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Spontaneous Waves in the Ventricular Zone of Developing Mammalian Retina
Abstract
Spontaneous rhythmic waves in the developing mammalian retina are thought to propagate among differentiated neurons in the inner retina (IR) and play an important role in activity-dependent visual development. Here we report a new form of rhythmic Ca(2+) wave in the ventricular zone (VZ) of the developing rabbit retina. Ca(2+) imaging from two-photon optical sections near the ventricular surface of the whole-mount retina showed rhythmic Ca(2+) transients propagating laterally as waves. The VZ waves had a distinctively slow Ca(2+) dynamics (lasting approximately 20 s) but shared a similar frequency and propagation speed with the IR waves. Simultaneous Ca(2+) imaging in VZ and multi-electrode array recording in the ganglion cell layer (GCL) revealed close spatiotemporal correlation between spontaneous VZ and IR waves, suggesting a common source of initiation and/or regulation of the two waves. Pharmacological studies further showed that all drugs that blocked IR waves also blocked VZ waves. However, the muscarinic antagonist atropine selectively blocked VZ but not IR waves at this developmental stage, indicating that IR waves were not dependent on VZ waves, but VZ waves likely relied on the initiation of IR waves. Eliciting IR waves with puffs of nicotinic or non-N-methyl-d-aspartate agonists in GCL produced atropine-sensitive waves in the VZ, demonstrating a unique, retrograde signaling pathway from IR to VZ. Thus differentiated neurons in the IR use spontaneous, rhythmic waves to send both forward signals to the central visual targets and retrograde messages to the developing cells in the VZ.
... We previously reported spontaneous waves in the VZ of E24-P0 rabbit retinas ( Syed et al. 2004). The waves Figure 8. Effects of gap junction blockers on stage II wave A, octanol-1 (100 µM) reversibly blocked the spontaneous waves in the ganglion cell layer of an E30 retina. ...
... Drugs that blocked IR waves, including hexamethonium and 18β-GA, also blocked VZ waves; however, atropine blocked only VZ, but not IR waves between E24 and P0 (stage II). We concluded that VZ waves were initiated in the IR and backpropagated to the VZ ( Syed et al. 2004). ...
... However, in contrast to stage I IR waves which were insensitive to muscarinic antagonists ( Fig. 2A), E22 VZ waves could be completely blocked by atropine (1 µm, n = 2) (Fig. 9C), suggesting that VZ waves required additional muscarinic communication. At stage II (P24-P1), VZ waves were inhibited by nicotinic (hexamethonium, 100 µm), muscarinic (pirenzepine, 2 µm, Fig. 9DE) and gap junction (18β-GA, 75 µm, data not shown) blockers, but not by CNQX (40 µm) (Fig. 9F) or CNQX (40 µm) + AP7 (100 µm), as we previously found (Syed et al. 2004). At stage III (>P2), the VZ waves were blocked by CNQX (25-40 µm) + AP7 (100 µm) (n = 3) and the muscarinic antagonist pirenzepine (2 µm, n = 2) (Fig. 9G). ...
We report here a systematic investigation of the dynamics, regulation and distribution of spontaneous waves in the rabbit retina during the course of wave development prior to eye opening. Three major findings were obtained in this longitudinal study. (1) Spontaneous retinal waves underwent three developmental stages, each of which displayed distinct wave dynamics, pharmacology and mechanism of generation and regulation. Stage I waves emerged prior to synaptogenesis and appeared as frequent, fast propagating waves that did not form spatial boundaries between waves. These waves could be inhibited by blockers of gap junctions and adenosine receptors, but not by nicotinic antagonists. Stage I waves lasted about one day (around embryonic day 22) and then switched rapidly to stage II, resulting in slower and less frequent waves that could be blocked by nicotinic antagonists and had a characteristic postwave refractory period and spatial boundaries between adjacent waves. Immediately after the transition from stage I to stage II, the waves could be reverted back to stage I by blocking nicotinic receptors, indicating the presence of mutually compensatory mechanisms for wave generation. Stage III waves emerged around postnatal day 3-4 (P3-4), and they were mediated by glutamtergic and muscarinic interactions. With age, these waves became weaker, more localized and less frequent. Spontaneous waves were rarely detected after P7. (2) GABA strongly modulated the wave dynamics in a stage- and receptor type-dependent manner. At stage I, endogenous GABAB activation downregulated the waves. The GABAB modulation disappeared during stage II and was replaced by a strong GABA(A/C)-mediated inhibition at stage III. Blocking GABA(A/C) receptors not only dramatically enhanced spontaneous stage III waves, but also induced propagating waves in >P7 retinas that did not show spontaneous waves, indicating a role of GABA inhibition in the disappearance of spontaneous waves. (3) Spontaneous retinal waves were found in both the inner and outer retina at all three stages. The waves in the outer retina (ventricular zone) also showed stage-dependent pharmacology and dynamics. Together, the results revealed a multistaged developmental sequence and stage-dependent dynamics, pharmacology and regulation of spontaneous retinal waves in the mammalian retina. The presence of retinal waves during multiple developmental stages and in multiple retinal layers suggests that the waves are a general developmental phenomenon with diverse functions.
... Third, cholinergic retinal waves are present prior to the formation of conventional synapses, as identified in electron microscopy studies (Fisher, 1979;Greiner & Weidman, 1981). Finally, in rabbit retina, calcium waves propagate through the ventricular zone that are correlated with waves in the ganglion cells (Syed et al., 2004a). The ventricular zone waves are blocked by muscarinic receptor antagonists as well as nAChR antagonists, implying that ACh released during waves in the ganglion cell layer diffuses tens of microns to the ventricular zone where it activates muscarinic receptors. ...
... Blocking muscarinic receptors in chick eyes leads to an increase in the size of the eye (Pearson et al., 2002). A probable source for ACh acting on muscarinic receptors is from cholinergic waves, which drive correlated calcium waves in the ventricular zone (Syed et al., 2004a). In other parts of the nervous system, elevation of intracellular calcium can also influence neuronal migration (reviewed in Komuro & Kumada, 2005) and neurotransmitter selection (reviewed in Spitzer et al., 2004). ...
In the few weeks prior to the onset of vision, the retina undergoes a dramatic transformation. Neurons migrate into position and target appropriate synaptic partners to assemble the circuits that mediate vision. During this period of development, the retina is not silent but rather assembles and disassembles a series of transient circuits that use distinct mechanisms to generate spontaneous correlated activity called retinal waves. During the first postnatal week, this transient circuit is comprised of reciprocal cholinergic connections between starburst amacrine cells. A few days before the eyes open, these cholinergic connections are eliminated as the glutamatergic circuits involved in processing visual information are formed. Here, we discuss the assembly and disassembly of this transient cholinergic network and the role it plays in various aspects of retinal development.
