Waves Are Swell Waves of Spontaneous Electrical Activity and Calcium Transients Occur in the Retina During Its Development

MARINA CATSICAS AND PETER MOBBS RETINAL DEVELOPMENT MARINA CATSICAS AND PETER MOBBS RETINAL DEVELOPMENT Waves are swell Waves of spontaneous electrical activity and calcium transients occur in the retina during its development. Recent work raises the question of how these waves are produced and propagated.

Spontaneous electrical activity is a feature of the devel- What is the function of these waves of activity? The opment of neurons in several regions of the embryonic axons of ganglion cells grow into the lateral geniculate nervous system. Since the first descriptions of sponta- nucleus (LGN), where their terminals at first form dif- neous activity in cells of the central nervous system, fuse connections with neurons throughout the structure. there has been growing interest in the relationship Later, these connections are remodelled so that the gan- between early electrical activity and the development of glion cell terminals lie in eye-specific layers, and the the complex and stereotyped patterns of connections rough topographic map of retinal space within the layers seen in the adult brain, extensively studied along the of the LGN formed at early times is refined [3]. The various relays constituting the vertebrate visual pathway. waves of action potentials that travel across the retina The sheet-like structure of the retina, combined with its during the period in which these refinements occur can highly ordered topographical organization (see Fig. 1), be prevented by treatment with the Na+-channel uniquely suit it for studies of the pattern of spontaneous blocker tetrodotoxin. When this is done in fetal cats, it electrical activity in development. This pattern can read- prevents the remodelling of ganglion cell axon terminals ily be determined in the neurons of the retinal ganglion within the LGN [4]. By contrast, a different type of cell layer by pressing the retina into contact with structural remodelling of ganglion cells occurs later electrodes fabricated in a two-dimensional array. Recent within the cat retina, when dendrites that initially ram- work has raised new questions concerning the mecha- ify throughout the inner plexiform layer are subse- nism by which spontaneous electrical activity is gener- quently trimmed such that the arborizations of ON and ated in the retina [1]. OFF ganglion cells - cells that, in the adult retina, increase and decrease, respectively, their frequency of When electrode arrays are used to record the pattern of action potential production when light falls on their action potentials generated amongst the ganglion cells of receptor field centres - are restricted to different sub- the ferret and cat retina [2], waves of electrical activity laminae [5]. This is not prevented by treatment with are found to travel between them at a time when mature tetrodotoxin, and thus does not depend upon action photoreceptors are absent. These travelling waves take potential activity, but instead relies upon the release of the form of bursts of several seconds of near-synchro- glutamate by bipolar cells [6]. nous action potential generation in neighbouring cells, followed by 1 or 2 minutes of silence. Each wave of Electrical recording with electrode arrays has the draw- neural activity sweeps across a patch of retina several backs that the spatial resolution is limited by the size and hundred microns wide, with the wave-front moving at separation of the electrodes, and that it cannot be used about 100 tum s- 1 and the signal taking about 0.2 seconds to identify the cell types involved in the production of to pass from cell to cell. retinal waves. Recording and dye-filling of cells with

Fig. 1. (a) The wiring of the mature retina. Light is turned into an electrical signal by the photoreceptor cells (PC). The signal passes along a vertical pathway comprised of bipolar cells (BC) and ganglion cells (GC), the axons of which run to the brain. The vertical flow of information is modu- lated by horizontal connections provided by horizontal cells (HC), between photoreceptors and bipolar cells, and amacrine cells (AC), between bipolar and ganglion cells. Abbreviations: POS, photoreceptor outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; FL, fibre layer. (b) Nissl-stained transverse section through the cat retina aligned so that the position of the retinal layers corresponds with that shown diagrammatically in (a). (Photograph courtesy Mitch Glickstein.)

