Acetylcholine

Acetylcholine (ACh) signaling plays a key role in the development of the retina. Prior to synapse formation, paracrine action of ACh is essential for regulating early developmental events, such as the regulation of the cell cycle (6) and the growth of neurites (7). In addition, cholinergic synapses are among the earliest to mature and thereby constitute the earliest functional circuits in the retina.

ACh in the retina is produced solely by one cell type, the starburst amacrine cell (SAC), a type of amacrine cell named for its radially symmetric processes (8). In the mature retina, released ACh acts on both muscarinic and nicotinic receptors to modulate the response properties of many different types of ganglion cells (9-13), but it does not affect the SACs themselves (14). However, during development, cholinergic signaling does occur between SACs (14). During the first week after birth in mice, ACh released from SACs activates nicotinic acetylcholine receptors (nAChRs) on neighboring SACs and thus gives rise to a cholinergic network. A key developmental function of this cholinergic network is the generation of retinal waves. This network appears at birth in mice and mediates the initiation and propagation of waves.

Aside from the effect ACh has on ganglion and amacrine cells via nicotinic acetylcholine receptors, it also acts on the muscarinic acetylcholine receptors (mAChRs) of many cells in the neuroblastic layer (15, 16) (Fig. 2). Figure 2 shows the effect of acetylcholine on progenitor cells in rabbit retina. A one-day old rabbit retina is loaded with Fura-2. Images show responses to bath application of 200μM nicotine (Nic, left), which activates nicotinic acetylcholine receptors, or 100μM carbachol (CCh, right), which activates muscarinic acetylcholine receptors. Red indicates cells that had increases in intracellular calcium whereas blue indicates cells without increases in intracellular calcium.

Figure 2.

Acetylcholine acts on muscarinic receptors in progenitor cells and nicotinic receptors in ganglion and amacrine cells. Adapted from Wong et al, 1995 (16).

The mAChRs are G-protein coupled receptors that lead to an increase in intracellular calcium via release of calcium from internal stores, as opposed to influx through ligand- or voltage-activated channels. Interestingly, the ACh released during retinal waves drives correlated mAChR-dependent calcium transients in undifferentiated cells of the ventricular zone (Fig. 2) (15). Hence, it is possible that ACh released by retinal waves induces signaling that is important for early phases of neurogenesis and for cell migration (17).

GABA

GABA is expressed in more cells during development than during adulthood, thus suggesting that it plays a transient role in circuit formation (18). During the first few postnatal days in rabbit, GABA displays a high transient expression in the ganglion cell layer. Moreover, the IPL of P0 ferret exhibits markers for enzymes involved in the synthesis of GABA (19).

During development, GABA initially serves to depolarize neurons. When activated, ionotropic GABA receptors, GABA_A and GABA_C, flux chloride (Fig. 3A). Since the chloride concentration within developing retinal neurons is high due to low expression of the potassium-chloride co-transporter, KCC2, receptor activation leads to an efflux of negatively charged chloride ions through the open channels and thus causes depolarization of the cell (Fig. 3A, B left). KCC2 expression gradually increases during the first two weeks after birth in mice (20). Thus there is a ‘switch’ from excitation to inhibition as the reversal potential for chloride drops below the threshold for firing action potentials (Fig. 3 A, B right).

Figure 3.

GABA switches from depolarizing to hyperpolarizing during development. From Ben-Ari et al. 2007 (71).

In turtle retina, the timing of the GABA switch correlates with a decrease in propagating spontaneous activity (21), suggesting that GABA depolarization plays a role in this propagation. In mammalian retina, GABA plays a minor role in correlated spontaneous activity during its excitatory period (1, 22, 23). However, after the GABA switch, GABA’s inhibitory action takes on a prominent role in shaping spontaneous activity. Blocking GABA_A receptors greatly increases the frequency of spontaneous retinal waves (22, 24).

What underlies the switch of GABA’s action from depolarizing to hyperpolarizing? Several environmental factors, including neural activity (25), have been implicated in the timing of this switch in the retina. A recent study used acutely isolated retinas from knockout mice and pharmacological manipulations in retinal explants demonstrates that the timing of the GABA switch in retinal ganglion cells is unaffected by blocking specific neurotransmitter receptors or global activity (26) (Fig. 4). Purified retinal ganglion cells remain depolarized by muscimol for at least two weeks in culture (Fig 4, right), indicating that the GABA switch is not cell-autonomous. Purified ganglion cells co-cultured with other retinal neurons also remain depolarized by muscimol (Fig. 4, middle). However, culturing purified ganglion cells with dissociated cells from the superior colliculus (Fig. 4, right), or treating purified ganglion cells with media conditioned by superior colliculus cultures (Fig. 4C, red), results in a switch to inhibition by muscimol after two weeks in culture, indicating that a diffusible signal independent of local circuit activity regulates the maturation of GABAergic transmission.