... The basis for wave propagation has been contentious: there is evidence that waves may be coordinated variously by gap-junctional coupling and neurochemical (excitatory GABAergic, acetylcholinergic and/or glutamatergic) transmission, and there is still controversy about the role of each (109). There is broad agreement, however, that the earliest waves in birds and mammals precede synaptogenesis and are susceptible to drugs that block gap junctions (13,103,121), whereas waves at later stages resist gap-junction blockers but succumb to drugs that block nicotinic cholinergic and/or glutamatergic transmission (108). ...
... However, the maturational shifts that underlie the emergence of inhibitory transmission and neurochemical wave propagation in the inner retina may also have implications for proliferation in the outer retina, where bipolar cells and rod photoreceptors, as well as Müller glia, are still being born. In a detailed study of the perinatal rabbit retina, Syed et al. (103) were able to combine multi-electrode-array analyses of ganglion-cell firing patterns in the inner retina with calcium imaging studies of proliferative progenitor cells in the ventricular zone. Spontaneous propagating waves were seen by both techniques and found to be spatiotemporally correlated, albeit with differing kinetics. ...
Gap-junction channels, the cytoplasmic proteins that associate with them, and the transcriptional networks that regulate them are increasingly being viewed as critical communications hubs for cell signaling in health and disease. As a result, the term "nexus," which was the original structural name for these focal intercellular links, is coming back into use with new proteomic and transcriptomic meanings. The retina is better understood than any other part of the vertebrate central nervous system in respect of its developmental patterning, its diverse neuronal types and circuits, and the emergence of its definitive structure-function correlations. Thus, studies of the junctional and nonjunctional nexus roles of gap-junction proteins in coordinating retinal development should throw useful light on cell signaling in other developing nervous tissues.
... One SP neuron makes on average 9 gap junction couplings with neighboring SP neurons and neurons in the CP (Dupont et al. 2006). Spontaneous network activity patterns in developing cochlea, retina, and neocortex can be suppressed by pharmacological blockade of gap junctions (Peinado et al. 1993;Syed et al. 2004;Tritsch et al. 2007). The sensitivity of spontaneous electrical activity to octanol may initially suggest the involvement of gap junction (Fig. 3A), but octanol is known for not differentiating between gap junctions and hemichannels. ...
Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1-P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 μM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1-P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.
... In mammals, waves are produced in three consecutive stages with unique cellular and pharmacological signatures. They are first mediated by gap junctions (Stage I: late gestation) (Catsicas et al. 1998;Bansal et al. 2000;Syed et al. 2004a), and then depend on cholinergic synaptic transmission (Stage II: until P9-10 in mouse) (Feller et al. 1996;Sernagor & Grzywacz, 1996, 1999Catsicas et al. 1998;Wong et al. 1998;Bansal et al. 2000;Sernagor et al. 2000;Zhou & Zhao, 2000;Sernagor et al. 2003;Syed et al. 2004b). GABAergic signalling becomes involved in modulating waves at around P4-5 in mouse (Zhang et al. 2006;Hennig et al. 2011) and at these early stages is depolarizing, as it is elsewhere in the developing central nervous system (CNS) (Ben-Ari et al. 2007), shifting polarity after P6 (Zhang et al. 2006; Barkis et al. 2010). ...
The immature retina generates spontaneous waves of spiking activity that sweep across the ganglion cell layer during a limited developmental period, before the onset of visual experience. The spatiotemporal patterns encoded in the waves are believed to be instructive for the wiring of functional connections throughout the visual system. However, the ontogeny of retinal waves is still poorly documented due to relatively low resolution of conventional recording techniques. Here, we have characterized the spatiotemporal features of mouse retinal waves from birth until eye opening with unprecedented detail using a large-scale, dense 4,096-channel multielectrode array that allowed us to record from the entire neonatal retina at near cellular resolution. We found that early cholinergic waves propagate with random trajectories over large areas with low ganglion cell recruitment. They become slower, smaller and denser when GABAA signalling matures, beyond postnatal day (P) 7. Glutamatergic influences dominate from P10, coinciding with profound changes in activity dynamics. At that time waves cease to be random, beginning to show repetitive trajectories confined to a few localised hotspots. These hotspots gradually tile the retina with time, and disappear after eye opening. Our observations demonstrate that retinal waves undergo major spatiotemporal changes during ontogeny. Our results support the hypothesis that cholinergic waves guide the refinement of retinal targets while glutamatergic waves may also support the wiring of retinal receptive fields. This article is protected by copyright. All rights reserved
... This adds to the dual pro-and anti-degenerative roles demonstrated for calcium (Verkhratsky and Toescu, 2003). A particular aspect of calcium signaling that also deserves attention is the occurrence of spontaneous calcium waves coordinated across both differentiated inner layers and the proliferative zone of the developing retina (Catsicas et al., 1998;Syed et al., 2004), which, nonetheless, have yet to be tested for a role upon PCD. ...
It has long been known that a barrage of signals from neighboring and connecting cells, as well as components of the extracellular matrix, control cell survival. Given the extensive repertoire of retinal neurotransmitters, neuromodulators and neurotrophic factors, and the exhuberant interconnectivity of retinal interneurons, it is likely that various classes of released neuroactive substances may be involved in the control of sensitivity to retinal cell death. The aim of this article is to review evidence that neurotransmitters and neuropeptides control the sensitivity to programmed cell death in the developing retina. Whereas the best understood mechanism of execution of cell death is that of caspase-mediated apoptosis, current evidence shows that not only there are many parallel pathways to apoptotic cell death, but non-apoptotic programs of execution of cell death are also available, and may be triggered either in isolation or combined with apoptosis. The experimental data show that many upstream signaling pathways can modulate cell death, including those dependent on the second messengers cAMP-PKA, calcium and nitric oxide. Evidence for anterograde neurotrophic control is provided by a variety of models of the central nervous system, and the data reviewed here indicate that an early function of certain neurotransmitters, such as glutamate and dopamine, as well as neuropeptides such as pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide is the trophic support of cell populations in the developing retina. This may have implications both regarding the mechanisms of retinal organogenesis, as well as pathological conditions leading to retinal dystrophies and to dysfunctional cellular behavior. (c) 2004 Elsevier Ltd. All rights reserved.
... In mammals, waves are produced in three consecutive stages with unique cellular and pharmacological signatures. They are first mediated by gap junctions (Stage I: late gestation) (Catsicas et al. 1998;Bansal et al. 2000;Syed et al. 2004a), and then depend on cholinergic synaptic transmission (Stage II: until P9-10 in mouse) (Feller et al. 1996;Sernagor & Grzywacz, 1996, 1999Catsicas et al. 1998;Wong et al. 1998;Bansal et al. 2000;Sernagor et al. 2000;Zhou & Zhao, 2000;Sernagor et al. 2003;Syed et al. 2004b). GABAergic signalling becomes involved in modulating waves at around P4-5 in mouse (Zhang et al. 2006;Hennig et al. 2011) and at these early stages is depolarizing, as it is elsewhere in the developing central nervous system (CNS) (Ben-Ari et al. 2007), shifting polarity after P6 (Zhang et al. 2006; Barkis et al. 2010). ...
