Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8057-8062, July 1996 Neurobiology Starburst amacrine cells change from spiking to nonspiking neurons during retinal development Z. JIMMY ZHOU* AND GORDON L. FAIN Departments of Physiological Science and Ophthalmology, 3836 Life Science Building, University of California, Los Angles, CA 90024-1527 Communicated by Carla J. Shatz, University of California, Berkeley, CA, April 22, 1996 (received for review February 23, 1996) ABSTRACT The membrane excitability of cholinergic autonomously, it has been thought that spike propagation in (starburst) amacrine cells was studied in the rabbit retina the starburst cell would be inappropriate. during postnatal development. Whole-cell patch-clamp re- Experimental results on starburst cell excitability have so far cordings were made from 110 displaced starburst cells in a remained inconsistent. Whereas previous studies using intra- thin retinal slice preparation of rabbits between postnatal cellular (9) and extracellular (24) recordings have suggested days P1 and P56 old. We report that displaced starburst cells spikes from starburst cells in the adult rabbit, recent patch- undergo a dramatic transition from spiking to nonspiking, clamp recordings have not been able to find any evidence for caused by a loss of voltage-gated Na currents. This change in regenerative activities in these cells (25, 26). Given the impor- membrane excitability occurred just after eye opening (P10), tance of this subject to our understanding of the functional role such that all of the starburst cells tested before eye opening of the starburst amacrine and other medium-field amacrine cell had conspicuous tetrodotoxin-sensitive Na currents and ac- types, it is important to determine if the starburst amacrine cell tion potentials, but none tested after the first 3 postnatal is indeed capable of generating somatic action potentials. weeks had detectable Na currents or spikes. Our results In addition to a possible role for action potentials in signal suggest that starburst cells use action potentials transiently processing, it seems possible that membrane excitability could during development and probably play a functional role in also play a role in the developing retina (27-29). Amacrine visual development. These cells then cease to spike as the cells and ganglion cells are the first cell types to differentiate retina matures, presumably consistent with their role in visual during development in rabbit (30, 31), and displaced starburst processing in the mature retina. cells in the rabbit retina already attain their adult-like dendritic morphology at birth (32). It is known that ganglion cells in the In the vertebrate retina, synaptic transmission by most inter- developing retina fire correlated spontaneous action potentials neurons is mediated by nonspiking, graded membrane poten- that are believed to play an important role in establishing tials (1). Action potentials so far have been found in only a precise neuronal connectivity in the visual pathway (33-35). relatively small population of retinal interneurons, predomi- There is some evidence that amacrine cells (35) and in nantly wide-field amacrine cell types (e.g., refs. 2-10). Many of particular starburst amacrines (52) may participate in gener- the nonspiking retinal interneurons are believed to be elect- ating spontaneous activity. rotonically compact and thus do not require action potential In this study, we examined the membrane excitability of propagation for synaptic integration or neurotransmitter re- starburst amacrine cells in the rabbit retina during postnatal lease. However, there are other retinal interneurons, e.g., some development. We report that displaced starburst cells undergo axon-bearing horizontal cells, that respond to illumination of a transition from spiking to nonspiking during a crucial period the retina with sustained graded potentials, but their electro- in visual development. This change in membrane excitability is tonic structure does not appear to be compact. It has been caused by a loss of Na channel current just after eye opening. suggested that action potential propagation in these rather ex- Thus, starburst cells may use action potentials transiently tensive interneurons may be inappropriate for specific functional during a specific period in development, as a part of a network reasons. For example, it has been thought that attenuation of that generates the spontaneous activity. They then become synaptic signals in some of these nonspiking cells may serve to nonspiking neurons as the retina matures, presumably consis- keep different parts of the cell functionally segregated (11). tent with their role in visual processing in the mature retina. The cholinergic (starburst) amacrine