intracellular microelectrodes, however, suggest that all of How do retinal waves propagate? One possibility is via the three classes of ganglion cell - , and y - found chemical synapses (see Fig. 2). Despite the fact that in the mammalian retina are likely to be involved [7]. conventional synapses are present in the inner plexiform Fluorescent probes have recently been used by Wong and layer of embryonic ferret retina at the time of wave pro- coworkers [1] to measure changes in intracellular Ca2+ duction [8], the slow rate at which the wave-front propa- ion concentration ([Ca 2+]i) in the neonatal ferret retina at gates makes it unlikely that fast synaptic transmission is a time when waves of electrical activity are known to involved. This does not, however, rule out an involve- occur. The results, as might be expected on the basis of ment of neurotransmitters, as we know little about either the electrical recordings, show transient increases in transmitter release at early times in development or the [Ca2+]i in retinal neurons. These last 10-15 seconds, and properties of immature synapses in the retina. The a- sometimes move from cell to cell in a wave that spreads at amino-3-hydroxy-5-methyl-4-isoxazole propionic acid rates, and over areas, similar to those of the electrical (AMPA)/kainate and N-methyl-D-aspartate (NMDA) waves seen with electrode arrays. classes of glutamate receptors are expressed early in development [9], but Wong et al. [1] have shown that The region over which Ca2 + transients occur varies, but glutamate receptors are unlikely to be involved in the in long periods of recording they appear in cells production of retinal Ca2 + transients because they throughout a wide area, suggesting that there are no continue to be triggered in the presence of the iono- physiological or anatomical barriers to their spread. The tropic glutamate receptor antagonists 6-cyano-7- Ca2 + signals are abolished by application of tetrodotoxin, nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5- as would be expected if the transients are initiated by phosphonovalerate (APV). As type A y-aminobutyric Na+-based action potentials. Calcium imaging of devel- acid (GABA) [10] and muscarinic acetylcholine [11] oping retina [1] has provided two interesting new obser- receptors are also known to appear in the retina at early vations. Firstly, not all ganglion cells within the active times, a detailed pharmacological examination is required zone through which a wave travels show increases in before all transmitter receptors can be excluded from [Ca 2+]i. Secondly, making use of the fact that amacrine having a role. cell bodies are smaller than those of ganglion cells, Wong and co-workers [1] have shown that some, but Another obvious route for the transmission of retinal not all, amacrine cells are involved in activity synchro- waves is via gap junctions. In the neocortex, Yuste and nous with that of ganglion cells. These two observations colleagues [12] have shown, in elegant experiments make it unlikely that the waves result from some non- employing Ca2 + imaging, that transient coupling via gap specific process, such as spreading depression. Wong et junctions allows Ca2 + waves to pass from cell to cell dur- al. [1] suggest that some kind of horizontal network is ing the period that circuits are formed in this region. formed between neurons in the inner plexiform layer to These waves differ, however, from those seen in the produce and propagate waves of electrical activity retina, as they survive treatment with tetrodotoxin and before the formation of the synaptic machinery of the thus do not result from Na+-based action potentials. retina is complete. Using Neurobiotin, a tracer that passes through most gap

Fig. 2. The three types of ganglion cell (a, and y) found in the mammalian retina are shown together with a pair of conventionally placed amacrine cells. a and y ganglion cells are coupled to ganglion cells of the same type (not shown) and to amacrine cells by gap junctions. Amacrine cells form a potential route for the passage of electrical activity and Ca2 + transients between a and y ganglion cells. ganglion cells may be involved in the production or transmission of waves through the action of a diffusible messenger, perhaps a neurotransmitter. Some of the ion channels involved in wave production are shown in the a ganglion cell. Depolarization in a trigger cell (identity unknown), perhaps resulting from the activation of a ligand-operated channel by a neurotransmitter, leads to the activation of Na+ channels, resulting in action potentials that can be blocked by tetrodotoxin. Depolarization during the action potential leads to the activation of Ca2 + channels, and the resulting influx of Ca2+ may lead to Ca2+- induced Ca2+ release (CICR) from intracellular stores to produce a Ca2+ transient. Depolarization may also lead to the release of trans- mitters from immature conventional synapses or perhaps via carriers (not shown). K+ released during action potentials may depolarize neighbouring cells and contribute to the spread of retinal waves. DISPATCH 979 junctions, Penn, Wong and Shatz [13] have shown that ot Much remains to be discovered concerning the triggering, ganglion cells are coupled to one another via gap junc- propagation and effects of retinal Ca2+ transients, and the tions, as are y ganglion cells, and that both at and y cells exact relationship between them and the waves of electri- are coupled to amacrine cells. Thus, a potential route cal activity that are thought to underlie their production exists by which Ca2+ waves might propagate between remains to be demonstrated. Pharmacological studies of these two populations of ganglion cells. the propagation of retinal waves are urgently required to uncover the mechanisms involved. Retinal waves are Although these intercellular junctions are present during riding on a swell of interest, but we are waiting for the time that the retina generates waves, and thus could detailed explanations of their transmission and purpose. contribute to the synchronization of action potential activity, they cannot be solely responsible for the progress Acknowledgements: We thank David Attwell, David Rossi, David Becker and Martine Hamann for their comments on the manuscript. of the wave-front. Indeed, 3 ganglion cells, which com- prise half the ganglion cell population in the ferret, are References not dye-coupled to one another, to amacrine cells or to 1. 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