Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

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13.2 Synapse formation

13.2.1Chemical synapses

Functional synaptic connections emerge once the plexiform layers are established through the formation of specific contacts between neurites originating from diverse cell types expressing different neurotransmitters (see previous chapter). Synapse formation proceeds in a centrifugal manner, from the inner to the outer retina, and it occurs first between cells that spread information laterally within the plexiform layers (amacrine cells (ACs)) and (horizontal cells (HCs)), followed by vertical connections (through bipolar cells (BCs)).

Ultrastructural studies

Substantial knowledge on the formation of retinal synaptic connections comes from ultrastructural studies (see Robinson, 1991, for a review). Synaptogenesis starts before eye opening, and even before birth, thus long before visual experience is even possible. Synaptic contacts are initially made in the inner plexiform layer (IPL) (Figure 13.1), and then they proceed towards the outer retina where the process continues well after eye opening. The time course of synapse formation in the IPL and in the outer plexiform layer (OPL) suggests important functional issues. First, the presence of early synaptic contacts in the inner retina coincides with the period during which the retina is spontaneously active, indicating the central role of retinal ganglion cells (RGCs) and ACs in generating this activity (this will be discussed in Section 13.3). Second, the fact that synaptic wiring in the outer retina continues after eye opening suggests that the refinement of circuitry in the outer layers is more likely to be influenced by visual experience.

The first connections observed in the IPL are amongst ACs and between ACs and RGCs (cat: Maslim and Stone, 1986; monkey: Nishimura and Rakic, 1985, 1987; mouse: Fisher, 1979; rat: Horsburgh and Sefton, 1987). Even before synaptic vesicles become detectable, apposed pairs of membrane specializations called junctions become apparent throughout the IPL. Nishimura and Rakic (1987) have performed a detailed analysis of the sequence of events occurring during the formation of synaptic contacts between ganglion and ACs in the monkey, showing that synaptic differentiation proceeds from the postsynaptic to the presynaptic side. Indeed, specialized areas of membrane thickenings occur first on the dendrites of RGCs. Then, they become apposed to amacrine processes, which in turn develop their own membrane specializations, including synaptic vesicles (Figure 13.1). These are the

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Figure 13.1 (cont.) expanded (and some now belong to rod BCs) and it is now possible to detect few synaptic vesicles in the richer neuropil. Immature, primordial ribbons (dark structures encircled by vesicles, indicated by arrows) can be seen ‘floating’ in neural processes, not yet attached to synapses. Conventional synapses are also appearing (asterisk). Postnatal day 6 (P6): the neuropil is much denser and shows mature conventional synaptic contacts (asterisks) (between ACs, RGCs and BCs). Immature ribbon synapses (triads) are also present (arrow). Adult: synaptic contacts are now completely mature, with conventional synapses (asterisk) as well as clearly delineated ribbon triads (arrow). Courtesy of Les Westrum and Anita Hendrickson (Les Westrum sadly passed away shortly after providing the micrographs. We will always remember his kindness and support).

first noticeable conventional synapses, and are followed by the appearance of conventional synapses between ACs. Ribbon synapses, characteristic of BCs, emerge much later, around birth (Figure 13.1), suggesting that functional contacts between BCs and ACs or RGCs do not emerge until late, at postnatal stages. The order of establishment of ribbon synapses is first between a BC and a single RGC or AC (dyads), followed by the formation of triads (Figure 13.1) through the addition of processes from another AC or RGC to dyads. The circuitry in the IPL is then completed by the addition of a feedback loop through amacrine or interplexiform cell processes onto bipolar axons. In primates, ribbon synapses appear after conventional synapses in the peripheral retina while the order is reversed in the fovea (Crooks et al., 1995). Issues related to primates are discussed further in Chapter 7.

