animals in this study each cell type is taken from Golgi-stained tissue. A glial cell (Gl) may have its soma in either the photoreceptor layer (PrL) or in the adhered to the ARVO Resolution on the Use of ganglion cell layer (GCL). Glial cells commonly contain melanin Animals in Research. Both side-blotched lizards (Uta pigment granules. Photoreceptor cells (Pr) send basal processes into stansburiana) and yucca night lizards (Xantusia vigilis) the plexiform layer (PL) where they contact other Pr's, the dendritic were used. They were killed by decapitation and the arbors of the ganglion cells (G), and perhaps interneurons. The parietal eyes, with a minimal amount of surrounding unidentified cell (?) that appears at the junction of the PrL and PL may be such an interneuron. tissue, were immediately dissected free. Two parietal eyes from a single species were pooled for each sample. The samples were incubated in 50 /x\ of beled precursors. Briefly, after 3 hr each sample was saline for 3 hr in the dark at room temperature. The washed in a saline solution, placed in 1 M formic incubation medium consisted of a saline solution acid in acetone, freeze disrupted, then extracted over- known to maintain the preparation5 in which was night. The following morning the samples were dried dissolved a previously dried (under vacuum) radio- under vacuum and resuspended in 20 /A of electro- labeled precursor to a specific neurotransmitter group. phoresis buffer (pH 1.9) containing nonradioactive To test for ACh production, the medium contained markers (2-5 n% each) for the precursors and antici- 3H-choline chloride (New England Nuclear [NEN]; pated products. The samples were subjected to high- Boston, MA, 80 Ci/mmol). For GABA, we used 3H- voltage paper electrophoresis for 1.5 hr at 6 KV. glutamic acid (NEN, 42.3 Ci/mmol); for the cate- After the paper was dried the marker compounds choleamines, 3H-tyrosine (NEN, 28 Ci/mmol); for were visualized and the electrophoretograms were the indoleamines, 3H-tryptophan (NEN, 8 Ci/mmol); processed for liquid scintillation counting as described 18 and for octopamine, 3H-tyramine (NEN, 8.8 Ci/ by Battelle. mmol). The precursors were used at a concentration of either 20 ^Ci/ml or 1 mCi/ml. Eserine sulfate (15 Verification of Synthesized Products jug/ml) was added as an AChE inhibitor in incubations Acetylcholine synthesis was verified by hydrolysis with choline chloride and pargyline (18 jug/ml) was with specific AChE from Electrophorus (Sigma; St. included as a monoamine oxidase inhibitor in the Louis, MO) and subsequent electrophoresis.17 Three incubations with tyrosine. parietal eyes incubated in 3H-choline chloride for 3.5 Procedures similar to those described in detail by hr were extracted with electrophoresis buffer. The Hildebrand et al17 were employed to extract and extract was divided and both halves were dried under separate the products synthesized from the radiola- vacuum. One half of the extract was resuspended in

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and pargyline (Sigma) to enhance serotonin levels in 3. the nervous system. In control experiments the 5-HT antibody was preabsorbed with its antigen, a bovine serum albumin conjugated to 5-HT, generously sup- plied by Immunonuclear Corporation. 2.

Results

The results described below are the same for both species except that 5-HT synthesis and localization 10 15 20 experiments were not carried out in X. vigilis. DISTANCE (cm)