... In mammals, waves are produced in three consecutive stages with unique cellular and pharmacological signatures. They are first mediated by gap junctions (Stage I: late gestation) (Catsicas et al. 1998;Bansal et al. 2000;Syed et al. 2004a), and then depend on cholinergic synaptic transmission (Stage II: until P9-10 in mouse) (Feller et al. 1996;Sernagor & Grzywacz, 1996, 1999Catsicas et al. 1998;Wong et al. 1998;Bansal et al. 2000;Sernagor et al. 2000;Zhou & Zhao, 2000;Sernagor et al. 2003;Syed et al. 2004b). GABAergic signalling becomes involved in modulating waves at around P4-5 in mouse (Zhang et al. 2006;Hennig et al. 2011) and at these early stages is depolarizing, as it is elsewhere in the developing central nervous system (CNS) (Ben-Ari et al. 2007), shifting polarity after P6 (Zhang et al. 2006; Barkis et al. 2010). ...
... In mammals, waves are produced in three consecutive stages with unique cellular and pharmacological signatures. They are first mediated by gap junctions (Stage I: late gestation) (Catsicas et al. 1998;Bansal et al. 2000;Syed et al. 2004a), and then depend on cholinergic synaptic transmission (Stage II: until P9-10 in mouse) (Feller et al. 1996;Sernagor & Grzywacz, 1996, 1999Catsicas et al. 1998;Wong et al. 1998;Bansal et al. 2000;Sernagor et al. 2000;Zhou & Zhao, 2000;Sernagor et al. 2003;Syed et al. 2004b). GABAergic signalling becomes involved in modulating waves at around P4-5 in mouse (Zhang et al. 2006;Hennig et al. 2011) and at these early stages is depolarizing, as it is elsewhere in the developing central nervous system (CNS) (Ben-Ari et al. 2007), shifting polarity after P6 (Zhang et al. 2006; Barkis et al. 2010). ...
Key points
Novel pan‐retinal recordings of mouse retinal waves were obtained at near cellular resolution using a large‐scale, high‐density array of 4096 electrodes to investigate changes in wave spatiotemporal properties from postnatal day 2 to eye opening.
Early cholinergic waves are large, slow and random, with low cellular recruitment.
A developmental shift in GABA A signalling from depolarizing to hyperpolarizing influences the dynamics of cholinergic waves.
Glutamatergic waves that occur just before eye opening are focused, faster, denser, non‐random and repetitive.
These results provide a new, deeper understanding of developmental changes in retinal spontaneous activity patterns, which will help researchers in the investigation of the role of early retinal activity during wiring of the visual system.
Abstract
The immature retina generates spontaneous waves of spiking activity that sweep across the ganglion cell layer during a limited period of development before the onset of visual experience. The spatiotemporal patterns encoded in the waves are believed to be instructive for the wiring of functional connections throughout the visual system. However, the ontogeny of retinal waves is still poorly documented as a result of the relatively low resolution of conventional recording techniques. Here, we characterize the spatiotemporal features of mouse retinal waves from birth until eye opening in unprecedented detail using a large‐scale, dense, 4096‐channel multielectrode array that allowed us to record from the entire neonatal retina at near cellular resolution. We found that early cholinergic waves propagate with random trajectories over large areas with low ganglion cell recruitment. They become slower, smaller and denser when GABA A signalling matures, as occurs beyond postnatal day (P) 7. Glutamatergic influences dominate from P10, coinciding with profound changes in activity dynamics. At this time, waves cease to be random and begin to show repetitive trajectories confined to a few localized hotspots. These hotspots gradually tile the retina with time, and disappear after eye opening. Our observations demonstrate that retinal waves undergo major spatiotemporal changes during ontogeny. Our results support the hypotheses that cholinergic waves guide the refinement of retinal targets and that glutamatergic waves may also support the wiring of retinal receptive fields.
... Hence, the correlation between ON and OFF ganglion cells, which have processes in distinct layers within the IPL, is evidence of diffuse action of ACh. Indeed, in rabbit retina, there is evidence that ACh released in the inner retina is able to activate muscarinic receptors on cells in the ventricular zone (Syed et al., 2004). Given that later in development, when waves are mediated by a glutamatergic circuit, there is direct evidence that waves are accompanied by volume release of glutamate (Blankenship et al., 2009), these data are consistent with the diffuse action of neurotransmitter as a characteristic feature of developing circuits that exhibit spreading depolarization (Allene et al., 2008). ...
Before vision, a transient network of recurrently connected cholinergic interneurons, called starburst amacrine cells (SACs), generates spontaneous retinal waves. Despite an absence of robust inhibition, cholinergic retinal waves initiate infrequently and propagate within finite boundaries. Here, we combine a variety of electrophysiological and imaging techniques and computational modeling to elucidate the mechanisms underlying these spatial and temporal properties of waves in developing mouse retina. Waves initiate via rare spontaneous depolarizations of SACs. Waves propagate through recurrent cholinergic connections between SACs and volume release of ACh as demonstrated using paired recordings and a cell-based ACh optical sensor. Perforated-patch recordings and two-photon calcium imaging reveal that individual SACs have slow afterhyperpolarizations that induce SACs to have variable depolarizations during sequential waves. Using a computational model in which the properties of SACs are based on these physiological measurements, we reproduce the slow frequency, speed, and finite size of recorded waves. This study represents a detailed description of the circuit that mediates cholinergic retinal waves and indicates that variability of the interneurons that generate this network activity may be critical for the robustness of waves across different species and stages of development.
... Later in development (P10-P13, Stage 3), waves are mediated by a glutamatergic circuit and are not affected by nAChR antagonists. Earlier in development (Stage 1), waves are partially mediated by cholinergic transmission, thus blocking nAChRs inhibits but not the retinal waves [5,9,10]. Remarkably, RWs are transmitted to the cortex and drive early patterns of spontaneous activity (spindle burst) in neonatal V1 cortex in rats [11,12]. ...
The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded, the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs) during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal). Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system.
... This artifact does not seem to arise for conventional fluorescence microscopy techniques (e.g. Syed et al., 2004). However, conventional imaging is often not feasible in the cortex where greater imaging depths require two-photon imaging. ...
A complete understanding of how brain circuits function will require measurement techniques which monitor large-scale network activity simultaneously with the activity of local neural populations at a small scale. Here we present a useful step towards achieving this aim: simultaneous two-photon calcium imaging and multi-electrode array (MEA) recordings. The primary challenge of this method is removing an electrical artifact from the MEA signals that is caused by the imaging laser. Here we show that artifact removal can be achieved with a simple filtering scheme. As a demonstration of this technique we compare large-scale local field potential signals to single-neuron activity in a small-scale group of cells recorded from rat acute slices under two conditions: suppressed vs. intact inhibitory interactions between neurons.
... Cassidy et al. [1994] have demonstrated that the simple spinal sensory reflexes of the limbs such as withdrawal, crossed extension and hopping cannot be elicited before the young naturally detaches from the mother at 3 weeks, and that they are strongly expressed only around weaning in the 7th postnatal week. A synchronization of dorsal horn neuron activity could also play a role in establishing somatotopy in the ascending sensory pathways, as has been shown for the establishment of retinotopy [Roering and Feller, 2000;Syed et al., 2004]. Ascending spinal pathways start growing prenatally but do not reach their brain targets before one to three postnatal weeks in opossums [Qin et al., 1993;Desrosiers, 1995]. ...
