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

The five subtypes of mAChR belong to the superfamily of metabotropic G-protein-coupled receptors. The M1, M3 and M5 receptors cause the release of Ca2+ from IP3-sensitive intracellular stores, whilst the M2 and M4 receptor subtypes reduce cAMP levels via the inhibition of adenylate cyclase.

In the chick, nAChR mRNA is present by E4.5 (Hamassaki-Britto et al., 1994). Its expression appears to be restricted to differentiating, rather than proliferating, cells; imaging studies have shown that nicotinic agonists are unable to evoke responses from progenitor cells (Wong, 1995a; Pearson et al., 2002), the responses instead arising from immature RGCs and ACs in the ganglion cell layer (GCL) and inner nuclear layer (INL) (Wong, 1995a). The expression of nAChRs by these cells is maintained in the adult (Keyser et al., 1988; Wada et al., 1989; Cauley et al., 1990). In the adult retina, mAChRs are expressed in AC, RGC and bipolar cells (BCs) and, in the case of M3, in the RPE (Fischer et al., 1998). They are also present in both the avian and mammalian retina from early in development (McKinnon and Nathanson, 1995; Wong, 1995a; Pearson et al., 2002), although the receptor subtypes expressed change with time. Immunoprecipitation and immunoblot analyses in the chick show that the M4 subtype predominates during the first week of development, whilst the expression of M3 increases moderately, and that of M2 dramatically, during the second week of development (E7 to E14) (Nadler et al., 1999).

Non-confocal Ca2+-imaging studies demonstrate that stimulation of mAChRs leads to marked increases in [Ca2+]i as early as E3 in the chick (Figure 6.2a; Yamashita and Fukuda, 1993; Yamashita et al., 1994; R.A.P, unpublished observations), and E20 in the rabbit (Wong, 1995a), retina. Non-confocal imaging does not permit the identification of the individual cells that respond to a given agonist since it also records changes in fluorescence from outside the focal plane. However, confocal-imaging studies have further demonstrated that mAChRs are expressed by cells throughout the immature neural retina, including dividing progenitor cells (Movie 6.1 online; Pearson et al., 2002). The activation of mAChRs leads to the release of Ca2+ from intracellular stores, causing oscillatory changes in [Ca2+]i (Yamashita et al., 1994; Pearson et al., 2002). The proportion of progenitor cells responding to muscarinic agonists is maximal during the peak of neurogenesis ( E6) but declines rapidly thereafter. The response is virtually absent by E8 in the chick (Figure 6.2a) (Yamashita et al., 1994; Pearson et al., 2002; R.A.P., unpublished results) and postnatal day (P)11 in the rabbit (Wong, 1995a), remaining only in differentiating neurons in the inner retina.

ATP receptors in the developing retina

Adenosine triphosphate and its derivatives stimulate P2 receptors, of which there are two classes, P2Y and P2X. The P2Y receptors are G-protein-coupled receptors, predominantly coupled to the IP3/Ca2+ cascade, although some isoforms are linked with cyclic adenosine monophosphate (cAMP) and the stimulation of tyrosine kinases and mitogen-activated protein kinases (MAPKs). The P2X receptors are ligand-gated ion channels, permeable to Na+, K+ and Ca2+.

Purines play important roles in development from the moment of fertilization. The ATP receptors are amongst the earliest functionally active membrane receptors, present in the