Figure 4.

The GABA switch in retinal ganglion cells is due to a diffusible factor. From Barkis et al., 2010 (26).

Glutamate

Glutamatergic signaling is the last to develop within the IPL. Glutamate is released primarily from bipolar cells and a small subset of amacrine cells (27, 28). In mice, the axons of bipolar cells first express VGLUT1, the enzyme responsible for packaging glutamate into vesicles, around one week after birth (29). Although, ribbon synapses between bipolar cells axons and the dendrites of amacrine and ganglion cells do not form until 11 days after birth (5), glutamatergic currents can be measured before this (24, 29).

State-of-the-art transgenic and imaging techniques have characterized the distribution of glutamatergic synapses onto ganglion cells and between particular subtypes of bipolar and ganglion cells (30, 31). Before the functional maturation of bipolar cell ribbon synapses and long before the structural maturation of the ganglion cell dendritic arbor, adult-like patterns of glutamatergic synapses can be seen. However, between some subtypes of bipolar and ganglion cells, there appears to be an activity dependent remodeling of these synapses (30).

Retinal Waves

Prior to photoreceptor maturation and eye opening, retinal ganglion cells periodically fire bursts of action potentials on the order of once per minute. This spontaneous rhythmic activity was first measured in fetal rat pups and was found to be highly correlated among neighboring ganglion cells (32).

Extracellular recordings using a multielectrode array (2) (Movie 1) and imaging of calcium transients (Movie 2) associated with bursts of action potentials (1, 33) have revealed that these spontaneous bursts propagate from one cell to the next in a wavelike manner. These retinal waves are an extremely robust phenomenon, observed in a large variety of vertebrate species, including chick, turtle, mouse, rabbit, rat, ferret and cat (34).

Movie 1.

Multi-electrode array recording of cholinergic waves. 512 electrode array recording from the mouse at 37°. Each dot represents multiunit activity recorded on an electrode at that site. The size of the dot is proportional to the amplitude of the (more...)

Movie 2.

Calcium imaging of cholinergic waves. P3 mouse retina loaded with Oregon Green-Bapta-1AM. Playback 10x, size 800x600 µm (unpublished).

Retinal waves persist for an extended period throughout development as shown in Figure 5. However, as the retina matures, the circuitry underlying these retinal waves changes. Thus the wave generating circuits progress through three distinct stages, reflecting the nature of the connections and the cells that are involved (Fig. 5).

Figure 5.

Retinal waves occur in three stages. Before birth in mice, waves are mediated by non-synaptic (NS) circuits. From around birth until 10 days after birth, waves are mediated by acetylcholine acting on nicotinic acetylcholine receptors (nAChRs). From 10 (more...)

Embryonic waves

The earliest waves recorded in mammals are thought to be propagated via gap junctions. Retinal waves before embryonic day 23 in rabbit persist in the presence of antagonists to ionotropic GABA, glycine, ACh, and glutamate receptors, but are completely blocked by 18β-GA, a blocker of gap junction coupling (22). Similarly, mice prior to birth exhibit waves that are insensitive to chemical transmission antagonists (35). How waves are initiated and propagated during this early stage of development is still not clear. During the first week after birth in ferret and mouse, neurobiotin coupling between ganglion cells of the same subtype and amacrine cells has been seen (3, 36). However, ganglion cell coupling is initially weak and becomes stronger with age, while the correlations generated by waves become weaker with age (37). In addition, tracer coupling is found primarily between retinal ganglion cells of the same subtype and many other ganglion cells are not gap junction coupled. Thus, the issue of wave propagation in this early network still remains to be explored.

Neurotransmitters play a modulatory role during early stage waves. At the earliest ages studied in chick retina (E8-E11), wave frequency decreases in the presence of an ACh antagonist and increases with an ACh agonist but waves are not blocked. These waves are unaffected by GABA_A and glutamate receptor antagonists (38). Similarly, embryonic waves in mouse are reduced in ACh receptor antagonists (35). In addition, in rabbit, early waves are blocked by the activation of GABA_B receptors and are increased in frequency by the inhibition of these receptors (22).