We use opossums Monodelphis domestica to study the development of mammalian motor systems. The immature forelimbs of the newborn perform rhythmic and alternating movements that are likely under spinal control. The hindlimbs start moving in the second week. Chemical synapses are scant in the spinal enlargements of neonatal opossums and the presence of electrochemical synapses has not been evaluated in this species or in other marsupials. As a first step aiming at evaluating the existence of such synapses in the neonatal spinal cord, we have investigated the presence of the exclusively neuronal gap junction protein connexin36 (Cx36) by immunohistochemistry in light microscopy. At birth, Cx36 immunoreactivity is moderate in the presumptive gray matter in both enlargements. Thereafter, it decreases gradually, except in the superficial dorsal horn where it increases to a plateau between P10 and P20. Cx36 labeling is detected in the presumptive white matter at birth, but then decreases except in the dorsal part of the lateral funiculus, where it is dense between P10 and P20. Cx36 has become virtually undetectable by P52. The presence of Cx36 in the spinal enlargements of postnatal opossums suggests that neurons might be linked by gap junctions at a time when chemical synapses are only beginning to form. The greater abundance of Cx36 observed transiently in the superficial dorsal horn suggests a stronger involvement of this protein in spinal sensory systems than in direct motor control of the limbs.
... Calcium influx from internal (through ryanodine receptors and IP 3 Rs) and external stores (via storeoperated Ca 2+ channels and voltage gated calcium channels) are involved in cell proliferation [5,789101112. Calcium wave-signaling is widespread, as it has been observed in cortical radial glia, in the ventricular zone [7,8], neurospheres [9], mesenchymal stem cells [10,13], mouse embryonic stem cells [11], mouse carcinoma stem cells [13] and cells in developing tissue [12,141516. Although calcium oscillations have been observed in numerous model systems, many questions about the role of these Ca 2+ events in cell cycle progression and cellular proliferation remain unanswered. ...
Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.
... It will also be interesting to determine if blocking the transients has any effect on the proliferation or differentiation of cells that generate different subclasses of motoneurons and interneurons. Propagating Ca 2+ transients have been observed in radial glial cells in the neocortex where they regulate cell proliferation (Weissman et al., 2004) and in the ventricular zone of the developing rabbit retina, where they were also dependent on neurally generated waves (Syed et al., 2004). ...
In the nervous system, spontaneous Ca(2+) transients play important roles in many developmental processes. We previously found that altering the frequency of electrically recorded rhythmic spontaneous bursting episodes in embryonic chick spinal cords differentially perturbed the two main pathfinding decisions made by motoneurons, dorsal-ventral and pool-specific, depending on the sign of the frequency alteration. Here, we characterized the Ca(2+) transients associated with these bursts and showed that at early stages while motoneurons are still migrating and extending axons to the base of the limb bud, they display spontaneous, highly rhythmic, and synchronized Ca(2+) transients. Some precursor cells in the ependymal layer displayed similar transients. T-type Ca(2+) channels and a persistent Na(+) current were essential to initiate spontaneous bursts and associated transients. However, subsequent propagation of activity throughout the cord resulted from network-driven chemical transmission mediated presynaptically by Ca(2+) entry through N-type Ca(2+) channels and postsynaptically by acetylcholine acting on nicotinic receptors. The increased [Ca(2+)](i) during transients depended primarily on L-type and T-type channels with a modest contribution from TRP (transient receptor potential) channels and ryanodine-sensitive internal stores. Significantly, the drugs used previously to produce pathfinding errors altered transient frequency but not duration or amplitude. These observations imply that different transient frequencies may differentially modulate motoneuron pathfinding. However, the duration of the Ca(2+) transients differed significantly between pools, potentially enabling additional distinct pool-specific downstream signaling. Many early events in spinal motor circuit formation are thus potentially sensitive to the rhythmic Ca(2+) transients we have characterized and to any drugs that perturb them.
... Two major questions in developmental neuroscience: how are these patterns generated, and what cellular processes are driven by these patterns? The first of these questions is addressed by Syed et al. (this issue, p. 1999 –2009). They focus on the embryonic rabbit retina, where they show that undifferentiated cells in the ventricular zone undergo spontaneous, highly correlated calcium transients that take the form of propagating waves. ...
There is growing evidence that even before synapses form, spontaneous calcium transients can influence many aspects of neurodevelopment (Spitzer 2002). These early spontaneous calcium transients can be patterned both temporally as periodic oscillations and spatially as propagating waves or as synchro- nized events across neighboring cells. Two major questions in developmental neuroscience: how are these patterns generated, and what cellular processes are driven by these patterns? The first of these questions is addressed by Syed et al. (this issue, p. 1999 -2009). They focus on the embryonic rabbit retina, where they show that undifferentiated cells in the ventricular zone undergo spontaneous, highly correlated calcium tran- sients that take the form of propagating waves. Note, these waves are distinct from previously described retinal waves (Feller 2002; Zhou 2001) because they propagate through the ventricular zone and not amacrine and retinal ganglion cells. The retina develops in roughly a layer-by-layer manner. The inner retina (the cell layers closer to the vitreal surface) devel- ops first when ganglion cells migrate to the ganglion cell layer and send their projections via the optic nerve to central brain structures. Ganglion cells then start to receive synaptic input from interneurons. At this stage, inner retinal waves (IR waves) emerge, involving amacrine cells and ganglion cells, and driv- ing patterning of ganglion cell axons (Feller 2002). VZ waves occur during this same stage of development.
... Transient increases in intracellular calcium regulate a wide range of cellular processes in developing and mature neurons, from rapid enhancement of neurotransmitter release to long lasting changes in gene expression (Carey and Matsumoto 1999; Emptage et al. 2001; Spitzer et al. 2004; Syed et al. 2004; Yuste et al. 1992). In Drosophila, GAL4 driving expression of the calcium-sensitive luminescent protein, apoaequorin, reveals slow rhythmic oscillations in intracellular calcium levels localized in mushroom bodies, a region of the insect brain required for olfactory associative learning (Rosay et al. 2001). ...
Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.
... The positive feedback signal amplification that occurs in the network provides an example of the kind of signal processing that can occur within neuropile. The similar results from invertebrates and vertebrates, and the increasing awareness of the importance of gap junctions in the vertebrate CNS, raise the possibility that this mechanism of signal amplification may be common; for example, retrograde chemical transmission has been found recently to play a role in cerebellar long-term depression (Duguid and Smart, 2004), in developing mammalian retina (Syed et al., 2004), and in the rat substantia nigra (Yanovsky et al., 2003). It seems reasonable to expect that other examples using electrical transmission exist and may soon be found. ...
