Intraocular Injections of Nipecotic Acid Produce a Preferential Block of Neuronal 3H-GABA Accumulation in Adult Rabbit Retina
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Intraocular Injections of Nipecotic Acid Produce a Preferential Block of Neuronal 3H-GABA Accumulation in Adult Rabbit Retina Paul Madres, Jr. and Dianna A. Redburn A procedure by which the activity of the retinal GABA uptake system can be manipulated in vivo has been developed. Intraocular injections of nipecotic acid, a proported GABA uptake blocker, were administered to adult rabbits every 48 hours for a two-week period. No behavioral or systemic changes were observed. Injections were well-tolerated with less than 10% loss of the tissue caused by physical damage or injection. Biochemical analyses demonstrated a dose-dependent inhibition of 14C-GABA uptake into retinal tissue. No effect on uptake was observed for saline-treated tissue. Autoradiographic analyses showed that in vivo treatment with nipecotic acid preferentially blocked accumulation of 3H- GABA into the amacrine cell bodies and processes in the inner plexiform layer. This treatment may be especially useful in assessing the functional significance of GABA transport in vivo. Invest Ophthal- mol Vis Sci 24:886-892, 1983 A specific, high affinity uptake system for 7-ami- rons, we wished to develop a procedure by which the nobutyric acid (GABA) has been well-characterized activity of the uptake system could be manipulated in a variety of neuronal tissues and is believed to serve in vivo. We chose to study a chronic treatment of several important functions. A portion of the GABA nipecotic acid, a proported GABA uptake blocker. transport sites is located on presynaptic terminals Our results demonstrated that multiple intraocular and serve to replenish releaseable pools of the neu- injections of nipecotic acid cause a preferential in- rotransmitter. In many instances, uptake also serves hibition of neuronal accumulation of 3H-GABA with to terminate the action of GABA after there has been no observable systemic side effects. This preparation a termination of release, thus allowing the system to may be potentially useful for assessing the functional return a resting state.1"3 significance of GABA transport systems in vivo. In the rabbit retina, GABA has been identified as a major inhibitory neurotransmitter45 and is released Materials and Methods from a population of amacrine cells to inhibit do- Adult (2-kg) New Zealand white rabbits were ob- paminergic6 and cholinergic transmission.7 Some of tained from a local breeder and housed singly for two the GABA appears to be released tonically in rabbit weeks in a light-controlled (12-hour light/dark cycle) retina,8 and thus the role of the GABA uptake system room. Each animal was maintained on commercial may be especially important. The concentration of rabbit chow. Fresh water was given daily. For ap- GABA in the synaptic cleft reflects the balance be- proximately 30 minutes each day, animals were han- tween release and uptake, and thus the activity of dled gently to increase environmental interaction. the uptake system can directly influence the duration and size of the postsynaptic response. Injection Because the uptake system appears to be a critical component of normal function in GABA-ergic neu- Each animal was anesthetized every 48 hours with an acepromazine-ketamine-rompun cocktail (0.7, 21.4, and 4.3 mg/kg, respectively) via an intramus- From the Department of Neurobiology and Anatomy, The Uni- versity of Texas Medical School, Houston, Texas. cular injection in the hind limb. Thirty minutes post- Supported by the National Institutes of Health grants 5 F32 EY injection, the orbit of the eye to receive nipecotic acid 05475-02 (P.M.), EY 0-1655 (DAR), and RCDA 1K04 EY 00088 treatment was bathed with 1% lidocaine hydrochlo- (DAR). ride. The eyelids were restrained by a lid speculum. Submitted for publication July 23, 1982. Using a 0.12 Castra Viejo forceps, the superior rectus Reprint requests: Paul Madtes, Jr., PhD, Department of Neu- robiology and Anatomy, The University of Texas Medical School, muscle was grasped gently to stabilize the globe. A Houston, TX 77025. 