Cholinergic waves

Several experimental results show that chemical synaptic transmission is a prerequisite for cholinergic wave propagation. 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 (1). Second, the compound postsynaptic currents measured from ganglion cells are blocked by bath application of Cd²⁺, a blocker of calcium channels that are associated with transmitter release (1). Third, the periodic Ca²⁺ increases, action potentials, and compound postsynaptic currents associated with waves can all be blocked by a variety of nAChRs antagonists (1, 39). Finally, genetic deletion of the beta2 subunit of the nAChR receptor (35) or deletion of the enzyme responsible for synthesis of ACh (40) results in the disruption of normal waves during the first week after birth.

The spatiotemporal properties of cholinergic retinal waves have been well-characterized by fluorescence imaging of calcium indicators, which are reliable markers of cell depolarizations. Figure 6 schematizes these waves (41). Waves initiate in small clusters of coactive neurons and then propagate over spatially restricted areas of the retina. 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 by repeated activation of the same clusters of neurons. Instead, the propagation boundaries of waves are determined in part by wave-induced refractory regions that last for 40-50 seconds. These observations have 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).

Figure 6.

Cholinergic retinal waves tile the retina. Pictured is a sequence of retinal waves that were measured using calcium imaging in a P2 ferret retina loaded with the calcium dye fura-2AM (read from left to right, top row first). This analysis is used to show (more...)

Recent studies in rabbit (Zheng et al., 2004; Zheng et al., 2006) and mouse (Ford et al. 2012) retina have revealed the cellular properties of starburst amacrine cells (SACs), the cell type that gives rise to cholinergic waves (Fig. 7) (42). Identifying SACs in mouse retinas was facilitated by the use of a line of mice in which GFP is expressed in SACs (mGluR2-GFP, Fig 7A). Using calcium imaging, spontaneous depolarizations of individual SACs were observed in the absence of synaptic excitation (Fig 7A), indicating that SACs themselves may be the source of initiation for waves. Paired recording between SACs revealed reciprocal cholinergic transmission (Fig 7C), indicating that waves are propagated via these slow, excitatory connections between neighboring SACs. Finally, wave boundaries are thought to arise from a slow after-hyperpolarization in the SACs that recovers over the course of tens of seconds following the depolarization during waves (Fig 7B).

Figure 7.

Cellular features of starburst amacrine cells underlie the spatiotemporal properties of retinal waves. (A) Fluorescence image of an mGluR2-GFP retina loaded with OGB. Right, Fluorescence image of GFP cells. Regions of interest are shown around each SAC. (more...)

Cholinergic waves may play a role in terminating early stage gap-junction mediated waves. Knock-out animals that lack nAChR receptor subunits still exhibit wave like activity under certain recording conditions, such as elevated temperature [alpha3(-/-) (35), beta2(-/-)(43, 44)]. This activity is likely mediated by gap junctions (44). Moreover, a study using a genetic model that eliminates ChAT in a large portion of the retina found normal cholinergic waves in the spared region, but compensatory waves in the region lacking ChAT (40). These studies suggest a sequential maturation of the retinal circuitry that relies on checkpoints to make transitions from one stage (gap-junction mediated waves) to the next (cholinergic transmission mediated waves). This sort of checkpoint model of neuronal development (45) is further supported by the disassembly of the cholinergic network to make way for glutamatergic signaling (46).

Glutamatergic waves

The synaptic circuitry that drives retinal waves changes postnatally. Though retinal waves early in development require cholinergic neurotransmission, studies in older ferret, rabbit, and mouse indicate that waves become insensitive to cholinergic antagonists and can be blocked by glutamate receptor antagonists (35, 47, 48). This switch in the requisite transmitter occurs at the age that bipolar cells make their initial synaptic connections with ganglion cells and conventional synapses between amacrine and ganglion cells become morphologically mature and numerous. This timing suggests that perhaps waves are mediated by neurotransmitters only when the synapses are first forming.

Glutamatergic waves have distinct spatial and temporal features. Unlike cholinergic waves that drive correlations in all neighboring ganglion cells regardless of subtype, glutamatergic waves occur more frequently in OFF cells than in ON cells (49) (Fig. 8). Waves occur in rapid clusters separated by periods of silence lasting about one minute (24). Within clusters, there is a distinct pattern of firing where ON and OFF cells alternate in firing bursts of action potentials (50) (Fig. 8). These spatial and temporal features are significantly shaped by inhibitory circuits. Blocking ionotropic GABA receptors increases wave frequency (51). Blocking glycine receptors does the same but also eliminates the asynchronous firing between ON and OFF cells.