Fig. 2. A histogram of radioactivity (DPM's) measured along a Choline portion of the electrophoretogram obtained from the acidic extract of two parietal eyes incubated with 3H-choline chloride. The labeled Figure 2 illustrates the distribution of radioactivity ellipsoidal spots delineate the boundaries of regions which stained in a portion of the electrophoretogram obtained positively for the nonradioactive ACh and choline standards added to the extract prior to electrophoresis. Note the colocalization of following electrophoresis of an acid extract of parietal 3 peaks of radioactivity and the standards. The origin of this run was eyes incubated with H-choline chloride. There are 45 cm to the left of the first bin. two peaks of radioactivity, one of which comigrated with the ACh standard. The other peak of radioactivity, which comigrated with the choline standard, is 3 0.1 M phosphate buffer containing AChE for 30 min; the H-choline taken up from the bath and retained the other half was treated similarly but without by the tissue. In Figure 2, and in succeeding histo- AChE. The two samples were then subjected to high- grams, the scale of ordinates represents raw counts voltage electrophoresis as described above. of disintegrations per minute (DPMs) for a single We verified GABA synthesis by electrophoresis experiment. We used two parietal eyes for each followed by ascending paper chromatography in two experiment. The experiments were replicated twice and yielded similar results except in the case of different solvent systems (n-butanol:acetic acid:H2O 18 tyrosine (see below). = 12:3:5, and n-butanol:pyridine:H2O = 1:1:1). We verified that the radioactive product which co- Localization of Cells which Accumulate GABA migrated with the ACh standard was indeed ACh by hydrolyzing the extract of a 3H-choline incubation Cells that take up GABA were localized by light with specific AChE and comparing the electropho- microscopic autoradiography. Parietal eyes were in- retograms of that sample and a control sample which cubated for 20 min in 3H-GABA, then fixed overnight received no AChE. Figure 3 shows the distribution at room temperature in 0.1 M cacodylate buffer (pH of radioactivity in the electrophoretograms obtained 7.4) containing 2% glutaraldehyde. The eyes were from the hydrolyzed and control samples. Treatment washed for 4 hr in cacodylate buffer, dehydrated in with AChE resulted in a decrease by 89% of the graded ethanols, and embedded in plastic. Sections radioactivity comigrating with the ACh standard. 1 urn thick were cut and coated with NTB2 emulsion These results suggest that most of the radioactive (Kodak; Rochester, NY). They were developed after substance which comigrated with the ACh standard an exposure period of 6 weeks, counterstained with was acetylcholine newly synthesized from 3H-choline. toluidine blue, and photographed. Glutamic Acid

Localization of Endogenous 5-HT-like 3 Immunoreactivity Our incubation of parietal eyes with H-glutamic acid resulted in the synthesis of a radioactive sub- We used a commercially available antibody to 5- stance, which comigrated with GABA in electropho- HT (Immunonuclear; Stillwater, MN) and both FITC- resis (Fig. 4). This same radioactive substance co- conjugated secondary antibody and peroxidase-anti- migrated with GABA when the electrophoresis was peroxidase (PAP) methods as previously described16 followed by ascending chromatography in two differ- to localize 5-HT-immunoreactive compounds. In ent solvent systems (Fig. 5). Thus the radioactive some experiments animals were injected the day compound associated with the GABA standard was before fixation with 100 mg/kg each of L-tryptophan most likely newly synthesized GABA.

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5 - 600. Without AChE

4. Choline 400.

0. Q 3 . 200.

10 15 20 DISTANCE (cm) Q 15 5 - b 5 10 With AChE DISTANCE (cm) Fig. 4. Histogram of radioactivity (DPMs) measured along a portion of the electrophoretogram obtained from the acidic extract J of two parietal eyes incubated with 3H-glutamic acid. The labeled 4 - ( ACh ) Choline ) spot indicates the boundaries of regions that stained positively for the nonradioactive GABA standard added to the extract. The — stained glutamic acid standard and the origin of this run were to the left of the first bin by 21 and 42 cm, respectively. i—i 3 .