Figure 6.2 Neurotransmitter-evoked changes in [Ca2+ ]i in the embryonic retina. (a) The neurotransmitters ACh, ATP, GABA and glutamate evoke increases in [Ca2+ ]i at different times during embryonic retinal development. Diagram illustrates when the early immature neural retina is responsive to these neurotransmitters, as determined from Ca2+ -imaging studies in the chick retina (see text). It should be noted that ACh and ATP play different and essential roles in the proper functioning of the adult tissue and may also modulate [Ca2+ ]i in differentiated cells. NB. Arrowheads indicate the latest time point in development studied in the current literature, for a given neurotransmitter receptor. It is possible that these responses continue later into development but await further investigation. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; KA, kainate; NMDA, N-methyl-D-aspartate; PCD, programmed cell death; UTP, uridine triphosphate. (b) Example traces of the changes in [Ca2+ ]i, as measured by a change in fluorescence intensity ( F/F), evoked in chick retinal progenitor cells by muscarinic (carbachol), purinergic (UTP), GABAergic and glutamatergic agonists. Adapted from Pearson et al. (2002).

embryo at the time of germ layer formation (Laasberg, 1990). However, there is little systematic data on the developmental expression patterns of the different receptor subtypes. Purine 2Y-like receptors are expressed throughout the proliferative period of development in the embryonic chick neural retina (Sakaki et al., 1996; Sugioka et al., 1996; Pearson et al., 2002). In the adult rat retina, glial cells express P2Y2 and P2Y4 receptors, and P2X2, P2X3, P2X4 and P2X7 receptors have been identified in RGCs (Greenwood et al., 1997; Wheeler-Schilling et al., 2001) and in the M¨uller cell population of the mammalian and human retina (Greenwood et al., 1997; Br¨andle et al., 1998; Pannicke et al., 2000; WheelerSchilling et al., 2001). Purinergic receptors are also present in the embryonic (Pearson et al., 2005a) and adult (Mitchell, 2001) RPE.

In the developing retina, ATP acts largely on progenitor cells, rather than immature neurons, evoking Ca2+ mobilization by activating P2Y receptors (Figure 6.2; Pearson et al., 2002; Sugioka and Yamashita, 2003). In the chick, the number of progenitor cells responding to ATP is largest at E3, declines dramatically towards E6 and is minimal from E8 onwards (Figure 6.2a; Sakaki et al., 1996; Sugioka et al., 1996; Pearson et al., 2002). This developmental decrease parallels the time course of proliferation, which is highest in the first few days of development and ceases around E8 in the chick retina (Prada et al., 1991), raising the possibility that purines may be involved in the regulation of proliferation.

GABA receptors in the developing retina

γ -Aminobutyric acid, the major inhibitory neurotransmitter of the CNS, acts on three receptor subclasses, GABAA, GABAB and GABAC. The GABAA and GABAC receptors are ligand-gated ion channels and demonstrate great pharmacological and functional diversity (Rudolph et al., 2001). In the adult CNS, GABA is an inhibitory neurotransmitter; activation of the GABAA receptor opens an integral Cl−-permeable channel, and causes the cell to hyperpolarize. In early development, [Cl−]i is high and GABAA receptor activation results in Cl− leaving the cell, causing a depolarization that activates L-type voltage-gated Ca2+ channels and Ca2+ influx (Segal and Barker 1984; Cherubini et al., 1991). γ -Aminobutyric acid is understood to exert trophic actions via this rise in [Ca2+]i. The high [Cl−]i occurs due to a delay in the maturation of the Cl− extrusion system, relative to Cl− uptake mechanisms (Nishi et al., 1974; Frambach and Misfeldt, 1983; Zhang et al., 1991; Reichling et al., 1994). The ability of GABA to cause increases in [Ca2+]i via secondary activation of voltage-gated Ca2+ channels decreases as the extrusion mechanism develops. Like the GABAA receptor, GABAC receptors are linked to a Cl− channel whilst GABAB receptors are linked indirectly to Ca2+ and K+ channels via G-protein activation.

All three GABA receptor subtypes are found in the adult retina. The GABAA receptors are expressed by subpopulations of BC, AC and RGCs (Greferath et al., 1994; Karne et al., 1997). They are also present during development, prior to synaptogenesis, and their abundance closely parallels neurogenesis (see Barker et al., 1998). The GABAB receptors are present by E8 in the chick and are located on RGCs and ACs (Catsicas and Mobbs, 2001). The GABAC receptor subunits are not detected until much later in development. In

the mouse, they appear at the end of neurogenesis (postnatal (P)6) and are functional from P9 (Greka et al., 2000). This correlates with the period of BC differentiation as well as eye opening.