27 gauge X 0.5 in gauge needle was inserted caudal 0146-0404/83/0700/886/$ 1.15 © Association for Research in Vision and Ophthalmology 886 Downloaded from iovs.arvojournals.org on 09/25/2021 No. 7 NIPECOTIC ACID CLOCK OF NEURONAL 3H-GADA UPTAKE IN VIVO / Modres and Redburn 887 to the muscle insertion, thus assuring that the lens buffer, 221.3 mM choline Cl was added in place of was not pierced during injection. One hundred mi- NaCl). AOAA was included in order to inhibit trans- croliters of one of the following were injected: saline, amination of GABA. 3H-dopamine (final concentra- 1 raM, 10 mM, or 100 raM nipecotic acid (Sigma) tion = 1 /iM; 20.1 Ci/mmole, Amersham) and 14C- dissolved in saline. The non-injected eye served as GABA (final concentration = 10 nM; 267 Ci/mmole, the control for each experimental animal. The orbit Amersham) were added and the samples were incu- was rinsed with gentamicin sulfate to reduce infection bated for 10 min. The samples then were poured onto after injection. The final concentration in the vitreal glass fiber niters (Whatman GF/A) and rinsed three chamber was calculated to be approximately a 10- times with the appropriate buffer (sodium-free or so- fold dilution of the injection solution. dium-containing). The filters containing the samples were removed, soaked in 1% SDS and 20 mM EDTA Observation solution overnight, and counted in a toluene-based solution with Triton X-100, by double label liquid A daily record of water and food intake, morning scintillation counting. activity level, feces appearance, response to sound Since specific uptake of GABA has been shown to and touch, and any irregular behavior was main- be sodium- and temperature-dependent, results were tained for each animal. Three hours after anesthesia, calculated by correcting for background and protein each animal was tested for pupillary light reflex and content. Specific uptake of 3H-GABA in retinal ho- contralateral closure. Each animal also was tested for mogenates was determined by subtracting nonspecific visual reflexes on days during which they did not re- uptake (the amount of uptake at 0 C in sodium-free ceive an injection. The pupillary light reflex was buffer) from total uptake (the amount of uptake at checked by placing the animal in total darkness and, 37 C in sodium-containing buffer) and dividing by after allowing acclamation, flashing a penlight source the amount of protein in each sample. The data are in one eye. Contralateral closure was followed under expressed as percentage ratios of experimental to con- identical conditions, shining a light in one eye and trol values. observing the change in pupil diameter in the con- tralateral eye. Autoradiography Retinal Isolation The second retinal piece was bisected and both In order to minimize the influence of acute effects halves were incubated at 37 C for 15 min in an ox- of nipecotic acid on this chronic study, the animals ygenated (95% O2/5% CO2) Ringer's buffer (118.3 were killed by decapitation under normal laboratory mM NaCl, 25.2 mM NaHCO3, 4.7 mM KC1, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and illumination 24 hours after the final injection, at 3 which time most of the 3H-nipecotic acid is cleared 11.1 mM glucose), pH 7.4 containing 10 nM. H- from the retina (Madtes, unpublished observations). GABA (57 Ci/mmole, Amersham). One-half then The eyes were enucleated, hemisected, and the vit- was fixed immediately in 2.5% glutaraldehyde in 0.05 reous discarded. Each eyecup then was divided into M sodium cocadylate buffer, pH 7.2. The remaining two pieces with a no. 21 scalpel blade for analysis. half was incubated an additional 30 min in fresh buffer containing no GABA, fixed for 30 min at room Each injected globe was inspected for signs of phys- temperature, and stored overnight at 15 C. Each sam- ical damage such as scleral scars and infection. Sim- ple then was post-fixed in 1% osmium tetroxide for ilarly, retinas were scrutinized for scarring, detach- 1 1/2 hr at 4 C. After osmication, the samples were ment, or hemorrhage. Any defective sample was dis- dehydrated in a graded alcohol series followed by carded. propylene oxide and embedded in Epon epoxy resin for sectioning. Thick sections (1 n) were cut with glass Biochemical Analysis knives and placed on glass slides cleaned with nitric The first retinal piece was homogenized in 10-fold acid. Representative sections were stained with Ri- 0.32 M sucrose with a Teflon® pestle in a glass ho- chardson's stain and assessed for morphologic integ- mogenizer tube (Thomas). Homogenate samples (ap- rity. The remaining slides were coated with Kodak® proximately 0.2 mg per tube) were incubated for 6 NTB-2 emulsion, allowed to dry, and placed in light- min in either a sodium-free (0 C) or sodium-con- tight boxes that contained dessicant for 4 weeks. The taining (37 C) modified HEPES buffer (20 mM sections then were developed in 1:1 Dektol for 2 min, HEPES, 150 mM NaCl, 6.2 mM KC1, 1.2 mM rinsed, fixed in Kodak® Fixer for 5 min, washed, and dried at 60 C. Some sections were stained with to- MgSO4, 3 mM CaCl2, 10 mM glucose, and 1 mM amino-oxyacetic acid (AOAA), pH 7.4; for Na-free luidine blue for comparison with unstained sections. Downloaded from iovs.arvojournals.org on 09/25/2021 888 INVESTIGATIVE OPHTHALMOLOGY 6 VJSUAL SCIENCE / July 1983 Vol. 24 Fig. I. Lack of effect of chronic nipecotic acid treatment on the morphology of adult rabbit retina. Adult rabbits were given intraocular injections of nipecotic acid (final concentrations = 10 mM) every 48 hr for a 2-week period.