Autoradiography of parietal eyes incubated in 3H- "TiT ~ ~hh GABA revealed radioactivity in three cell populations. 10 15 20 DISTANCE (cm) Rather heavy labeling was found over lens cells (Fig. 6a). In addition, somata in the photoreceptor layer Fig. 3. Histograms of radioactivity (DPMs) measured along (PrL) and the ganglion cell layer (GCL) and narrow portions of electrophoretograms obtained from the acidic extract of three parietal eyes incubated with 3H-choline chloride. The processes traversing the sensory epithelium were la- labeled spots indicate the boundaries of regions that stained positively beled by uptake of the radioactive GABA. Most of for the nonradioactive ACh and choline standards added to the the narrow processes (Fig. 6a) are undoubtedly those extract prior to electrophoresis. a, No AChE was added to this half of glial cells. The parietal eye glial cells commonly of the extract, b, Specific AChE from Elect rophorus was added to contain numerous melanin granules, and we found this sample and only 11% of the expected radioactivity remains in the ACh spot compared with sample a. Origins of these runs were that processes with such granules were heavily labeled well to the left of the first bin. in the autoradiographs (Fig. 6b). The labeled somata

4. 4. Butanol Pyridine : H Fig. 5. Histograms of radio- 2 Butanol : Acetic Acid : activity (DPMs) measured along portions of chromatograms from (JGAI/T) ascending paper chromatography. The origins were the loca- 3. — 3. tions of GABA after electrophoresis as in Figure 4. The labeled Q spots indicate the boundaries of O O the stains for the nonradioactive GABA standards added prior to 2. 2. electrophoresis. Left, Chro- 1 matographic solvent system was n-butanol:pyridine:H2O = 1:1:1. Right, Solvent system was n-butanol:acetic acid:H2O = 12:3:5. 1 10 10 DISTANCE (cm)

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Fig. 6. Autoradiographs of a l-/tm section of a parietal eye of Uta stansburiana incubated in 3H-GABA. a, Heavy autoradiographic label is over cells of the lens (L) and radially oriented glial processes (long arrow). Short arrow indicates a cell which is similar to the cells in which 5-HT-like immunoreactivity was localized (eg, Fig. 8a). Calibration line = 50 urn. b, Silver grains label glial cells in the ganglion cell layer (long arrow) and in the photoreceptor layer (short arrow). Pholorecep- tors (P) and ganglion cells (G) are not labeled. A population of cells at the junction of the photoreceptor and plexiform layers was labeled (arrowhead). These cells are unique and may be neurons (see text). Calibration mi?mi line = 20 ^m.

in the GCL (Fig. 6b) are also glial. The neurons granules characteristic of the parietal eye glial cells. (ganglion cells) of the GCL exhibited no evidence of In cross-sections near the middle of the eye there 3H-GABA uptake. were up to five such cells and they appeared to be In the PrL only a few somata were labeled by 3H- somewhat regularly spaced along the junction of the GABA uptake (Fig. 6b). Some of the labeled cells PrL and the plexiform layers (PL). Using the intensity were undoubtedly glial. Some, however, appear not of toluidine blue staining and the presence of pigment to be glial. These cells stained lighter with toluidine granules as criteria for separating the two populations, blue than did the glial cells of both the GCL and the an analysis of variance revealed no significant differ- PrL. Also, they did not contain the melanin pigment ence (F,28 = 0.495) between the mean cross-sectional

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diameters of the glial cell profiles (5.9 ^m) and these 5_ other labeled profiles (6.3 ^m). The nonglial profiles differed from the mean diameters of the photoreceptors (7.7 ixm) (Fi)40 = 7.029) and the ganglion cells (11.3 /um) (F,,7 = 28.129) in the same section. It appears then, that GABA is synthesized in this eye 4_ C 5-HT ) 5 and that lens cells, glial cells and a small, as yet Q. unidentified, population of cells can take up GABA. Q