In the OPL, the first synapses to appear are ribbon synapses between photoreceptors and HCs (rabbit: McArdle et al., 1977; cat: Maslim and Stone, 1986). Contacts are first established between cones and HCs (mouse: Rich et al., 1997), closely followed by wiring between rods and HCs. Vertical functional connections between photoreceptors and BCs arise later, once again starting with cones, and followed by rods.

Markers for various synaptic release-associated proteins

Synaptic transmission requires several important steps. First, neurotransmitters must be packed into vesicles, and then these vesicles must be docked at the active zone where they fuse with the membrane of the terminal, releasing their content into the synaptic cleft, through the universal mechanism of exocytosis. Exocytosis is Ca2+-dependent, and it is under the control of a vast family of proteins associated either with the membrane of the vesicle or with the membrane of the terminal. Invaluable information can therefore be gathered on the ontogeny of synaptic vesicular release by studying the development of mechanisms controlling vesicular neurotransmitter transport, or by following the developmental expression of synaptic release proteins in different types of retinal synapses.

A recent developmental study of the expression of the vesicular transporter for glutamate, VGLUT1, in the mouse retina reports that vesicular glutamatergic neurotransmission appears in cone circuits before it does in rod circuits, both in the IPL and OPL (Sherry et al., 2003). The vesicular transporter VGLUT1 also appears in OFF circuits before it does in ON circuits in the IPL (see Chapter 14).

The developmental expression of VGAT, the vesicular transporter for γ-aminobutyric acid (GABA) and glycine, precedes that of VGLUT1 by several days in the postnatal rodent retina (Johnson et al., 2003). Indeed, VGAT is already present in the IPL by postnatal day (P)1. On the other hand, VGLUT1 does not appear until several days later (rat: P5 to P7 in the OPL and P7 in the IPL; mouse: P3 in the OPL and P5 in the IPL), reaching its adult pattern by P14. Its expression precedes the formation of mature ribbon synapses (P11 in mouse IPL and P13 in rat IPL), but is already in place when spontaneous excitatory postsynaptic responses begin (P7 in mouse, see Section 13.3). These observations point towards several interesting conclusions. First of all, they suggest that vesicular GABAergic and/or glycinergic neurotransmission at conventional synapses occurs at early developmental stages in the

IPL, before glutamatergic synapses become functional. This is in accordance with several physiological studies demonstrating an important role for GABAergic neurotransmission in the generation of early retinal spontaneous activity (see Section 13.3.3). The second important conclusion is that there is vesicular glutamatergic release in the IPL before the maturation of ribbon synapses. One possible explanation for the presence of this early glutamatergic neurotransmission is that it originates in photoreceptors, known to make transient connections in the IPL at early stages, during the period of spontaneous activity (Johnson et al., 1999). Second, it may originate from axon collaterals of RGCs projecting back into the IPL. Such collaterals have been observed in the immature retina (discussed in Sernagor et al., 2001).

Many proteins involved in synaptic transmission, either at the presynaptic or at the postsynaptic level, have been identified in various parts of the nervous system. Synapse formation can thus be assessed using various approaches to identify these proteins because their expression during development is prone to be involved in the functional maturation of synaptic circuitry. In general, they appear at early stages, long before synapses mature morphologically, which indicates that the establishment of the basic membrane synaptic machinery is an early step during synaptogenesis. The fact that these proteins are present before synapses are functionally formed suggests that they play a role in modulating synaptic maturation, perhaps through trafficking synaptic vesicles to growing neurites.

Synaptogenesis can be followed at the light microscope level using immunocytochemical labelling of synaptophysin or syntaxin, transmembrane proteins found on synaptic vesicles, believed to be involved in neurosecretion. Both are present in the IPL at the time synapse formation starts in the central retina (human: Nag and Wadhwa, 2001). In the outer retina, synaptophysin or syntaxin staining appears once the OPL is fully differentiated (rat: Dhingra et al., 1997; human: Nag and Wadhwa, 2001; mouse: Sharma et al., 2003). The expression of both these proteins follows a central-to-peripheral gradient.