Figure 8.

Glutamatergic waves occur in clusters of asynchronous ON/OFF bursts. Multi-electrode array recording of glutamatergic waves from a 12 day old mouse. ON ganglion cells precede OFF ganglion cells during burst clusters. Adapted from Kerchensteiner and Wong, (more...)

Similar to the transition from gap-junction to cholinergic waves, the onset of glutamatergic waves may play an active role in the disassembly of the cholinergic circuits (46). Mice lacking the vesicular glutamate transporter VGLUT1 lack glutamate release from bipolar cells. However, these mice still have retinal waves during the time that littermate control animals have glutamatergic waves. Interestingly, these waves in the VGLUT1 knockout mice are unaffected by glutamate receptor antagonists but are blocked by nAChR antagonists (24). Thus, glutamate signaling from bipolar cells seems necessary for dismantling the cholinergic network.

There is some evidence that the transitions between the different circuits that mediate waves are linked. Prior to birth waves are thought to propagate via gap-junctions between ganglion cells (Fig. 9A, left). From postnatal day 1-10 waves are propagated via SAC release of acetylcholine onto other SACs (Fig 9A, Middle, black box). Acetylcholine also depolarizes ganglion cells. During this period of development, the gap-junction signaling between ganglion cells is reduced (Fig 9A, Middle, red box). From P10-P15 bipolar cells release glutamate to propagate waves in a mechanism that is thought to involve spillover of glutamate to excite neighboring bipolar cells (Fig. 9A right, black box). Cholinergic signaling between SACs is reduced (Fig. 9A right, red box).

Figure 9.

A. Transitions between waves stages are homeostatically regulated. Different circuits mediated waves at different ages. B. In wild type mice gap-junction mediated waves (gray) are followed by cholinergic waves (blue) starting at P0, then glutamatergic (more...)

Genetic disruption of cholinergic or glutamatergic waves result in an extended action of the previous wave generating circuit (Fig. 9B). In wild type mice gap-junction mediated waves (gray) are followed by cholinergic waves (blue) starting at P0, then glutamatergic waves (green) at P10. In mice lacking the Beta2 subunit of the nicotinic acetylcholine receptor, gap-junction mediated waves persist until ~P8. In mice lacking vesicular glutamate transporter VGLUT1, cholinergic waves persist through the second postnatal week.

Gap junctions and waves

Gap junctions are thought to play a role in the generation of embryonic waves, as described above. At later ages in mammals, however, gap junctions also play a minor role in the propagation of retinal waves. Gap junction antagonists reduce or partially block retinal waves after birth in mice (36) and at later stages in rabbit (22) when waves depend critically on chemical transmission. However, these antagonists also have several non-specific effects so these results are inconclusive. A different approach is to study mice in which specific connexins are genetically deleted (Fig. 10). Knockouts of connexins 36 and 45, the two major gap junction forming connexins in the IPL, have normal wave propagation (Fig. 10 A) but the firing between waves at later stages is increased (Fig 10B). Glutamatergic waves and the firing between waves is eliminated by the application of bipolar cell synapse antagonists DNQX and AP5, indicating that bipolar input is important for the generation of waves as in wild type retinas (52).

Figure 10.

Spatiotemporal properties of retinal waves are similar in control, Cx45, and Cx36/45 double knockout mice. A, Examples of waves recorded in different genotypes. Each grayscale value represents an active area in one frame, with darker shades corresponding (more...)

In contrast, in the chick retina, gap junctions were found to be involved in wave generation at all ages. Octanol, a significant inhibitor of waves in E8 chick retina, restricts tracer coupling between ganglion cells and other cell types but not between ganglion cells themselves, indicating that the circuitry mediating these waves involves cells other than ganglion cells (38).

ON/OFF circuitry within the retina

Two sub-circuits that have been well characterized in the adult retina are the ON and OFF pathways. The classes of bipolar cells that transmit responses to the onset of light (ON responses) are distinct from those that transmit responses to the cessation of light (OFF responses). These ON and OFF circuits have ganglion cell dendrites, amacrine cell processes and bipolar cell inputs that are physically segregated from each other into what are called the ON and OFF layers of the IPL. The formation of these ON or OFF circuits involves the dendritic maturation of ganglion cell types.