Tryptophan 3j

Figure 7 illustrates the results of the incubation of parietal eyes in 3H-tryptophan. A peak of radioactivity comigrated with the 5-HT standard suggesting that 5-HT was synthesized by the parietal eye. When we 2. attempted to localize 5-HT in our preparation using immunocytochemical methods we found no evidence of its presence within the margins of the parietal eye. 10 15 We did, however, find 5-HT-like immunoreactivity DISTANCE (cm) sparsely distributed around the margin of the eye Fig. 7. A histogram of radioactivity (DPMs) measured along a (Figs. 8a-b) and occasionally in a plexus of fine (<0.5 portion of the electrophoretogram obtained from the acidic extract ^m diameter) fibers at the junction of the cornea-like of two parietal eyes incubated with 3H-tryptophan. The labeled dermis and the lens (Fig. 8c). Note that the immu- spots indicate the boundaries of regions which stained positively noreactivity is localized to profiles that are outside for the nonradioactive tryptophan and 5-HT standards added to the extract prior to electrophoresis. The origin of the run was 14 the boundaries of the sensory epithelium. These cells cm to the left of the first bin. were not labeled in control experiments in which the primary antibody was preabsorbed with antigen (Fig. 8d). The distribution and location of some of the 5- Tyramine HT-immunoreactive cells along the lateral and ventral margins of the eye are similar to those of what appear We found no evidence for octopamine synthesis to be endothelial or meningeal cells that are scattered from tyramine. along that margin (eg, Fig. 6a, short arrow). In other sections we observed 5-HT-like immunoreactivity in Discussion neuron-like processes along the external margin, but these processes remained outside the capsule of the Three substances, GABA, ACh, and 5-HT, which eye throughout their observable course (Fig. 8b). are generally accepted as neurotransmitters in other Attempts to follow the immunoreactive fibers of systems, are synthesized by the parietal eye. Of these the dermis-lens junction (Fig. 8c) from that immediate three possible neurotransmitters, 5-HT was localized area were unsuccessful. None of the labeled fibers in sites outside the sensory portion of the parietal eye 1314 appeared to leave the plexus. Nor did we see any proper (Figs. 8a-b). Meiniel reported that the immunoreactive somata in the region of the plexus parietal eye does not incorporate the 5-HT precursor of fibers. Taken together, the above results imply that 5-hydroxytryptophan and that no histofluorescence 5-HT was synthesized by cells around, but not in, indicative of 5-HT could be detected in eyes processed the parietal eye. with the Falck-Hillarp method. One would expect to observe these phenomena if serotonergic cells were Tyrosine present and, in fact, she did observe both phenomena in pineal glands from the same animals. Despite our There was no clear evidence of catecholeamine results suggesting that 5-HT is synthesized by the synthesis from tyrosine. A small peak of radioactivity parietal eye, the evidence from Meiniel's laboratory comigrated with a norepinephrine standard in one and our localization of 5-HT-like immunoreactivity experiment but the result could not be duplicated so force us to conclude that 5-HT is not involved in the it remains of questionable significance. We found no processing of visual information within the parietal evidence of dopamine synthesis. eye. The remote possibility remains that 5-HT is so

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Fig. 8. Photomicrographs of 5-HT-like immunofluorescence around the margin, but not within, the parietal eye of Uta stansburiana. a, Soma-like staining (arrow), b, Fine fibers resembling neuronal processes (arrow). These fibers remained outside the sensory epithelium of the eye for their observable course, c, A plexus of fine labeled fibers (arrow) at the interface between the lens (L) and dermis. d, A control section treated with an antibody to 5-HT, which had been preabsorbed with its antigen (X260).

involved but that the levels of 5-HT present in this parietal eye. GABA is synthesized in the eye from its eye are below the detection limits of the methods precursor glutamic acid (Figs. 4 and 5). Also, GABA used. This is most likely not the case because our is accumulated by cells of the eye. Figure 6 shows attempts to elevate the endogenous 5-HT level with cells of the lens and cells in both the PrL and the tryptophan and pargyline failed to reveal 5-HT-like GCL which are autoradiographicully labeled after immunoreactivity within the parietal eye but did incubation in 3H-GABA. In the lateral eye retina,19 increase the intensity of labeling of the cells around as well as elsewhere in the nervous system,20 both the eye. glial cells and neurons accumulate GABA from the Engbretson and Lent5 demonstrated that 5-HT can extracellular milieu. In the retina of the parietal eye stimulate action potentials in efferent fibers to the glial cells and perhaps some neurons also accumulate parietal eye and that the photoresponsiveness of the GABA. eye is affected by such efferent information. Given In the GCL only glial cells took up GABA; no these results it seems logical to conclude that 5-HT neurons were labeled. The somata of these glial cells is not directly involved in the peripheral coding of are located in the GCL at the proximal margin of neural impulses in response to light but may be the eye and stain quite darkly. They extend processes involved in the more centrally controlled modulation across the PL and through the PrL (Fig. 1) in a of peripheral coding. manner analogous to the Muller cells of lateral eye The possibility remains that GABA and/or ACh retinas. These same processes contain melanin gran- are involved in visual information processing by the ules and we found these melanin granule-laden pro-

