Formation of Early Retinal Circuits in the Inner Plexiform Layer
Marla Feller
[Introduction] [Neurotransmitters and Early Retinal Development] [Spontaneously Active Synaptic Circuits] [Retinal Waves] [Chemical Synaptic Transmission] [Gap Junctions] [Role of Activity in Formation of ON and OFF Circuitry] [References] [Author]
Introduction
The mammalian retina has long been a model system for study of development of neural circuits in the CNS because the adult network is well organized into cell-type specific layers, and the anatomy, physiology and function of many of the retinal cell types is well characterized. The development of the retina requires several steps. The first step is to create the right proportion of the 7 cell types that comprise the retina. This process occurs primarily through genesis of the correct number of each cell types. Only ganglion cells have their final number regulated by cell death, which reduces the number of ganglion cells by as much as 50% in some species. The second step is for cells to migrate into the correct location. Third, once the correct number of neurons have settled in their proper location, they start to form synaptic connections with other retinal neurons. Finally, these groups of synaptically coupled cells evolve into the circuits that comprise the adult retina.
Progenitor cells in the neurepithelium lining the surface of the neural tube, later become the ventricular zone of the the optic vesicles, optic cup and early retina. Postmitotic cells leave the ventricular zone to migrate to one of three cell layers in the retina remaining attached radially from one side of the retina to the other. This is shown in figure 1 which is from Golgi staining studies of Cajal done at the end of the last century. The neural cells lie at different levels in the retina and when in correct position lose their anchoring radial connections. (Fig. 1). Then polarity of the differentiating cells occurs and dendrites and axons grow out appropriately. The ganglion cells are the first to emerge as recognizable neurons with axons passing to the optic nerve and central brain structures (Fig. 1, a, b ). Then amacrine cells (Fig. 1c), Muller cells (Fig. 1, d) bipolar, and horizontal cells form in the correct layer. Finally photoreceptors remain to line the top layers (Fig. 1e and f).
Mechanisms that govern the formation of neural circuits during development traditionally have been divided into activity-independent and activity-dependent categories. In this view, the early organization of the circuits, namely the phenotype, location and early connections of neurons, are governed by activity independent mechanisms, e.g. the genetic profile of the cell. Neural activity acts independently of these genetic cues to refine initially crude connections into the detailed circuits that characterized the adult nervous system. Since many stages of retinal development occur before there is any light-induced neural activity they are thought to be part of the genetic profile of the cell. However, recent evidence suggests that environmental cues, mediated by chemical and electrical activity within the cell, influence the earliest stages of development.
If the retina is unable to respond to light, what is the source of this neural activity? Recent experiments in a wide variety of vertebrate species have demonstrated that early synaptic circuits in immature retinas can lead to both chemical and electrical signaling in developing neurons. This chapter will provide a review of the sources of this spontaneous activation of cells and the possible role this activity plays in the development of the retina.
Neurotransmitters and Early Retinal Development
There is good evidence now that neurotransmitters can be found at the earliest stages of retinal development and these neurotransmitters can function in the absence of traditional synapses (Redburn and Rowe-Rendleman, 1996). For instance, markers of cholinergic neurons (such as antibodies to choline acetyl transferase and acetylcholine esterase) can be observed in the neuroblastic layer as early as embryonic day 3 in chick and P0 in the ferret and mouse (Feller et al., 1996; Bansal and Feller, unpublished observations), the developmental period during which amacrine cells are being generated. These cells are presumably starburst amacrine cells, the only source of acetylcholine in the adult retina.
By monitoring intracellular calcium concentrations using fluorescence imaging, Wong (1995) showed that during these initial stages of retinal development muscarinic acetylcholine receptor (mAChR) agonists cause substantial increases in intracellular calcium of many cells in the neuroblastic layer (see Figure 2, above). M-AChRs are cGMP gated channels that lead to increases in intracellular calcium by causing a release of calcium from internal stores, as opposed to influx though ligand- or voltage-activated channels. After the cells were postmitotic and began to migrate out of the ventricular zone, this responsiveness to mAChR agonists was reduced. Amacrine and ganglion cells still had responses to cholinergic agonists, but they were mediated via nicotinic receptors, as they are in the adult. Hence, it seems possible that even before cholinergic neurons have left the ventricular zone, and long before these neurons have formed synaptic connections, they could be inducing signaling that is important for early phases of neurogenesis and also cell migration.
As discussed below, activation of nAChRs is also vital for generating spontaneous activity in the IPL at later stages of retinal development.
GABA is expressed in more cells during development than during adulthood. Figure 3 shows the number of amacrine cells that are immunoreactive to GABA between E38 and P16 in the developing ferret retina compared with the adult ferret retina. This exuberance of GABA positive neurons suggests that like ACh, GABA may play a transient role in circuit formation (for review, see Sandell, 1998). For instance, GABA is thought to play a role in synaptogenesis between cones and horizontal cells early in postnatal development of the OPL in rabbit retina (Messersmith and Redburn, 1993).