cell is a medium-field amacrine cell type found in all mammalian retinas examined so MATERIALS AND METHODS far (for review, see refs. 12-14). Starburst cells exist in the rabbit retina as two mirror-symmetric populations in the inner Whole-Cell Patch-Clamp Recording in Rabbit Retinal Slices. plexiform layer (IPL), each having radially symmetric den- Retinal slices were prepared from pigmented rabbits aged be- dritic fields that extend from 200 to 800 ,im in diameter tween P1 and P56 (P0, day of birth) as described (36). Briefly, 3- depending on eccentricity (15-17). These cells receive input to 5-mm wide strips of retina, typically from areas within 6 mm from cone bipolar cells and other amacrine cells and make from the visual streak in the inferior retina, were isolated on direct output synapses onto ganglion cells and amacrine cells, nitrocellulose filter paper (Millipore type HAWP) and sliced at including starburst cells themselves (18-21). It has been a thickness of 150-200 ,um in a cold (4°C) physiological saline proposed that starburst cells may be involved in the production containing 137 mM NaCl, 4.3 mM KCl, 1.7 mM CaCl2, 1.2 mM of directional selectivity and in the regulation of the respon- MgCl2, 10 mM Hepes, 5 mM glucose (pH 7.4 with NaOH). The siveness of ganglion cells in the retina (e.g., refs. 13, 22, and 23). slices were subsequently incubated in Ames medium (Sigma; ref. It has been further postulated that the starburst dendritic tree 37) that was equilibrated with carbogen (95% 02/5% C02) for consists of semiisolated local processing units, which may serve 1-10 hr at room temperature (21-25°C). as a substrate for synaptic interactions crucial for directional selectivity (23). In order for these local units to function Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; DAPI, 4',6-diamidino-2-phenylindole; TTX, tetrodotoxin. *To whom reprint requests should be addressed at: Department of The publication costs of this article were defrayed in part by page charge Physiology and Biophysics, University of Arkansas for Medical payment. This article must therefore be hereby marked "advertisement" in Sciences, 4301 West Markham Street, Mail Slot 505, Little Rock, AR accordance with 18 U.S.C. §1734 solely to indicate this fact. 72205. 8057 Downloaded by guest on October 1, 2021 8058 Neurobiology: Zhou and Fain Proc. Natl. Acad. Sci. USA 93 (1996) Whole-cell patch-clamp recordings were made from slices to those stained with DAPI (see Results), though fine dendritic under a fixed-stage upright microscope (Zeiss) equipped with spines were observed predominantly from cells in young epifluorescence and a 40X water-immersion objective lens animals during the first postnatal week as previously reported configured for Hoffman modulation optics (Modulation Op- (32). In general, we found the dendritic diameter of starburst tics, Greenvale, NY). Cells were recorded in carbogenated cells increased with animal age during the first 2-3 postnatal Ames medium at room temperature (21-25°C) with 2-5-Mfl weeks, although the dendrites as revealed by the staining of resistance electrodes containing 95 mM KCI, 10 mM NaCl, 0.5 Lucifer yellow varied considerably in length from cell to cell, mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM Hepes, 2 mM presumably due to differences in eccentricity and in the degree adenosine 5'-triphosphate (ATP-sodium salt, Sigma), 0.5 mM of dendritic damage after slicing. In young retinas without the guanosine 5'-triphosphate (GTP-sodium slat, Sigma), and 1 DAPI label, we occasionally also encountered some axonless mM ascorbate (pH 7.2 with KOH). Series resistance, typically cells in the GCL, with a somal size similar to that of the between 10 and 15 Mfl, was compensated by 40-70% with the starburst cells, and with dendrites stratifying rather narrowly in series resistance compensation circuitry in the patch-clamp the sublaminar b of the IPL, perhaps corresponding to the amplifier (Axopatch 200A, Axon Instruments, Burlingame, nonstarburst amacrine cell previously described (32). How- CA). The liquid junction potential was corrected as described ever, this cell type had a much larger dendritic field and (36, 38). Data acquisition and analysis were done with the somewhat thicker proximal dendrites than those of starburst software programs PCLAMP (Axon Instruments) and ORIGIN cells and was, therefore, quite easily distinguished from dis- (MicroCal, Northampton, MA). placed starburst cells. Although we cannot exclude the possi- Identification of Displaced Starburst Cells in the Slice. bility that in young rabbits without DAPI injection some other Identification of a displaced starburst cell was based on its axonless nonstarburst cells but with a similar dendritic mor- round, 7-10-,um cell