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cesses ensheathing the photoreceptors to be heavily ACh was synthesized from its precursor choline labeled in our autoradiographs (Figs. 6a-b). (Fig. 2). Although we have not yet attempted to Only a small number of somata in the PrL took identify the site of ACh synthesis, photoreceptors up 3H-GABA and they were not photoreceptors. and/or ganglion cells seem the most likely candidates. Some of these autoradiographically labeled somata Histochemical and immunocytochemical localization were those of glial cells. Others, however, appeared of the ACh-synthesizing enzyme, choline acetyltrans- different from the glial cells. Though they were similar ferase, would help determine which cells are likely to in cross-sectional diameter to the positively identifiable be cholinergic. glial cells, these other labeled cells were consistently In summary, 5-HT is most likely not involved in more lightly stained by toluidine blue and they did visual information processing within the parietal eye. not contain pigment granules. In addition, they are The synthesis of GABA and ACh and the uptake of consistently located at the interface between the PrL 3H-GABA by a small population of cells in the and the PL whereas the somata of the labeled glial parietal eye makes these two compounds candidates cells were seen at many levels in the PrL. We believe for neurotransmitter substances there. We wish to that the profiles in the PrL which labeled with 3H- emphasize that the requirements for establishing these GABA constitute two populations of cells, one glial substances as neurotransmitters in this visual system and the other nonglial. In l-/im sections, these nonglial have not been met. Rather, we have established a cells resemble the unidentified cell type illustrated in basis for investigating further whether they play a Figure 1. They correspond in position to the neurons role in information processing. If they prove to act described from electron micrographs by Jenison and as neurotransmitters in this simple eye, the well his co-workers.23 The PrL neurons which they de- known pharmacology of cholinergic and GABA-ergic scribed varied in distribution and number with animal neurotransmission will be valuable in understanding species but were always at the PrL-PL boundary and the manner in which they function to produce the were regularly spaced. This small group of cells that neural code that is the output of the parietal eye. we find in the PrL seems unique in structure, location, Key words: parietal eye, neurotransmitters, 7-amino butyric distribution, and by the fact that they accumulate acid, acetylcholine, serotonin GABA from their surroundings. We have shown that something in the parietal eye Acknowledgments did synthesize GABA from its normal precursor The authors thank Kathy J. Anderson for her technical glutamic acid (Figs. 4 and 5). There is no convincing assistance and Dr. Steven C. Chamberlain for his helpful evidence that glial cells synthesize GABA. The avail- comments and assistance with the manuscript. able information indicates that glial cells normally break down the GABA which they take up from their References surroundings. Neurons also take up GABA and they 1. Petit A: Ultrastructure de la retine de Toeil parietal d'un are generally accepted to be the site of GABA syn- Lacertilian, Anguis fragilis. Z Zellforsch 92:70, 1968. 2. Jenison G and Nolte J: The fine structure of the parietal retinas thesis. Let us make the reasonable assumption that ofAnolis carolinensis and Iguana iguana. Cell Tissue Res 199: glia and neurons in the parietal eye are similar 235, 1979. biochemically to the glia and neurons of other com- 3. Jenison GL, Eldred WD, and Nolte J: A second class of ponents of the nervous system. Given this assumption, neurons within the retinas of the parietal eyes of Anolis our synthesis experiments suggest that there are neu- carolinensis and Iguana iguana. Brain Res 168:615, 1979. 4. Engbretson GA: Neuronal connectivity of parietal eye ganglion rons in the parietal eye which synthesize GABA. Any cells. ARVO Abstracts. Invest Ophthalmol Vis Sci 25(Suppl): such neurons should also accumulate GABA from 64, 1984. their surroundings. The cells along the boundary 5. Engbretson GA and CM Lent: Parietal eye of the lizard: between the photoreceptor and plexiform layers, which neuronal responses and feedback from the pineal gland. Proc accumulated 3H-GABA, constitute the only possible Natl Acad Sci USA 73:654, 1976. 6. Engbretson GA, Reiner A, and Brecha N: Habenular asymmetry population of such neurons. The possibility remains and the central connections of the parietal eye of the lizard. J that the GABA synthesis which we found was not Comp Neurol 198:155, 1981. neuronal or glial. If such is the case, then the locus 7. Korf H-W and Wagner U: Nervous connections of the parietal of synthesis might be the lens cells that also labeled eye in adult Lacerta s. sicula Rafinesque as demonstrated by autoradiographically. At this point we simply cannot the anterograde and retrograde transport of horseradish peroxidase. Cell Tissue Res 219:567, 1981. distinguish between the two alternatives but we are 8. Dodt E and Scherer E: Photic responses from the parietal eye encouraged to pursue the possibility that a type of of the lizard Lacerta sicula campeslris (DeBetta). Vision Res GABA-ergic neuron exists in this eye. 8:61, 1968.