The precise timing of when retinal cells become responsive to GABA is unclear. Responses to GABA appear early in retinal development, during the proliferative period, but the evidence for their presence on progenitor cells is equivocal. Non-confocal imaging of whole retina has shown that GABA, acting via GABAA receptors, can evoke changes in [Ca2+]i from as early as E3 in the chick retina (Yamashita and Fukuda, 1993), although the response peaks at around E8 (Allcorn et al., 1996; Catsicas and Mobbs, 2001; R.A.P. unpublished observations) before disappearing by E14 (Figure 6.2; Allcorn et al., 1996; Catsicas and Mobbs, 2001), when the Cl− extrusion mechanisms mature and GABA begins to exert a hyperpolarizing, rather than depolarizing action. From E6, the GABA-evoked Ca2+ response is largely restricted to immature neurons in the GCL and INL (Allcorn et al., 1996; Catsicas and Mobbs, 2001), suggesting that GABA sensitivity may be a property of differentiated neurons. However, in studies of the rat neocortex, whole-cell electrophysiological recordings of VZ cells (presumed to be progenitors, although this was not determined) indicated that they too respond to GABAergic stimulation (LoTurco et al., 1995), as do progenitor-like cells of the regenerating newt retina (Ohmasa and Saito, 2004). It is possible that GABAA receptors are expressed by progenitor cells in both tissues but only shortly prior to terminal division. Consistent with this, more recent studies in the cortex have demonstrated that GABA-evoked changes in membrane potential and [Ca2+]i are confined to cells either in the final round of division or undergoing differentiation; only a very small fraction of the responses arose from cells identified as neural progenitor cells (Maric et al., 2001). Similarly, combined confocal and immunohistochemical studies of the embryonic retinal VZ suggest that, whilst a subpopulation of VZ cells do respond to GABA, it is comprised largely of postmitotic, rather than progenitor, cells (Pearson et al., 2002).

Glutamate receptors in the developing retina

Glutamate, the predominant fast excitatory neurotransmitter of the CNS, activates metabotropic (mGluRs) and ionotropic (N-methyl-D-aspartate (NMDA) and non-NMDA) receptors. Non-NMDA receptors are further divided into α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA)-preferring and kainate-preferring subtypes. The metabotropic mGluR1 and mGluR5 receptors are linked to the IP3/Ca2+ signalling cascade (Abe et al., 1992; Aramori and Nakanishi, 1992), whilst NMDA receptor channels are directly permeable to Na+, K+ and Ca2+. The Ca2+ permeability of the NMDA receptor underlies its roles in development, cell cytotoxity and in learning and memory. Magnesium blocks the channel in a voltage-dependent manner and is relieved by depolarization (Bliss and Collingridge, 1993). The AMPA receptors are permeable to Na+ and K+ and usually have low permeability to Ca2+, although certain isoforms may be highly permeable to Ca2+; AMPA receptors lacking an edited version of the GluR2 subunit, or made up of GluR1, GluR3 and GluR4 subunits, are Ca2+ permeable (Murphy and Miller, 1989; Pruss et al., 1991;

Burnashev et al., 1992). Interestingly, the Ca2+-permeable form of the AMPA receptor is prevalent during early development but decreases with maturation (Pellegrini-Giampietro et al., 1992), suggesting that this isoform may have a specific role(s) in development.