Lateral excitation is a mechanism for amplifying coordinated input to postsynaptic neurons that has been described recently in several species. Here, we describe how a postsynaptic neuron, the lateral giant (LG) escape command neuron, enhances lateral excitation among its presynaptic mechanosensory afferents in the crayfish tailfan. A lateral excitatory network exists among electrically coupled tailfan primary afferents, mediated through central electrical synapses. EPSPs elicited in LG dendrites as a result of mechanosensory stimulation spread antidromically back through electrical junctions to unstimulated afferents, summate with EPSPs elicited through direct afferent-to-afferent connections, and contribute to recruitment of these afferents. Antidromic potentials are larger if the afferent is closer to the initial input on LG dendrites, which could create a spatial filtering mechanism within the network. This pathway also broadens the temporal window over which lateral excitation can occur, because of the delay required for EPSPs to spread through the large LG dendrites. The delay allows subthreshold inputs to the LG to have a priming effect on the lateral excitatory network and lowers the threshold of the network in response to a second, short-latency stimulus. Retrograde communication within neuronal pathways has been described in a number of vertebrate and invertebrate species. A mechanism of antidromic passage of depolarizing current from a neuron to its presynaptic afferents, similar to that described here in an invertebrate, is also present in a vertebrate (fish). This raises the possibility that short-term retrograde modulation of presynaptic elements through electrical junctions may be common.
... This adds to the dual pro-and anti-degenerative roles demonstrated for calcium (Verkhratsky and Toescu, 2003). A particular aspect of calcium signaling that also deserves attention is the occurrence of spontaneous calcium waves coordinated across both differentiated inner layers and the proliferative zone of the developing retina (Catsicas et al., 1998; Syed et al., 2004), which, nonetheless, have yet to be tested for a role upon PCD. Nitric oxide (NO) is produced by both neurons and glia, and the exact physiological roles of this messenger from either source are still a matter of debate. ...
It has long been known that a barrage of signals from neighboring and connecting cells, as well as components of the extracellular matrix, control cell survival. Given the extensive repertoire of retinal neurotransmitters, neuromodulators and neurotrophic factors, and the exhuberant interconnectivity of retinal interneurons, it is likely that various classes of released neuroactive substances may be involved in the control of sensitivity to retinal cell death. The aim of this article is to review evidence that neurotransmitters and neuropeptides control the sensitivity to programmed cell death in the developing retina. Whereas the best understood mechanism of execution of cell death is that of caspase-mediated apoptosis, current evidence shows that not only there are many parallel pathways to apoptotic cell death, but non-apoptotic programs of execution of cell death are also available, and may be triggered either in isolation or combined with apoptosis. The experimental data show that many upstream signaling pathways can modulate cell death, including those dependent on the second messengers cAMP-PKA, calcium and nitric oxide. Evidence for anterograde neurotrophic control is provided by a variety of models of the central nervous system, and the data reviewed here indicate that an early function of certain neurotransmitters, such as glutamate and dopamine, as well as neuropeptides such as pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide is the trophic support of cell populations in the developing retina. This may have implications both regarding the mechanisms of retinal organogenesis, as well as pathological conditions leading to retinal dystrophies and to dysfunctional cellular behavior.
Vision begins in the retina, whose intricate neural circuits extract salient features of the environment from the light entering our eyes. Neurodegenerative diseases of the retina (e.g., inherited retinal degenerations, age-related macular degeneration, and glaucoma) impair vision and cause blindness in a growing number of people worldwide. Increasing evidence indicates that homeostatic plasticity (i.e., the drive of a neural system to stabilize its function) can, in principle, preserve retinal function in the face of major perturbations, including neurodegeneration. Here, we review the circumstances and events that trigger homeostatic plasticity in the retina during development, sensory experience, and disease. We discuss the diverse mechanisms that cooperate to compensate and the set points and outcomes that homeostatic retinal plasticity stabilizes. Finally, we summarize the opportunities and challenges for unlocking the therapeutic potential of homeostatic plasticity. Homeostatic plasticity is fundamental to understanding retinal development and function and could be an important tool in the fight to preserve and restore vision.
The thalamus is a key structure in the mammalian brain, providing a hub for communication within and across distributed forebrain networks. Research in this area has undergone a revolution in the last decade, with findings that suggest an expanded role for the thalamus in sensory processing, motor control, arousal regulation, and cognition. Moving beyond previous studies of anatomy and cell neurochemistry, scientists have expanded into investigations of cognitive function, and harness new methods and theories of neural computation. This book provides a survey of topics at the cutting edge of this field, covering basic anatomy, evolution, development, physiology and computation. It is also the first book to combine these disciplines in one place, highlighting the interdisciplinary nature of thalamus research, and will be an essential resource for students and experts in biology, medicine and computer science.
Synopsis
This chapter discusses the developmental processes and mechanisms through which the intricate circuitry of the retina arises from a uniform pool of progenitor cells. The chapter is divided into conceptually sequential but developmentally overlapping steps in the assembly of the mature retina and focuses, when possible, on evidence gathered in mice.
In this chapter, we turn to neural network models of wave propagation in the cortex and other parts of the nervous system. There has been a rapid increase in the number of computational studies of network dynamics, which are based on biophysically detailed conductance-based models of synaptically (and possibly electrically) coupled neurons. These models provide considerable insights into the role of ionic currents, synaptic processing, and network structure on spatiotemporal dynamics, but they tend to be analytically intractable. This has motivated an alternative approach to network dynamics, involving simplified neuron models that hopefully capture important aspects of wave phenomena, while allowing a more concise mathematical treatment. In the case of oscillatory networks, such a simplification can be achieved by reducing a conductance-based neuron model to a phase model. Alternatively, one can use a simplified spiking neuron model such as integrate-and-fire in order to investigate waves in excitable and oscillatory neural media. Both of these approaches are considered in this chapter, which also provides a summary of various wave phenomena in cortical and subcortical structures.
Chapter 7 develops the theory of waves in excitable neural fields, where the fundamental network element is a local population of cells rather than a single neuron. It is shown how many of the PDE methods and results from the analysis of waves in reaction–diffusion equations considered in Chap. 2 can be extended to the nonlocal equations of neural field theory. First, the existence and stability of solitary traveling fronts and pulses in one-dimensional excitatory neural fields are considered. In the case of traveling pulses, it is necessary to include some form of local negative feedback mechanism such as synaptic depression or spike frequency adaptation. Two approaches to analyzing wave propagation failure in inhomogeneous neural media are then presented: one based on averaging methods and the other on interfacial dynamics. Finally, wave propagation in stochastic neural fields is analyzed, and oscillatory waves in two-dimensional neural media are briefly discussed.
Chapter 1 provides a detailed introduction to the working parts of a neuron, including conductance-based models of action potential generation, synaptic and dendritic processing, and ion channels. Two important mathematical topics are also introduced. First, the dynamics of a periodically forced neural oscillator is used to introduce phase-reduction and averaging methods, phase-resetting curves, and synchronization. These are later applied to the study of waves in oscillatory neural media. Second, a detailed account of stochastic ion channels and membrane voltage fluctuations is given, which also provides background material on stochastic processes. A major theme is how to model and analyze stochastic hybrid systems, in which a continuous variable (e.g., voltage) couples to a discrete jump Markov process (e.g., number of open ion channels). Spontaneous action potential generation is formulated as a first passage time problem, which is solved using perturbation methods such as WKB and matched asymptotics. These methods are later used to analyze related problems such as the generation of calcium sparks and bistability in populations of spiking neurons.