Synaptic vesicle protein 2 (SV2) is another important protein involved in the regulation of Ca2+-stimulated synaptic vesicle exocytosis (Buckley and Kelly, 1985). Three isoforms (SV2A, SV2B, SV2C) encoded by different genes show differential spatial and temporal patterns of expression in the developing and adult mouse retina (Wang et al., 2003). At maturity, SV2A is present in cone terminals and in the IPL, SV2B in ribbon synapses between rods/cones and BCs, and SV2C in starburst ACs and other conventional synapses in the IPL. All three show a central-to-peripheral gradient of developmental expression and are already present before the onset of synaptogenesis. Synaptic vesicle protein 2A shows a transient high level of expression in HCs, at the time when they start forming contacts with cone photoreceptors.

In the last decade, studies on the SNARE complex have advanced our understanding of molecular mechanisms of synaptic release. The SNARE complex is a family of proteins that is essential for the fusion of synaptic vesicles with the terminal plasma membrane (Sollner et al., 1993). According to the SNARE hypothesis, vesicles fuse with the membrane of the terminal through the binding of a VAMP (vesicle-associated membrane protein) with two proteins on the terminal membrane, SNAP-25 (synaptosomal-associated protein of 25 kDa)

and syntaxin (Rothman and Warren, 1994). The SNARE complex then triggers additional events that lead to vesicular fusion. Hence, studying the expression of SNARE complex proteins during development provides important insight about mechanisms of synapse formation. Prior to eye opening, cholinergic ACs exhibit transient high levels of SNAP-25 (Brazilian opossum Monodelphis domestica: West Greenlee et al., 1998; rat: West Greenlee et al., 2001), but not of syntaxin and VAMP (West Greenlee et al., 2001). As development proceeds, towards eye opening SNAP-25 expression decreases to basal levels. The transient high expression of SNAP-25 temporally correlates with the period during which cholinergic ACs participate in the generation of spontaneous waves of activity (see next section), suggesting that the regulation of SNARE protein expression is important for retinal wiring. Perhaps high levels of SNAP-25 facilitate interactions between acetylcholine-containing synaptic vesicles and amacrine terminal membranes, thereby causing a transient increase in the probability of release. Syntaxin and SNAP-25 expression precedes the expression of VAMP both in the IPL and OPL (West Greenlee et al., 2001), suggesting a role for these two proteins during synaptogenesis.

A detailed study of the chronological appearance of 11 membrane and membraneassociated synaptic proteins in mouse ribbon synapses shows that several synaptic membrane proteins appear before ribbon synapses maturation, and that some are initially found at extra-synaptic locations (Kriegstein and Schmitz, 2003). However, all proteins cluster at ribbon synapses within a short temporal window, between P4 and P5, several days earlier than ribbon synapses mature morphologically, between P7 and P12.

Ribbon synapses contain scaffolds of highly specialized proteins associated with synaptic release at the active zone. Bassoon is one of these structural proteins, and it is found in photoreceptor ribbon synapses (Brandst¨atter et al., 1999). Bassoon is important for the formation of these synapses during development. Indeed, in mice lacking a functional Bassoon protein, few ribbon synapses develop, and they do not appear normal. Ribbons fail to anchor to the presynaptic active zone, resulting in impaired neurotransmission at these synapses as well as in the formation of ectopic synapses and abnormal dendritic branching in second-order neurons (Dick et al., 2003).

The extracellular matrix influences synapse formation

Many factors undoubtedly play a role in the appropriate wiring of retinal circuits. Laminin, which is part of the extracellular matrix, is one of these factors. Laminins are a complex family of extracellular glycoproteins with α, β and γ chains (Timpl, 1996). Beta2containing laminins are expressed on the apical surface of the retina and in the OPL and they are important in retinal development, including synapse formation (Libby et al., 1999). Indeed, laminin β2 chain-deficient mice exhibit abnormal synaptic contacts between rods and second-order neurons while synapses in the IPL appear normal. Triads are rare in these animals, whereas dyads are more common. The most striking malformation is the presence of floating ribbons, presynaptic specializations not apposed to any postsynaptic structure. Another type of laminin, containing the α4 chain, is present in the inner retina (Libby

et al., 2000), suggesting a role for extracellular matrix components in synapse formation in the IPL as well.