Glutamate is the main neurotransmitter of RGCs, BCs and PRs in the vertebrate retina. The distribution of glutamate receptors has been described for the adult retina of several species, although the precise distribution may vary between species. In the postnatal chick, all subunits of the AMPA/kainate receptors and the NR1 subunit of the NMDA receptor are expressed in the INL and the GCL, predominantly by ACs and RGCs (Santos Bredariol and Hamassaki-Britto, 2001). Expression of the majority of glutamate receptor subtypes is low prior to synaptogenesis, increasing as development progresses (Cristovao et al., 2002a,b). However, Western blot analyses have shown that a number of glutamate subunits are also present much earlier in development (E5) (Santos Bredariol and Hamassaki-Britto, 2001), although their expression is largely restricted to immature neurons.

Interestingly, electrophysiological recordings from retinal cell cultures, and Ca2+- imaging studies in whole-mount retinas, have shown that the Ca2+-permeable forms of the AMPA/kainate receptor are functional from E5 to E6 in the chick retina (Allcorn et al., 1996; Sugioka et al., 1998). The AMPA/kainate receptor-mediated Ca2+ rises are maximal at E9 to E10, before declining towards E12 and the onset of synaptogenesis (Figure 6.2; Sugioka et al., 1998). The cells that respond to glutamate at these early stages are largely immature RGCs and ACs, located in the presumptive INL and GCL (Rorig and Grantyn, 1994; Wong, 1995b; Allcorn et al., 1996; Sugioka et al., 1998). Studies of transdifferentiating RPE (which can grow to reform the neural retina) and retinal re-aggregation models have similarly found that a response to glutamate is only prominent once differentiation has commenced (Naruoka et al., 2003).

Glutamate-evoked Ca2+ changes have been observed in some cells in the VZ of the E6 chick (Allcorn et al., 1996; Pearson et al., 2002), and E20 rabbit (Wong, 1995b), retina. However, the response to glutamate, like that to GABA, appears to be mainly restricted to a population of non-dividing cells (Pearson et al., 2002). Maric et al. (2000) have shown, using whole-cell patch clamping and Ca2+ imaging, that glutamate-evoked inward currents and associated increases in [Ca2+]i are only seen in differentiating cortical neurons. In contrast, ionotropic agonists failed to evoke changes in voltage in progenitor cells. However, a subpopulation of cells that stained positively for both 5 -Bromo-2 -deoxy-uridine (BrdU; a marker of proliferation) and TuJ-1 (an antibody against neuron-specific β-tubulin and a marker of differentiated neurons) did show increases in [Ca2+]i in response to AMPA, suggesting that functional ionotropic glutamate receptors are expressed at the time of terminal cell division and early differentiation (Maric et al., 2000). A similar situation may pertain in the retina, although further studies will be required to confirm this. In contrast, NMDA receptor-mediated responses to glutamate are absent during early retinal development (Allcorn et al., 1996; Sugioka et al., 1998; Pearson et al., 2002) and appear later, in differentiated cells, near the onset of synaptogenesis (Somahano et al., 1988; Wong, 1995b; Duarte et al., 1996; Catsicas et al., 2001). On the basis of these findings, it seems

likely that NMDA and non-NMDA glutamate receptors adopt different roles during retinal development, and that non-NMDA receptors alone are involved in early developmental processes.

6.2.6 The role of neurotransmitters in early retinal development

Regulation of progenitor cell cycle by cholinergic and purinergic neurotransmitters