GABA has a particularly high and transient expression in the ganglion cell layer during the first few postnatal days of rabbit. In addition, markers for enzymes involved in the synthesis of GABA can be found on either side of the IPL early in development in ferret retina as shown in figure 3 (Karne et al., 1997). So, similarly to the role that GABA may play in development of the OPL, a role may be in formation of circuits in the IPL.
For a review on the early role of glutamate, see Redburn et al., (1992).
Spontaneously Active Synaptic Circuits
Retinal Waves
The first synaptically connected circuits that appear in the developing IPL are between amacrine and ganglion cells (Greiner and Weidman, 1981; Karne et al., 1997). Prior to photoreceptor maturation and eye opening, retinal ganglion cells, the projecting neurons of the retina, periodically fire bursts of action potentials. This spontaneous rhythmic activity was first measured in fetal rat pups. This activity was found to be highly correlated among neighboring ganglion cells (Galli and Maffei, 1988). Both extracellular recording using a multielectrode array (Meister et al., 1991) and imaging of calcium transients associated with bursts of action potentials (Feller et al., 1997; Wong et al., 1995) have revealed that these spontaneous bursts propagate from one cell to the next in a wavelike manner. Recent experiments demonstrate that blockade of spontaneous retinal activity disrupts the normal pattern of retinal ganglion cell axons in its primary target, the lateral geniculate nucleus of the thalamus (Penn et al, 1998), indicating that spontaneous activity in the retina plays a critical role in the normal development of the adult visual system.
The spatiotemporal properties of retinal waves have been well characterized by fluorescence imaging of calcium indicators, a reliable marker of cell depolarizations (Feller et al., 1997). Waves initiate in small clusters of coactive neurons from which they then propagate over spatially restricted areas of the retina (Figure 4). Initiation sites and wave boundaries are distributed randomly across a given retina, indicating that the global patterns of waves are not determined by fixed structures such as pacemaker cells or repeated activation of the same clusters of neurons. Instead, wave boundaries arise in part from a refractory period (Figure 5). These observations led to the hypothesis that every region of the retina is equally likely to initiate or propagate a wave, and therefore the global spatial patterns of waves are determined by the local history of retinal activity (Feller et al., 1997).
Retinal waves are an extremely robust phenomenon, observed in a large variety of vertebrate species, including chick, turtle, mouse, rabbit, rat, ferret and cat (Wong, 1999). Though wave periodicity and velocity in all species are comparable, the circuitry underlying the propagation may be substantially different. Waves are first seen around the time that neurons residing in the inner retina are starting to form circuits while the outer retinal neurons have not made synaptic connections, and photoreceptors are not yet functional (Greiner and Weidman, 1981; Mey and Thanos, 1992). At this stage of development, ganglion cells have migrated into the ganglion cell layer and their axons have reached their primary targets, the lateral geniculate nucleus in mammals, and the tectum in chick.
Chemical Synaptic Transmission
In postnatal ferret and mice retinas, chemical synaptic transmission is a prerequisite for wave propagation, as indicated by several experimental results. First, simultaneous whole cell voltage clamp recordings from ganglion cells demonstrate that increases in [Ca2+]i correlated across cells are driven by compound synaptic inputs (Figure 5, Feller et al., 1996) (Fig. 5 and 6). Second, the compound postsynaptic currents measured from ganglion cells are blocked by bath application of Cd2+, a blocker of voltage-activated calcium channels, including those associated with transmitter release (Feller et al., 1996). Third, the periodic Ca2+ increases, action potential, and compound postsynaptic currents associated with waves can all be blocked by a variety of nAChRs antagonists (Feller et al., 1996; Penn et al., 1994). Note, in the adult retina, acetylcholine acts as a modulator of ganglion cell firing, while glutamate is the primary excitatory transmitter. However, at the earliest ages studied, glutamatergic blockers do not affect wave generation (Wong, 1995).
Is acetylcholine involved only in the initiation of waves or is it also involved in the propagation of activity from one area of the retina to another? To address this question, a large number of cells within the ganglion cell layer can be depolarized directly by pressure ejection of potassium from a pipette (Figure 7) (Feller et al., 1996). Such a large-scale depolarization reliably initiates waves. However, in the presence of nicotinic AChR antagonists, pressure ejection of potassium still causes a large depolarization of cells around the pipette but waves cannot be generated. These experiments indicate that cholinergic synaptic transmission is required for propagation of the activity away from a local area of depolarization.