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9. Hamasaki DI: Spectral sensitivity of the parietal eye of the 15. Wake K, Ueck M, and Oksche A: Acetylcholinesterase-con- green iguana. Vision Res 8:591, 1969. taining nerve cells in the pineal complex and subcommissural 10. Jenison G and Nolte J: An ultraviolet-sensitive mechanism in area of the frogs Rana ridibunda and Rana esculenta. Cell the parietal eye. Brain Res 194:506, 1980. Tissue Res 154:423, 1974. 11. Quay WB: Retinal and pineal hydroxyindole-O-methyl trans- 16. Engbretson GA, Brecha N, and Reiner A: Substance P-like ferase activity in vertebrates. Life Sci 4:983, 1965. immunoreactivity in the parietal eye visual system of the lizard 12. Joss JMP: A rhythm of hydroxyindole-O-methyltransferase Via stansburiana. Cell Tissue Res 227:543, 1982. (HIOMT) activity in the scincid lizard, Lampropholas guichen- 17. Hildebrand JG, Barker EH, and Kravitz EA: Screening for oti. Gen Comp Endocrinol 36:521, 1978. neurotransmitters: a rapid radiochemical procedure. J Neurobiol 13. Meiniel A: L'epiphyse et l'oeil parietal de l'embryon de Lacerta 2:231, 1971. vivipara J: recherche qualitative des monoamines en fluorescence 18. Battelle BA: Neurotransmitter candidates in the visual system ultraviolette et incorporation de 5-hydroxytryptophane-3H (=5- of Limulus polyphemus: synthesis and distribution of octopa- HTP-3H) au niveau des photorecepteurs rudimentaires secre- mine. Vision Res 20:911, 1980. toires. Arch Anat Histol Embryol 56:111, 1973. 19. Ehinger B: Glial and neuronal uptake of GABA, glutamic acid, 14. Meiniel A, Collin JP, and Roux M: Pineale et 3eme oeil de glutamine and glutathione in the rabbit retina. Exp Eye Res l'embryon de Lacerta vivipara: etude qualitative et quantitative, 25:221, 1977. en microscopie photonique de l'incorporation de 5-hydroxy- 20. Hokfelt T and Ljungdahl A: Cellular localization of labeled tryptophanne-3H, au cours de l'ontogenese. J Neural Transm gamma-aminobutyric acid (3H-GABA) in rat cerebral cortex: 36:249, 1975. an autoradiographic study. Brain Res 22:391, 1970.

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