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

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Figure 6.1 Mechanisms of neurotransmitter release during development. A number of potential mechanisms could be involved in the release of neurotransmitters prior to synaptogenesis. These include vesicular release, diffusion or ‘leakage’ across the cell membrane, carrier proteins and reversed action of uptake transporters and transient opening of gap-junction hemichannels.

vesicles, which transiently fuse with the cell membrane, discharging their contents into the synaptic cleft. Neurotransmitters diffuse across the cleft to the postsynaptic terminal of a target neuron and activate the appropriate receptors. However, at the stage of development considered in this chapter, synaptic connections have yet to form, which raises the question of how neurotransmitters are released. There are, in fact, several mechanisms that could enable neurotransmitter release prior to the maturation of synapses (Figure 6.1).

Elements of the presynaptic exocytotic machinery are expressed very early in development. In the chick, synaptotagmin, syntaxin, synaptic vesicle-2 and dynamin, all components of the vesicular release mechanism, are expressed in immature retinal ganglion cells (RGCs) as early as embryonic day (E)4 (Bergmann et al., 1999, 2000; Grabs et al., 2000). Specific vesicular transporters for GABA (VGAT) and glutamate (VGLUT1 and VGLUT2), which are required to package GABA or glutamate into vesicles ready for release, are also expressed before synapse formation in the rodent retina (Johnson et al., 2003; Sherry et al., 2003). Thus, differentiating cells in the immature retina express many components of the vesicular release mechanism and may be capable of vesicular release prior to synaptogenesis.

Neurotransmitters can also be released by non-vesicular mechanisms. These include ‘leakage’, or diffusion, of neurotransmitter molecules across the plasma membrane (Katz and Miledi, 1977), transport on carrier proteins and release through membrane channels (Figure 6.1). In the mature nervous system, diffusion across the plasma membrane has little effect on neuronal signalling since this ‘leaked’ transmitter is largely restricted to the synaptic cleft and rapidly broken down by enzymes or sequestered by uptake mechanisms.

However, during development, when the mechanisms that terminate transmitter action are still maturing, this leakage may represent a significant source of release into the extracellular environment.

Non-vesicular release of GABA and glutamate has been observed in several preparations and is thought to occur via the reversed action of uptake transporters (Attwell et al., 1993; Schwartz, 1987) or exchangers (Warr et al., 1999), which normally act to terminate neurotransmitter action by removing it from the extracellular space and that are expressed during early retinal development (see above). Another non-vesicular GABA-release mechanism has been described for the immature cortex (Demarque et al., 2002). Here, release was unaffected by traditional blockers of vesicular release, such as voltage-gated Na+ and Ca+ channel blockers and botulinium toxin. Demarque et al. (2002) also considered reverseduptake and exchange-mediated release improbable, since inhibition of GAT-1 had no effect on release. However, since other transporters are expressed at these early times, release mediated by exchangers should not be ruled out yet and more work is required to determine the precise mechanisms involved. Recently, Yang and Kunes (2004) demonstrated that, in the developing Drosophila retina, ACh is also released via a non-vesicular mechanism and is important in the regulation of photoreceptor (PR) axon guidance, although the details of the release mechanism were not determined.

A novel mechanism of transmitter release, involving gap-junction hemichannels, has been revealed by recent investigations. Gap junctions are large intercellular channels formed by the docking of two connexin hemichannels, one contributed by each cell. The opening of ‘undocked’ hemichannels permits communication between a cell’s cytoplasm and the extracellular space, and the passage of a variety of molecules, including ATP (Cotrina et al., 1998, 2000; Stout et al., 2002; Bennett et al., 2003). We now know that hemichannels open spontaneously under physiological conditions during retinal and cortical development. In the embryonic chick retina, hemichannels on the retinal-facing surface of the retinal pigment epithelium (RPE) open and release ATP into the subretinal space, which subsequently stimulates proliferation of the underlying progenitor cells (Pearson et al., 2005a). Similarly, in the embryonic cortex, radial glial cells release ATP via a hemichannel-dependent mechanism, which acts to promote their proliferation (Weissman et al., 2004).

6.2.2 Sources of neurotransmitters in development

The immature retina is responsive to neurotransmitters from very early stages of development. For example, the embryonic chick retina can respond to cholinergic stimulation by E3 (see Section 6.2.5). However, for these responses to be physiologically relevant, neurotransmitters must be present at the same stage. Where might these neurotransmitters come from? One major potential source is the population of immature neurons. Neurogenesis begins as early as E2 in the chick retina and, as described above, neurotransmitters can be released prior to synapse formation. Immunocytochemical and biochemical studies have shown that ACh is the predominant neurotransmitter in a subpopulation of amacrine cells (ACs)

called ‘starburst’ amacrines (Baughman and Bader, 1977; Hayden et al., 1980), which are involved in the processing of direction and movement and born from E3 in the chick. During development, immature horizontal cells (HCs) may also release ACh, as cholinergic markers are transiently expressed in the outer mammalian retina prior to synaptogenesis (Kim et al., 1999, 2000). Other markers of cholinergic neurons, such as acetylcholinesterase, can also be found by E3 in the chick (Layer, 1991).