As mentioned above, the sole source of the ACh in the retina is a subclass of amacrine cells, called starburst amacrine cells. Immunohistochemical stains for choline acetyltransferase (CHAT) reveal that this population of cells exist early in the developing ferret retina. Zhou (1998) simultaneously recorded from rabbit starburst amacrine and ganglion cells and found that starburst cells undergo spontaneous depolarizations that are correlated with the depolarizations of neighboring ganglion cells (See Fig. 8). These experiments also show that, like ganglion cells, starburst amacrine cells are excited via synaptic input during waves. This is consistent with recent findings from Stellwagen et al. (1999) that the compound postsynaptic currents associated with waves are not decreased in the presence of tetrodotoxin. Though starburst cells have the ability to fire action potentials early in development (Zhou and Fain, 1996), they do not reach action potential threshold during waves (Zhou, 1998), indicating that they release ACh in an action potential-independent, graded fashion, characteristic of adult amacrine cells. Although the source of synaptic input to starburst amacrine cells during waves is unknown, these results indicate that a complex network of amacrine and ganglion cells, connected via excitatory chemical synapses, mediates retinal waves.
The synaptic circuitry that drives retinal waves changes postnatally. Though cholinergic neurotransmission is required and GABA contributes to the depolarization of cells during retinal waves early in development in the ferret, recent studies in older ferrets indicate that waves are insensitive to cholinergic antagonists and can be blocked by glutamate receptor antagonists (Wong, 1999). This switch in the requisite transmitter occurs at the age that bipolar cells are making their initial synaptic connections with ganglion cells and when conventional synapses between amacrine and ganglion cells become morphologically mature and numerous. This leads to the hypothesis that perhaps waves are mediated by neurotransmitters only when the synapses are first forming (Fig. 9).
GABA has a modulatory role in retinal waves. Imaging of the spontaneous increases in Ca2+ associated with waves has shown that GABA-A receptor antagonists can dramatically alter the amount of wave-induced depolarization (Fischer et al., 1998). GABA is the primary transmitter of most amacrine cells in the retina, and, at the youngest ages studied, it provides excitatory input for ganglion cells (Fischer et al., 1998). Unlike ACh, however, GABA does not influence wave periodicity since GABA blockers do not alter either the frequency of the cholinergic barrages that are associated with waves measured in ganglion cells (Feller et al., 1996), or other properties of wave propagation at ages less than P10 (Fischer et al., 1998; Stellwagen et al., 1998).
Waves persist after GABA becomes inhibitory (Fischer et al., 1998). However, these changes in the circuitry mediating waves with development leads to a changes in the frequency of events occurring in different subsets of ganglion cells (see below).
Gap Junctions
Gap junctional coupling between ganglion cells and between ganglion and amacrine cells in postnatal ferret retinas has been assessed by neurobiotin injection experiments (Penn et al., 1994). As can be seen in figure 10, ferret, like rabbit, cat and primate, retinas show signs of dye coupling between cells of the same type (homotypic coupling) and of different types (heterotypic coupling),when neurobiotin is injected into a single cell. This dye coupling is particularly strong between large populations of the same type of amacrine cell, although , as far as we know at present, this does not include the adult acetylcholine-containing cells (Vaney, 1994) (Fig.10).
Gap junctions appear to be involved in wave generation in the chick retina. At all ages, agents that are known to inhibit transmission through gap junctions can significantly inhibit waves (Catsicas et al., 1998; Wong et al., 1998). Octanol, which inhibits waves in E8 chick retina, restricts tracer coupling between ganglion cells and amacrines, but not ganglion-ganglion cell coupling, indicating that wave generation involves cells other than ganglion cells. Neurotransmitters also affect wave propagation and, as in the ferret, the modulatory role of different transmitter systems changes with age. At the earliest ages studied (E8-E11), wave frequency decreases in the presence of an ACh antagonist and increases in an ACh agonist, but waves are not blocked. Waves are unaffected by GABA-A and glutamate receptor antagonists (Catsicas et al., 1998). In contrast, at the older ages (past E11), waves are blocked completely by a combination of glutamate antagonists, increase in frequency in the presence of a GABA-A antagonist, but are unaffected by ACh antagonists (Wong et al., 1998).
Does electrical transmission play a role in generating retinal waves in mammals in addition to active chemical (cholinergic) transmission? Gap junction transmission can actually be influenced by ACh, as has been shown in the developing neocortex (Roerig and Katz, 1998). Figure 10 shows the state of dye coupling between ganglion cells and amacrine cells in postnatal ferret retinas. It is thought that gap junctions between ganglion cells are unlikely to be the substrate of wave propagation though. Tracer coupling is restricted to ganglion cells of the same subtype, and a major class of ganglion cells, the beta cells in ferrets (analogous to X cells in cat), show no evidence of coupling (see Fig. 10). In addition, in development, ganglion cell coupling is initially weak, then becomes stronger with age. The reverse correlations occur with waves because they become weaker with age (Wong et al., 1993). One idea is that gap junction coupling between amacrine cells (possibly the cholinergic amacrine cells) is a transient early phase of transmission employed before the chemical synapses are completely mature. However, tracer coupling between amacrine cells at the earliest ages is yet to be defined. Moreover, it could equally well be postulated that the first transient gap junctions between ganglion and amacrine cells could provide excitation from the ganglion cell layer back to the amacrine cell layer.