13.2.2 Electrical synapses

Gap junctions allow electrical and metabolic communication between adjacent cells. They are formed by the apposition of two hemichannels or connexons, each made of six large transmembrane proteins called connexins. Connexins exist in many different forms and they are subdivided in four groups, α, β, γ and δ. In the vertebrate retina, connexins 26, 32 and 43 have been identified (in addition to connexin 35 in fish and 36 in mammals). Both the specific cell and connexin types determine particular roles of gap junctions. Extensive networks of gap junctions are present between most cell types in the adult retina, where they are important for the modulation of responses to changing light levels, when network activity shifts from cone pathways in daylight to rod pathways at night.

Gap junctions play an important role during CNS development, including neurogenesis, cell death, pattern formation and synchronization of impulses across neuronal populations during synapse formation (synapse elimination or strengthening). The precise role of gap junctions during these major developmental events and the exact nature of the signals that are transmitted between cells through these channels remain to be elucidated.

It is known that gap junctions are absent during the early stages of synaptogenesis in the monkey IPL, not appearing until the density of chemical synapses has reached mature levels (Nishimura and Rakic, 1985). Immature A and B type HCs in the rabbit retina show homologous coupling (i.e., coupling between cells of the same type) at birth, long before synapse formation is completed, suggesting that direct communication between these cells is essential for OPL maturation (Johnson et al., 2000).

Most of our knowledge of the role of gap junctions during retinal development comes from studies on the developing chick retina (reviewed in Becker et al., 1998). Connexins 26, 32 and 43 are all expressed in the immature chick retina, but their expression changes with development. As a general rule, these connexins are expressed at transient high levels during embryogenesis, while cells proliferate and migrate, and during the period of spontaneous correlated activity (see Section 13.3). Connexin 43 is the first to be detected in the neuroepithelium of the eyecup, followed by connexins 32 and 26 at embryonic day (E)4 to E4.5, when the first RGCs are born. By E5, ganglion cells are connected to few other cells, and coupling increases until E11, when clusters of about a dozen cells are connected, with their soma in the ganglion cell layer (GCL) or in the INL (Becker et al., 2002). High levels of connexin 32 correlate with coupling between RGCs, and it is suggested that this type of communication between ganglion cells is involved in the regulation of new RGC differentiation. Connexin 43 is the only one of the connexins expressed in the pigment epithelium, and it is suggested that it regulates neuronal cell proliferation. Indeed, when its expression is knocked down eyes become smaller than normal (Becker et al., 1998). In support, the rate of cell proliferation is slower in albino animals (Ilia and Jeffery, 1996). A new study reports that ATP, released from the retinal pigment epithelium onto the neural retina via

connexin 43 hemichannels, regulates cell proliferation in the neural retina (Pearson et al., 2005). Connexin 43 and 26 levels decrease at the onset of cell death, which in the chick retina is between E9 and E12.

Of greater relevance to the theme of this chapter, is the fact that connexin levels decrease to normal between E14 and E18, during the period of synapse formation. Connexin 26 shows a delimited transient increase (lasting about one day) on dendritic processes of starburst ACs in the OFF sublamina of the IPL during synaptogenesis. As yet, however, its role in synapse formation is not understood. By E14, coupling between RGCs decreases to clusters of three to four cells, all of the same morphological type (Becker et al., 2002).