In the adult retina, GABA is synthesized from glutamate by the enzyme, glutamic acid decarboxylase, and released by ACs and HCs. da Costa Calaza et al. (2000) examined the developmental profile of glutamic acid decarboxylase expression in chick retina. This enzyme is present at low levels between E3 and E6, as revealed by immunohistochemical staining, but increases rapidly after E6, a time that corresponds with the peak of AC and HC birth. Similarly, autoradiographic studies of GABA uptake (the mechanism by which GABA is removed from the extracellular space) has shown that cell bodies and processes in the central regions of the retina label at E6, but no labelling occurs in the ventricular zone (VZ) (Frederick, 1987), and immunolabelling studies have shown that the GAT-1 GABA transporter is present at E8 (Catsicas and Mobbs, 2001). Consistent with these findings, Frederick further demonstrated that HC, AC and RGCs all label positively for GABA uptake by E8, although progenitor cells remain unlabelled (Frederick, 1987). Thus, the expression of GABA transporters and synthesizing enzymes appear to be features of differentiating, but not proliferating, cells.

Purine nucleotides play important roles as neurotransmitters and neuromodulators in the mature CNS. Potential sources of extracellular purines include both neurons and glia. In the adult nervous system, ATP is co-released with other neurotransmitters from adrenergic, cholinergic, glutamatergic and GABAergic neurons (for review, see Burnstock, 2004). Adenosine triphosphate is also released via a synaptic-independent mechanism (see Section 6.2.1) from the embryonic RPE (Pearson et al., 2005a) and M¨uller glia (Newman, 2001) and, in the embryonic cortex, from radial glia (Weissman et al., 2004).

6.2.3 Modes of action of neurotransmitters

Neurotransmitters exert their actions largely by activating one of two families of membranebound receptors, ionotropic and metabotropic receptors. Ionotropic receptors are predominantly involved in fast synaptic transmission and the receptor-binding site is directly coupled to an ion channel. Neurotransmitter binding causes the ion channel to open, allowing the passage of particular ions, such as Na+, K+, Ca2+ or Cl−. Depending on which ions pass through the channel, a neurotransmitter may depolarize the cell (e.g. ACh acting on the nicotinic ACh receptor (nAChR)) and hence be excitatory, or hyperpolarize it (e.g. GABA acting on the GABAA receptor), and exert an inhibitory effect. Metabotropic receptors are G-protein- coupled receptors. Transmitter binding leads to the activation of a G-protein that, depending on the type of G-protein, activates one of several effector systems including the phospholipase C/inositol(1,4,5)-triphosphate (IP3)/Ca2+ system, the adenylate cyclase/cAMP system, or by direct action on ion channels such as K+ and Ca2+ channels.

6.2.4 Neurotransmitters, [Ca2+]i and development

How do neurotransmitters exert their effects on early developmental events? Neurotransmitters act to alter a number of aspects of cell function including membrane voltage, enzyme activity and [Ca2+]i. The latter is of particular interest since all the early embryonic transmitter systems act to modulate [Ca2+]i. It is also one of the responses most amenable to study and manipulation, and thus much of the research into the role of neurotransmitters in retinal development has focused on these transmitter-evoked [Ca2+]i changes and their downstream consequences. Intracellular Ca2+ transients are frequent in the developing retina and can be patterned temporally, as periodic oscillations, and spatially, as transients occurring in single cells, as synchronized events occurring in small groups of neighbouring cells, or as large-scale propagating waves (see below and Figure 6.4).

Calcium has a key influence on many developmental events in the CNS and is implicated in the regulation of proliferation, migration, differentiation and circuit formation. Intracellular Ca2+ transients may be required for progression through several steps in the proliferative cell cycle including the G1/S-phase transition, S-phase itself, entry into mitosis and key points within mitosis including the metaphase–anaphase transition and induction of cytokinesis (see Berridge, 1995; Santella, 1998; Santella et al., 1998; Whitaker and Larman, 2001). Changes in [Ca2+]i are also required for the movement of progenitor cells between the VZ and vitreal surface in G1 and G2 of the cell cycle (Pearson et al., 2005b).

Intracellular Ca2+ levels play a major role in the regulation of neuronal differentiation. Imaging studies have shown that multiple spontaneous [Ca2+]i transients occur during the maturation of neurons (Gu and Spitzer, 1993, 1995; Gu et al., 1994) and that they control a variety of developmental events via information encoded in their frequency, amplitude and duration. These include the maturation of K+ currents, neurotransmitter synthesis and receptor expression (Spitzer et al., 1993), and the regulation of the rate of neurite extension, the outgrowth of which is inversely related to the frequency of growth cone-restricted [Ca2+]i transients. Growth-cone stalling, axon retraction and growth-cone turning have all been found to be associated with subtle changes in the frequency and amplitude of [Ca2+]i transients (Gu and Spitzer, 1995; Dolmetsch et al., 1998; Zheng et al., 1994, Zheng, 2000) although the details of the molecular mechanisms underlying these processes remain to be determined. Changes in [Ca2+]i are also required for the translocation of immature neurons, as described in the developing cortex (Komuro and Rakic, 1998). The possibility that [Ca2+]i transients might provide a route by which neurotransmitters could influence retinal development is considered in greater detail later in this chapter (see p. 111).

6.2.5 Early expression of neurotransmitter receptors in the embryonic retina

ACh receptors in the developing retina

Acetylcholine, the classic fast excitatory neurotransmitter of the peripheral nervous system, acts at (1) nAChRs, which are ligand-gated ionotropic receptors that are selectively activated by nicotine-like ligands, and permeable to Na+ and K+, and (2) muscarinic (m) AChRs.