During early development, progenitor cells divide rapidly to expand the pool of retinal cells. The onset of neurogenesis overlaps with this proliferative period (Prada et al., 1991). That muscarinic and purinergic neurotransmitters are expressed at these times (see Section 6.2.2) raises the possibility that they might modulate proliferation and/or differentiation. Interestingly, the response to purinergic stimulation is maximal during the proliferative period (Sakaki et al., 1996; Sugioka et al., 1996; Pearson et al., 2002), whilst the muscarinic response, although prevalent at early times, is maintained throughout the peak of neurogenesis (Yamashita et al., 1994; Wong, 1995a; Sakaki et al., 1996; Pearson et al., 2002), suggesting that these two neurotransmitters might play different roles in development. Consistent with this, activation of purinoceptors increases both retinal progenitor cell DNA synthesis (a measure of the number of cells re-entering the cell cycle) (Sugioka et al., 1999; Sanches et al., 2002) and speeds their mitosis, effects that lead to enhanced proliferation and bigger eyes (Pearson et al., 2002, 2005a) (Figure 6.3). In contrast to the proliferative actions of ATP, muscarinic stimulation appears to act as a brake on mitosis, almost doubling the time it takes for cells to divide (Pearson et al., 2002). Muscarinic agonists similarly reduce DNA synthesis in embryonic rat retinal cultures (Santos et al., 2003), whilst in the chick, more prolonged exposure to muscarinic agonists or antagonists leads to smaller or larger eyes, respectively, and corresponding changes in the number of proliferating cells (Pearson et al., 2002). However, there are reports that earlier exposure to muscarinic agonists can increase eye size in the chick (Angelini et al., 1998) and affect nuclear status in the sea urchin (Harrison et al., 2002). The reasons for these apparently opposing effects of ACh are not yet clear.

In the embryonic cortex, GABA and glutamate have been shown to influence proliferation by changing the duration of the progenitor cell cycle; both GABA and glutamate increase the size of cortical VZ clones (Haydar et al., 2000). In contrast, LoTurco et al. (1995) have shown that GABA and glutamate decrease the number of dissociated, embryonic cortical cells synthesizing DNA. When applied alone, GABAA and AMPA/kainate receptor antagonists increase proliferation by increasing DNA synthesis (suggesting that GABA or glutamate might act to repress proliferation), but decrease it when applied together. LoTurco and colleagues suggest that glutamate and GABA bring about their actions through depolarization-evoked [Ca2+]i increases, similar to those evoked by these transmitters in the embryonic retina (Yamashita and Fukuda 1993; Wong, 1995b; Pearson et al., 2002). However, in contrast to findings in the cortex, the Ca2+ influxes evoked by GABA and glutamate do not appear to regulate mitosis in the retina (Pearson et al., 2002), a finding perhaps consistent with the idea that GABA and glutamate receptor expression might be

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Control UTP

Figure 6.3 Neurotransmitters can modulate proliferation in early retinal development. ATP acts at P2Y receptors to stimulate the proliferation of neural retinal progenitor cells during early development. (A) Example images from a confocal time-lapse series of mitosis in the chick retinal VZ. The chromatin of the mitotic cell progresses from prophase (0 s), through metaphase (90 to 1500 s) and into anaphase (1650 s onwards). Scale bar 5 µm. (B) Mitosis in the embryonic neural retinal VZ is faster in the presence of purinergic agonists. Histogram shows the time spent in mitosis by progenitor cells in either control solution or in the presence of the purinergic P2Y-R agonist, UTP (left). The proliferative actions of purinergic agonists decrease with age. Histogram (right) shows that the time progenitor cells spend in mitosis is no longer affected by purinergic agonists at E8. N, number of retinas; n, number of cells. (C) Purinergic agonists increase proliferation in the early embryonic neural retina. Histogram shows the % of BrdU-positive cells found following 8 hour exposure to UTP, compared with controls, in the E5 retina. (D) Increased proliferation following exposure to purinergic agonists can lead to increases in eye size, compared with controls. Scale bar 2 mm. Adapted from Pearson et al. (2005a).

correlated with differentiation, rather than proliferation (Wong, 1995b; Maric et al., 2000, 2001; Pearson et al., 2002). An interesting possibility is that, in the neocortex, the actions of GABA and glutamate may be to depolarize differentiated neurons and bring about the release of some other factor, such as ACh, which then acts as a brake on the cell cycle. This does not appear to be the case in the retina, however, for both glutamatergic, and GABAergic, stimulation and blockade were without effect on mitosis or eye development (Pearson et al., 2002). Nevertheless, since transmitter systems interact to regulate other aspects of development (Catsicas et al., 1998; Wong et al, 2000), this possibility is worthy of further investigation.