Chapter describes the construction of population-based rate models under the assumption that the spiking of individual neurons is unimportant. The issue of how stochasticity at the single-cell level manifests itself at the population level is discussed, introducing topics such as balanced networks, Poisson statistics, and asynchronous states. Stochastic methods are then used to analyze bistability in a stochastic population model. Finally, the transition from spatially structured neural networks to continuum neural fields is highlighted. The latter take the form of nonlocal integrodifferential equations, in which the integral kernel represents the distribution of synaptic connections.
Finally, in this chapter, a variety of topics regarding wavelike phenomena in the developing and diseased brain are presented. First, the possible role of calcium and retinal waves in early development is summarized. There is then a detailed description and analysis of cytoskeletal waves involved in neurite growth and cell polarization. This introduces another interesting phenomenon, namely, wave pinning. Three distinct examples of waves in the diseased brain are considered: spreading depression and migraine auras, epileptic waves, and the spread of neurodegenerative waves due to protein aggregation. In order to tackle the latter two phenomena, a review of complex network theory is included, covering topics such as small-world networks, scale-free networks, neuronal avalanches, branching processes, and epidemics on networks.
This chapter gives a comprehensive review of calcium wave modeling, with an emphasis on their role in neuronal calcium signaling. Two models of intracellular waves are considered in some detail: a reaction–diffusion model of calcium dynamics and the fire–diffuse–fire model of calcium release. The latter is formally very similar to the spike–diffuse–spike model of spiny dendrites and is analyzed accordingly. Stochastic models of spontaneous calcium release (calcium puffs and sparks) are then analyzed using the stochastic methods introduced in the first chapter. Finally, several models of intercellular calcium waves in astrocytes are presented. Traditionally, astrocytes were thought to be physiologically passive cells that only play a supporting role in the central nervous system by regulating and optimizing the environment within which neurons operate. However, there is an increasing amount of empirical data indicating that astrocytes play a more active role in modulating synaptic transmission and neuronal signal processing.
Chapter 2 covers the classical problem of waves in one-dimensional excitable media, as exemplified by the FitzHugh–Nagumo model of action potential propagation along an axon. Standard methods for analyzing front and pulse solutions of PDEs are described, including phase-plane analysis, singular perturbation methods and slow–fast analysis, and Evans functions for wave stability. In addition, the problem of wave propagation failure in myelinated axons is considered, where an averaging method is used to determine the effects of spatial discreteness on wave speed. This method is later used to analyze wave propagation failure in inhomogeneous neural fields. Finally, stochastic traveling waves are considered, where formal perturbation methods are used to show how to separate out fast fluctuations of the wave profile from the slow diffusive-like wandering of the wave.
Introduction Once various cell types have migrated to their final location, they start synthesizing neuro–transmitters and extend neurites. At that stage, they are ready to begin forming synaptic connections with other retinal neurons. We have already seen in Chapter 6 that neurotransmitters are present before the formation of functional synapses, before electrical activity can be detected, suggesting that they play a trophic role during retinal development. However, retinal visual processing, the conversion of light into electrical signals and the relaying of these signals to the visual centres of the brain, cannot occur until neurons have established synaptic contacts with each other. The first part of this chapter describes the formation of synaptic connections in the various retinal layers. In the last decade, important issues regarding the establishment of synaptic connections have been resolved thanks to the advent of genetic engineering and to the development of powerful specific cellular or subcellular markers, some of which are reviewed here. We put a particular emphasis on the formation of synapses between photoreceptors and second-order neurons because photoreceptors are involved in many various types of hereditary retinal degenerations. We review studies using transgenic mouse models because they provide invaluable knowledge about factors influencing photoreceptor synaptogenesis (Farber and Danciger, 1997). Understanding factors that affect synapse formation between photoreceptors and retinal neurons is crucial for reaching better insights into these devastating diseases that often lead to blindness. We will also discuss briefly the formation of gap junctions.
Introduction In addition to intrinsic control mechanisms (see Chapter 5 and Cepko et al., 1996), the production of neurons by progenitor cells and the determination of their fate are regulated via an array of diffusible factors, two families of which are considered in this chapter: neurotransmitters and neurotrophins. Neurotrophins are now known to play an essential role in both the formation and the maintenance of the nervous system throughout development and adult life. There is growing evidence that besides their role as molecules mediating communication between nerve cells in the mature nervous system, a variety of both slow and fast neurotransmitters also play important roles during neuronal development. This chapter reviews recent evidence that demonstrates that a number of non-synaptic neurotransmitter release mechanisms, together with many neurotransmitters and their receptors, are present in the developing retina prior to the onset of synapse formation and that these early neurotransmitters act to modulate a range of events in neural development. Their precise mechanisms of action are still being elucidated but, as described here, the ability to modulate [Ca2+]i is one feature common to these early neurotransmitter systems, and is thought to underlie a number of their developmental actions. It is becoming clear that both neurotransmitters and neurotrophins play important regulatory roles in the early stages of retinal development, including the modulation of proliferation, differentiation, cell survival and circuit formation.
The developing vertebrate retina is characterized by periodic waves of activity spreading across the ganglion cell layer. These waves are present only during perinatal weeks, undergoing substantial changes in their dynamic properties until they disappear. This chapter reviews the cross-species similarities and differences in the neural circuitry underlying retinal waves during development and presents the current theoretical models explaining the mechanisms of wave generation and propagation.
Chapter 3 presents two different models of traveling waves along spiny dendrites: a spike–diffuse–spike model of propagating voltage spikes mediated by active dendritic spines and a reaction–diffusion model of Ca2+–calmodulin-dependent protein kinase II (CaMKII) translocation waves. The former model introduces methods that are later used to analyze solitary waves propagating in spiking neural networks. The latter model turns out to be identical in form to the diffusive susceptible–infected (SI) model of the spread of epidemics, which is a generalization of the scalar Fisher–KPP equation of population genetics. One characteristic feature of such equations is that they support traveling fronts propagating into an unstable steady state, in which the wave speed and longtime asymptotics are determined by the dynamics in the leading edge of the wave—so-called pulled fronts. In particular, a sufficiently localized initial perturbation will asymptotically approach the traveling front solution that has the minimum possible wave speed. Hence, pulled fronts have very different properties from propagating action potentials. Homogenization methods are also presented, which allow one to approximate the discrete distribution of spines by a smooth distribution.