Although gap junctions do not appear to mediate correlated spontaneous activity in the immature inner retina after the onset of synaptogenesis (see Section 13.3), they seem to mediate vertically propagating spontaneous Ca2+ waves that travel throughout the thickness of the chick embryo retina prior to synapse formation (Catsicas et al., 1998). This indicates that gap junctions are important for communication between retinal layers during early development, but the role of these vertical waves remains to be unravelled. Gap junctions have also been recently reported to mediate early lateral waves in the rabbit, prior to synapse formation (Syed et al., 2004a).

13.3Early synaptic network-driven neural activity

13.3.1Spontaneous bursting activity in immature ganglion cells and amacrine cells

Once conventional synaptic contacts have been established between ACs and RGCs in the IPL, long before vision is even possible, spontaneous network-driven activity begins in the inner retina, occurring first between ACs and RGCs (reviewed in Wong, 1999; Sernagor et al., 2001). Intracellular recordings reveal the presence of spontaneous excitatory and inhibitory synaptic responses in ganglion cells before eye opening in mammals (rat: Rorig and Grantyn, 1993; mouse: Johnson et al., 2003); and in turtle, spontaneous synaptic events are present even at early embryonic stages (Sernagor et al., 2001). In mice, the frequency of these early events increases after eye opening, and this is due to a higher probability of release from presynaptic BCs and ACs (Tian and Copenhagen, 2001).

These early spontaneous synaptic responses give rise to action potentials in RGCs. Indeed, in vitro extracellular recordings from the neonatal rabbit retina first revealed that developing RGCs periodically fire bursts of action potentials in the absence of light stimulation (Masland, 1977). Subsequently, Galli and Maffei (1988) (see also Maffei and Galli-Resta, 1990) succeeded in demonstrating that spontaneous bursting activity is present in vivo in the fetal rat retina. In mammals, these discharge patterns gradually switch from brief periodic bursts to more sustained firing with increasing age (Tootle, 1993; Wong et al., 1993). In comparison, RGCs in turtle exhibit a wide variety of spontaneous discharges throughout development (Sernagor and Grzywacz, 1995; Grzywacz and Sernagor, 2000; Sernagor et al., 2001). Rhythmic bursting activity therefore appears to be a general characteristic of the immature retina. It has been recorded in other parts of the nervous system

Figure 13.2 Spontaneous waves of activity in the developing retina. The upper panels depict an array of 60 neighbouring extracellular electrodes (each dot represents one electrode) used to record spontaneous bursting activity from the GCL (the retina is placed with the GCL facing down onto the electrodes). The activity (not from real data) on one electrode is represented by a small waveform, and the amount, by the size of the waveform. Each panel represents activity from the same retina at 1 s intervals. At time 0, spontaneous activity is detected on two electrodes in the upper left side of the array. The activity propagates diagonally to the bottom right. The curved arrow in the last panel depicts the trajectory of the wave from start to end. The three lower panels of the figure illustrate the trajectory of the centre of mass of retinal waves recorded at different developmental stages in turtle, with the asterisk representing the point of origin of the activity. At S22 (three weeks before hatching, when waves emerge), the waves propagate in a direct line, illustrated by the relatively straight trajectory. Two weeks later, at S25, the waves are much more winding (and slower). In the particular example depicted here, the wave goes first downward and then turns left and upward. At hatching (S26), waves stop propagating, and become replaced by stationary patches of coactivated cells. Consequently, it is not possible to detect a clear wave trajectory anymore at that time.

(O’Donovan, 1999), suggesting that such activity may serve a fundamental role in neural development.

13.3.2 Retinal waves: how and why?

An important and rather unique feature of this spontaneous bursting activity is that it is temporally correlated between neighbouring cells, as first reported by Maffei and GalliResta in 1990. Using a multielectrode array that allowed them to record simultaneously from large populations of RGCs (in cat and ferret), Meister et al. (1991) discovered that this bursting activity propagates across the retina as waves (Figure 13.2). These waves are rather slow, occurring on the order of every few minutes; they can originate at any point in the retina and then propagate in random directions and patterns (Meister et al., 1991;