The dendrites of most retinal ganglion cells arborize diffusely within the IPL before restricting their dendrites to distinct lamina (35, 53-57) (Fig. 11). Some studies have suggested that this segregation of ganglion cell dendrites into ON and OFF layers involves bipolar cell activity. First, this segregation is prevented by applying APB to hyperpolarize ON bipolar cells during the period of glutamatergic retinal waves (Fig. 12) (58, 59). Second, mice that lack the MHCI receptor CD3zeta, and thus display altered glutamatergic retinal waves, have ganglion cells with reduced dendritic motility and more diffuse dendrites within the IPL (60) (Fig. 11). However, not all manipulations of spontaneous retinal activity during development alter dendritic stratification. Preventing synaptic release of glutamate from ON bipolar cells by expressing tetanus toxin does not prevent the stratification of ganglion cell dendrites, but it does reduce synapse formation onto the ON bipolar cells (30).

Figure 11.

Retinal waves drive refinement of some retinal ganglion cell dendrites.

Figure 12.

Activity dependent maintenance of ganglion cell dendritic stratification in the developing cat retina. From Bodnarenko and Chalupa, 1993 (58) .

Is there a role for cholinergic waves in this ganglion cell stratification process? Several studies suggest that there is. First, blocking nAChRs during the period of cholinergic waves reduces the motility of filipodia on the dendrites of ganglion cells (61), demonstrating that cholinergic waves can drive structural changes in dendrites. Second, studies in turtle demonstrate that blocking cholinergic waves with nAChR antagonists reduces receptive field sizes (62). Finally, mice lacking the beta2 subunit of the nAChR exhibit a delay in, but not an absence of, the fine stratification of ganglion cell dendrites (35). These findings indicate that cholinergic waves do influence the outgrowth of ganglion cell dendrites but they are not the primary factor that dictates their final organization.

Refinement of retinal projections

Retinal axons undergo a period of refinement before vision begins. At birth in mice, axons from both eyes reside in overlapping regions of the dorsal lateral geniculate nucleus of the thalamus. By about two weeks after birth, the axon terminals from the ganglion cells of each eye separate into non-overlapping regions. Similarly, within the superior colliculus, retinal axons at birth extend over the entire area of the colliculus. However, over the course of about one week, these axons retract to their appropriate retinotopic regions and extensively arborize within a small target zone. These processes of eye-specific segregation and retinotopic map refinement occur during the period of retinal waves. Thus, the hypothesis has emerged that waves might provide cues within their activity pattern to instruct these developmental processes.

The refinement of retinal projections to the brain is thought to be driven by the precise initiation, propagation and termination properties of cholinergic waves (63). The periodic initiation of waves induces depolarizations and calcium transients that may be tuned to drive axon guidance (64, 65) and plasticity mechanisms (66, 67). Propagation speed sets the time scale over which neighboring cells are correlated, and thus may be critical for retinotopic map refinement (68). The spatial extent of wave propagation has been shown to be important for establishing eye-specific segregation of retinal inputs within the thalamus (69).

Retinal waves determine the final size of termination zones of retinal projections to the superior colliculus (SC, Figure 13A). Focal labeling of retinal ganglion cells using DiI labels in retina (Retina) gives small target zones in superior colliculus (WT, Fig. 13A, left). In knockout mice lacking normal cholinergic waves (β2-nAChR KO, Fig. 13A middle), termination zones are less compact. Rescue of the β2-nAChR gene in a subset of retinal cells (Fig. 13 β2 tg) produce small waves, which are sufficient to rescue normal retinotopic map refinement.

Figure 13.

Retinal waves drive refinement of central projections. A. Schematic showing results of several experiments based on DiI anterograde labeling to determine the retinotopic projection of retinal ganglion cells to the superior colliculus. Results for WT mice, (more...)

Retinal waves also play a role in eye-specific segregation of retinal ganglion cell projections to the lateral geniculate nucleus of the thalamus (Fig. 13b). In wild type mice, there is little overlap between ipsi- and contra-lateral projections. Mice lacking cholinergic waves (β2 KO) and mice with small waves (β2 tg) have significantly overlapping projections from either eye.

Retinal waves also play a role in eye-specific segregation of retinal ganglion cell projections to the lateral geniculate nucleus of the thalamus (Fig. 13b). In wild type mice, there is little overlap between ipsi- and contra-lateral projections. Mice lacking cholinergic waves (β2 KO) and mice with small waves (β2 tg) have significantly overlapping projections from either eye.