The initial structural and functional development of visual circuits in reptiles, birds, and mammals happens independent of sensory experience. After eye opening, visual experience further refines and elaborates circuits that are critical for normal visual function. Innate genetic programs that code for gradients of molecules provide gross positional information for developing nerve cells, yet much of the cytoarchitectural complexity and synaptogenesis of neurons depends on calcium influx, neurotransmitter release, and neural activity before the onset of vision. In fact, specific spatiotemporal patterns of neural activity, or 'retinal waves', emerge amidst the development of the earliest connections made between excitable cells in the developing eye. These patterns of spontaneous activity, which have been observed in all amniote retinae examined to date, may be an evolved adaptation for species with long gestational periods before the onset of functional vision, imparting an informational robustness and redundancy to guide development of visual maps across the nervous system. Recent experiments indicate that retinal waves play a crucial role in the development of interconnections between different parts of the visual system, suggesting that these spontaneous patterns serve as a template-matching mechanism to prepare higher-order visually associative circuits for the onset of visuomotor learning and behavior. Key questions for future studies include determining the exact sources and nature of spontaneous activity during development, characterizing the interactions between neural activity and transcriptional gene regulation, and understanding the extent of circuit connectivity governed by retinal waves within and between sensory-motor systems.
During the first two weeks of mouse postnatal development, transient retinal circuits give rise to the spontaneous initiation and lateral propagation of depolarizations across the ganglion cell layer (GCL). Glutamatergic retinal waves occur during the second postnatal week, when GCL depolarizations are mediated by ionotropic glutamate receptors. Bipolar cells are the primary source of glutamate in the inner retina, indicating that the propagation of waves depends on their activation. Using the FRET based optical sensor of glutamate, FLII81E-1µ, we found that retinal waves are accompanied by a large transient increase in extrasynaptic glutamate throughout the inner plexiform layer. Using two-photon calcium imaging to record spontaneous calcium transients in large populations of cells, we found that despite this spatially diffuse source of depolarization, only a subset of neurons in the GCL and inner nuclear layer (INL) are robustly depolarized retinal waves. Application of the glutamate transporter blocker, DL-TBOA (25 µM) led to a significant increase in cell participation in both layers, indicating that the concentration of extrasynaptic glutamate affects cell participation in both the INL and GCL. In contrast, blocking inhibitory transmission with the GABA-A receptor antagonist Gabazine and the glycine receptor antagonist Strychnine increased cell participation in the GCL without significantly affecting the INL. These data indicate that during development, glutamate spillover provides a spatially diffuse source of depolarization, but that inhibitory circuits dictate which neurons within the GCL participate in retinal waves.
Intercellular calcium (Ca(2+)) waves (ICWs) represent the propagation of increases in intracellular Ca(2+) through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca(2+) from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs.
Over the last two decades, technological advances in the fields of microchip and electronics manufacturing have enabled an increase in the production and use of silicon-based multi-electrode arrays. (Singer, 2000; Morin et al., 2005) These multi-electrode arrays or MEA for short, have come in a variety of shapes and materials, but fall into two broad classes: thin and sharp (implantable) or dishbased (planar). Although many investigations are currently undertaking research in vivo with implantable versions, this chapter focuses on applications of planar MEAs (pMEA), which are very well suited for in vitro experiments with slice or dissociated cells preparations. This chapter illustrates the utility and advantages of pMEAsin electrophysiological investigations with acute hippocampal slices, while introducing a new generation of conformally designed higher-density pMEAs as an adjuvant approach to facilitate and enhance MEA-based research. Currently, the research being undertaken on pMEAs ranges from studying processes of neuronal plasticity underlying learning and memory, to tracking activity development in networks, and also pharmacological drug screening and testing. These diverse applications can be classified, based on the intricacy of their methodology, into the following nonmutually exclusive categories: (1) MEAs can be used as a multitude of single independent electrodes for rapid high-throughput experiments; (2) the spatial relations between electrode tips can be used synergistically to map electrical activity to tissue location; (3) recording simultaneously from multiple electrodes allows correlation of temporal information, which is not possible with many recordings from single electrodes; (4) the combination of spatial and temporal monitoring reveals the spatiotemporal dynamics of the neuronal network; (5) the ability to maintain cultured preparations on pMEAs allows longterm physiological investigations; and (6) recording and stimulating through the pMEA creates two-way communication with the tissue that is indispensable for investigating and developing neuroprosthetic applications. High-throughput applications involve sampling several electrodes out of the total number on the MEA and selecting a representative one, or treating subgroups statistically as multiple samples from a homogeneous population. Electrodes within a particular cytoarchitectural region of a slice usually record similar neural responses. This redundancy of observed signals can be used to enhance the statistical significance of results by grouping responses into larger sample sizes. Similar time savings are achieved in cell cultures, where the multitude of electrodes records the activity of numerous cells at the same time, thereby decreasing the number of individual experiments needed to reach a significant population sample. Such high-throughput use of MEAs as biosensors has been applied to drug screening using cell culture (Pine, 1980; Gross et al., 1995) and hippocampal slice rhythmic activity (Shimono et al., 2000). In the first case, drugs are classified according to changes in the firing activity of neuronal cells cultured on MEAs (Gross et al., 1995, 1999). In the second case, changes in the frequency of carbachol-induced theta rhythmic oscillations in hippocampal slices are correlated with specific drug properties (Shimono et al., 2000). In both cases, the MEAs provided multiple sample points in different regions of the network, which enabled either a quick selection of an optimal site or averaging several channels for greater statistical accuracy. In contrast to using array electrodes as individual and independent streams of data, the spatial arrangement of electrodes can be used to generate spatial maps of the activity in a slice. Any parameter of the recorded potentials can be plotted in a color-coded matrix according to the relative spatial positions of the electrodes in order to generate topographic activity maps. Such spatial activity maps can be matched to a picture of the slice showing the actual electrode positions in order to visualize the activity in relation to the subregions of a slice (Shimono et al., 2000) or map the spatial extent of a response along a network (Jimbo and Robinson, 2000). In addition, if electrodes are close enough to each other, they enable current source density (CSD) analysis, which can elucidate the origins and meaning of the complex field potentials recorded (Wheeler and Novak, 1986). The ability to simultaneously record from all the MEA electrodes over time enables correlation of activity between different parts of a network in order to study their patterns and plasticity in cell and tissue preparations. The temporal sequence of firing of ensembles of cells can provide information on network states. Beggs and Plenz (2003) analyzed cell bursting avalanches to describe the stability of the network. Jimbo et al. (1999) reported on time-dependent synaptic plasticity in networks of cultured cells in observing that connections between cells that fired within 20 msec before the other were potentiated after tetanus, whereas connections between cells negatively correlated within 20 msec were depressed. pMEAs combine spatial and temporal information and enable the conversion of static spatial activity maps into dynamic spatiotemporal map sequences. These series of maps can be joined as frames of amovie to visually trace the propagation of spontaneous, evoked, or rhythmic activity across the slice. For example, Novak and Wheeler (1989) studied the temporal propagation of seizure activity, and Shimono et al. (2000) localized and spatiotemporally followed the origin of theta rhythm generated by carbachol in a slice, both using CSD analysis of the signals recorded from the MEA. The surface of pMEAs is ideal for long-term tissue cultures of both slices and dissociated cells, as it provides a flat, biocompatible, and sterilizable support with embedded electrodes that can continuously monitor culture activity without disrupting the closed system. Longer-term experiments can track changes in activity and plasticity of developing cultures and networks (Gross and Schwalm, 1994; Stoppini et al., 1997; Thiebaud et al., 1997; Jahnsen et al., 1999) under different chronic pharmacological treatments (Shimono et al., 2002). Although several neuroprosthetics, such as cochlear, cortical (Chapin et al., 1999), and retinal (Humayun et al., 2003) devices rely on implantable in vivoMEA technology, pMEAs still play a major role in understanding network connectivity and dynamics (Meister et al., 1994; Warland et al., 1997). pMEAs are being used as an in vitro testing platform to first characterize the information processing of the target neuronal network, before undertaking in vivo experiments. In our current goal to replace the CA3 hippocampal area with a microchip (FPGA/VLSI) implementation of a nonlinear model of CA3 (Berger et al., 2001), we are using pMEAs to provide a functional proof-of-principle.pMEAexperiments allowus first to generate nonlinear models, then to test hardware implementations in order to change parameters and conditions rapidly, cost effectively, and with fewer animals. Similarly, the retinal prosthesis project relies on understanding underlying network dynamics and plasticity of the retina in order to transform incident light into an electrical stimulation pattern that will produce correct visual percepts (Humayun and Weiland, personal communications). Retinal stimulation and recording experiments are thus currently being undertaken on pMEAs to develop a nonlinear mathematical model of the retinal network that will be implemented in the next generation of retinal prostheses (Chichilnisky and Kalmar, 2003; Frechette et al., 2005). © 2006 Springer Science+Business Media, Inc. All rights reserved.