Neurotransmitters and the modulation of Ca2+ transients in the proliferating retina

It is possible, as mentioned above, that neurotransmitters exert their effects on the cell cycle via their ability to modulate [Ca2+]i. Both [Ca2+]i transients in single cells and locally synchronized events between adjacent progenitors in the retina (Pearson et al., 2002, 2004; Sugioka and Yamashita, 2003), and the cortex (Owens and Kriegstein, 1998), occur during the proliferative period of development (Figure 6.4). Spontaneous [Ca2+]i transients in single cells (Figure 6.4b, Movie 6.2 online) are independent of action potentials, as demonstrated by their insensitivity to the Na+ channel blocker tetrodotoxin (Owens and Kriegstein, 1998; Pearson et al., 2002), but are modulated by neurotransmitters. In the chick retina, both muscarinic and purinergic receptor antagonists reduce the frequency of these transients (Pearson et al., 2002) suggesting that, in vivo, endogenous purines and ACh normally act to evoke [Ca2+]i transients in progenitor cells. Further, exogenous ATP and ACh generate conspicuous changes in [Ca2+]i in retinal progenitor cells and, since their actions are prevented by buffering [Ca2+]i changes, their actions on cell division (described above) are thought to be mediated via a Ca2+-dependent mechanism (Pearson et al., 2002; Sugioka and Yamashita, 2003). In glial cultures, ATP-induced proliferation is similarly accompanied by [Ca2+]i oscillations (Morita et al., 2003), the duration of which regulates cell division (Moll et al., 2002).

Thus, it is possible that the [Ca2+]i signal evoked in progenitors by neurotransmitters, such as ATP, exerts a direct effect on the cell cycle. If this were so, it would provide a mechanism by which molecules such as neurotransmitters could regulate cell division and modulate both cell number and the timing of the production of the different types of CNS cells. For example, progenitor cells initially cycle rapidly, dividing to increase their number. The cell cycle then slows down as neurogenesis proceeds (Caviness et al., 1995). As we have seen, ATP acts to stimulate proliferation in the embryonic retina (Sugioka et al., 1999; Pearson et al., 2002, 2005a), whilst ACh has the opposite effect (Pearson et al., 2002; Santos et al., 2003). Thus, during early retinal development, ATP may act to promote proliferation and expansion of the progenitor pool, whilst later on a decrease or down-regulation of the purinergic signal (see Figure 6.3B) could allow the muscarinic signal to exert a stronger effect, producing a slowing of the cell cycle. Glutamatergic and GABAergic antagonists also caused a reduction in [Ca2+]i transients but, interestingly, only in a population of

time sec

50% ∆F/F

Figure 6.4 Spontaneous [Ca2+ ]i transients in the embryonic chick retina. (A) Schematic diagram of the immature retina showing (left) the proliferative cell cycle and (right) types of Ca2+ activity. (B) spontaneous [Ca2+ ]i in single cells in the VZ. (i) Confocal section through the VZ of an E5 chick retina labelled with the Ca2+ indicator, Fluo-4, (ii) an individual mitotic cell undergoing a single transient, (iii) traces showing transient changes in [Ca2+ ]i in interphase (cells 1, 2 and 4) and a mitotic cell (cell 3) (chromatin labelling not shown). Scale bar 10 µm. (C) Coordinated spontaneous [Ca2+ ]i in groups of adjacent cells. (i) Confocal section through the VZ of an E5 chick retina labelled with Fluo-4, (ii) traces showing the coordinated rise in [Ca2+ ]i in neighbouring cells. Scale bar 10 µm. (D) spontaneous [Ca2+ ]i waves propagate both laterally through differentiating neurons and back into the depth of the retina towards the VZ. (i) Spontaneous [Ca2+ ]i waves in the prospective GCL of an E8 retina, (ii) propagation of a Ca2+ wave across the retina. Fluorescence (non-confocal) images taken through the GCL in an E11 retina loaded with Fura-2. Scale bar 200 µm, (iii) Spontaneous [Ca2+ ]i waves propagate throughout the depth of the retina. The three-dimensional pseudo-colour line-image graph shows how [Ca2+ ]i changes through the depth of a transverse retinal slice from an E11 embryo as a function of time. Warmer colours reflect higher [Ca2+ ]i. Two fast [Ca2+ ]i transients (white arrows) can be seen spanning the depth of the retina from the GCL to the VZ (labelled here PRL, or prospective PR layer). % F/F corresponds to the percentage change in the average normalized fluorescence over the field of view. Adapted from Pearson et al. (2002, 2004) and Catsicas et al. (1998). For colour version, see Plate 6.