Recent experiments have demonstrated that agents known to influence coupling through gap junctions by modulation of intracellular levels of cAMP (Hampson et al., 1992; Mills and Massey, 1995) have dramatic effects on the propagation of waves (Stellwagen, 1998). As in the developing chick retina (Catsicas et al., 1998), forskolin, dopamine and adenosine agonists all lead to bigger, faster and more frequent waves in mammalian retinas. All in all, it appears that gap junctions play a more important role in wave generation at earlier stages, before chemical synapses have started to form, than at later stages of development in the mammalian retina.
Role of Activity in Formation of ON and OFF Circuitry
Two examples of sub-circuits that have been well-characterized in the adult retina are the ON and OFF pathways. Different classes of bipolar cells transmit responses to the onset of light (ON responses) through a distinct circuit from the cells that transmit the cessation of light (OFF responses). A diagram of this well known circuitry and division of labor in the cat retina is shown below (Nelson et al., 1978).
Fig. 11. ON/OFF circuitry in the adult retina (Nelson et al., 1978). (78K jpeg image)
Ganglion cell dendrites, amacrine cell processes and bipolar cell inputs for these two circuits are physically segregated from each other in what are called the ON and OFF layers of the IPL (Figure 11). The formation of the ON and OFF circuits involves the dendritic maturation of ganglion cell types. In an extensive study done by Chalupa and colleagues (Figure 12, below), it was shown that developing ganglion cells start initially with a diffuse dendritic branching pattern and then later segregate into the two types with characteristic restricted branching to the ON and OFF layers of the IPL (Fig. 12). They showed that neural activity is required for the formation of this segregation, since intraocular injections of APB, a glutamate analog, disrupts the normal timecourse of dendritic stratification in cats. Presumably APB is acting by inhibiting ON- cone and rod bipolar cells that have direct (cone pathways) or indirect (rod pathways) glutamatergic inputs to the ganglion cells. Note, that Bodnarenko and coauthors (1995) demonstrated that other pathways activity was also required for maintenance of the stratification, since APB treatment only delays stratification, and does not permanently arrest it . Interestingly, it has recently been demonstrated that transgenic mice lacking the mGluR6 receptors have normal ON/OFF dendritic stratification (Tagawa et al., 1999). These results indicate that either the APB signaling responsible for disrupting the ON/OFF segregation was not through mGluR6, or simply that mice are different than cats.
Do retinal waves play a role in driving this segregation? Retinal waves are modulated by the formation of ON and OFF layers in the developing IPL. In ferrets, ganglion cell dendrites are stratified by P14 and before bipolar cells are present (Wong and Oakley, 1996). At ages less than P14, all waves involve ON and OFF ganglion cells. At ages older than P21, however, OFF cells continue to participate in waves with the same frequency, but the ON cells participate in only 30% of the waves (Wong and Oakley, 1996). The circuitry underlying these different temporal patterns has not yet been identified, although they are differentially modulated by GABA (Fischer et al., 1998). It remains to be determined whether spontaneous activity via retinal waves plays a role in the establishment of these circuits.
The Table below summarizes our present knowledge of the timecourse of events occurring during development of the circuits of the IPL in a variety of species. This is an exciting and dynamic field of research and our knowledge of the powerful events that shape our visual information processing at the first synaptic levels is continuously being updated. We look forward to a time when the Table below is complete
Summary of the developmental events in the retina described in this chapter. (117K jpeg image)
References
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The author Dr. Marla Feller was born in Red Bank, New Jersey. She received her B.A. and Ph.D. in Physics from University of California at Berkeley in 1985 and 1992 respectively. She headed a laboratory at the National Institutes of Neurology from 1994-2000 and most recently has moved to the Neuroscience department at UC San Diego. Marla initiated outstanding research with Dr. Carla Shatz to study the mechanisms and function of spontaneous activity in the developing mammalian retina. Together they were able to determine that these highly correlated activity patterns were generated by a cholinergic network that functions long before the retina itself can respond to light. Currently Marla is investigating the mechanisms underlying the generation of this highly patterned activity and exploring the role it plays in the development of the retina itself. |
[Introduction] [Neurotransmitters and Early Retinal Development] [Spontaneously Active Synaptic Circuits] [Retinal Waves] [Chemical Synaptic Transmission] [Gap Junctions] [Role of Activity in Formation of ON and OFF Circuitry] [References]
July 29, 1999