non-dividing cells (Pearson et al., 2002). This is consistent with the findings from the pharmacological studies described earlier, that these receptors appear to be expressed by differentiating, rather than proliferating cells (Maric et al., 2000, 2001; Pearson et al., 2002).

Transient changes in [Ca2+]i may also be involved in the regulation of another phase of the cell cycle, a process called interkinetic nuclear migration, where progenitor cell nuclei move between the VZ and the vitreal surface during G1 and G2. Like migrating immature cortical neurons (Komuro and Rakic, 1996), translocation of the nucleus in progenitor cells requires transient changes in [Ca2+]i (Pearson et al., 2005b). Glutamate and GABA receptors have been shown to regulate the migration of immature neurons in the cortex (Komuro and Rakic, 1993; Rakic and Komuro, 1995). Whilst it seems likely that neurotransmitters could modulate the spatiotemporal features of [Ca2+]i events in interkinetic nuclear migration, this possibility remains to be investigated.

One signal, but opposing actions?

The Ca2+ signals generated by both muscarinic and purinergic stimulation arise from the release of Ca2+ from intracellular stores (Sugioka et al., 1996; Pearson et al., 2002). How then might they exert different effects on proliferation? Such differences may be attributable to one or more of several factors; they may be linked to the production of different second messengers or effectors in addition to, or downstream of, Ca2+. Alternatively, different receptors may couple to Ca2+ stores in different locations within the same cell (Short et al., 2000). It is also possible that the ACh and ATP released endogenously produce [Ca2+]i signals of a different magnitude or frequency that code their different effects (Dolmetsch et al., 1997). Support for the first hypothesis comes from studies that demonstrate that MAPK, which is downstream of the IP3/Ca2+ second messenger cascade, is activated in ATP-induced retinal proliferation (Sanches et al., 2002) but is not involved in the muscarinicmediated inhibition of proliferation (Santos et al., 2003).

Neurotransmitters and the modulation of local Ca2+ events in the proliferating retina

In addition to the [Ca2+]i transients in single cells, small groups of adjacent progenitor cells also undergo synchronized rises in [Ca2+]i during the proliferative period of development in the retina (Figure 6.4C; Pearson et al., 2004, 2005b) and the cortex (Owens and Kriegstein, 1998). These local [Ca2+]i waves are likely mediated via a combination of neurotransmitter release and gap-junction coupling between neighbouring cells (Owens and Kriegstein, 1998; Pearson et al., 2002, 2004; Weissman et al., 2004). Recently, Kriegstein and colleagues have shown that waves with similar spatiotemporal characteristics occur in proliferating radial glial cells (which act as progenitor cells during early development). These [Ca2+]i waves are mediated by the spread of ATP released via gap-junction hemichannels from individual glia and act to modulate radial glial proliferation (Weissman et al., 2004). A similar mechanism may exist in the proliferating retina since synchronized [Ca2+]i transients also occur between retinal progenitors (Figure 6.4C; Pearson et al., 2002, 2004). The importance of local synchronization of Ca2+ changes in cell proliferation has yet to be determined.