The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.
Neural progenitor cells and developing neurons show periodic, synchronous Ca(2+) rises even before synapse formation, and the origin of the synchronous activity remains unknown. Here, fluorescence measurement revealed that the membrane potential of the nuclear envelope, which forms an intracellular Ca(2+) store, changed with a release of Ca(2+) and generated spontaneous, periodic bursts of fluctuations in potential. Furthermore, changes in the nuclear envelope's potential underlay spike burst generations. These results support the model that voltage fluctuations of the nuclear envelope synchronize Ca(2+) release between cells and also function as a current noise generator to cause synchronous burst discharges.
Increases in intracellular [Ca(2+)] occur synchronously between cells in the neuroepithelium. If neuroepithelial cells were capable of generating action potentials synchronized by gap junctions (direct current electrical coupling), the influx of Ca(2+) through voltage-activated Ca(2+) channels would lead to a synchronous increase in intracellular [Ca(2+)]. However, no action potential is generated in neuroepithelial cells, and the [Ca(2+)] increase is instead produced by the release of Ca(2+) from intracellular Ca(2+) stores. Recently, synchronous fluctuations in the membrane potential of Ca(2+) stores were recorded using an organelle-specific voltage-sensitive dye. On the basis of these recordings, a capacitative [alternating current (AC)] electrical coupling model for the synchronization of voltage fluctuations of Ca(2+) store potential was proposed [Yamashita M (2006) FEBS Lett580, 4979-4983; Yamashita M (2008) FEBS J275, 4022-4032]. Ca(2+) efflux from the Ca(2+) store and K(+) counterinflux into the store cause alternating voltage changes across the store membrane, and the voltage fluctuation induces ACs. In cases where the store membrane is closely apposed to the plasma membrane and the cells are tightly packed, which is true of neuroepithelial cells, the voltage fluctuation of the store membrane is synchronized between the cells by the AC currents through the series capacitance of these membranes. This article provides a short review of the model and its relationship to the structural organization of the Ca(2+) store. This is followed by a discussion of how the mode of synchronization of [Ca(2+)] increase may change during central nervous system development and new molecular insights into the synchronicity of [Ca(2+)] increase.
Multipotent retinal progenitors undergo a varied number of divisions to produce clones of heterogeneous sizes and cell types. We describe the transition from a proliferating progenitor to a differentiated postmitotic cell and discuss how controls of proliferation operate within individual cells as well as in the whole tissue. We discuss how extracellular and intracellular signaling, transcriptional regulation, cell cycle kinetics, interkinetic nuclear migration, orientation of cell division, and epigenetic modifications all interact to regulate a progenitor's transition from division to differentiation. We also propose some directions for future research.
Electrical synaptic transmission through gap junctions underlies direct and rapid neuronal communication in the CNS. The diversity of functional roles that electrical synapses have is perhaps best exemplified in the vertebrate retina, in which gap junctions are formed by each of the five major neuron types. These junctions are dynamically regulated by ambient illumination and by circadian rhythms acting through light-activated neuromodulators such as dopamine and nitric oxide, which in turn activate intracellular signalling pathways in the retina.The networks formed by electrically coupled neurons are plastic and reconfigurable, and those in the retina are positioned to play key and diverse parts in the transmission and processing of visual information at every retinal level.
The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.
Radial glial proliferation is a critical step in the construction of cerebral cortex. In this issue of Neuron, Weissman and colleagues use time-lapse calcium imaging techniques to demonstrate that spontaneous calcium waves sweeping through cohorts of radial glia in the ventricular zone can modulate their proliferation during cerebral cortical development.
A characteristic feature of developing neural networks is spontaneous periodic activity. In the developing retina, retinal ganglion cells fire bursts of action potentials that drive large increases in intracellular calcium concentration with a periodicity of minutes. These periodic bursts of action potentials propagate across the developing inner retina as waves, driving neighboring retinal ganglion cells to fire in a correlated fashion. Here we will review recent progress in elucidating the mechanisms in mammals underlying retinal wave propagation and those regulating the periodicity with which these retinal waves occur. In addition, we will review recent experiments indicating that retinal waves are critical for refining retinal projections to their primary targets in the central visual system and may be involved in driving developmental processes within the retina itself.
The retinal pigment epithelium (RPE) plays an essential role in the normal development of the underlying neural retina, but the mechanisms by which this regulation occurs are largely unknown. Ca2+ transients, induced by the neurotransmitter ATP acting on purinergic receptors, both increase proliferation and stimulate DNA synthesis in neural retinal progenitor cells. Here, we show that the RPE regulates proliferation in the underlying neural retina by the release of a soluble factor and identify that factor as ATP. Further, we show that this ATP is released by efflux through gap junction connexin 43 hemichannels, the opening of which is evoked by spontaneous elevations of Ca2+ in trigger cells in the RPE. This release mechanism is localized within the RPE cells to the membranes facing the neural retina, a location ideally positioned to influence neural retinal development. ATP released from RPE hemichannels speeds both cell division and proliferation in the neural retina.
At specific stages of development, nerve and muscle cells generate spontaneous electrical activity that is required for normal maturation of intrinsic excitability and synaptic connectivity. The patterns of this spontaneous activity are not simply immature versions of the mature activity, but rather are highly specialized to initiate and control many aspects of neuronal development. The configuration of voltage- and ligand-gated ion channels that are expressed early in development regulate the timing and waveform of this activity. They also regulate Ca2+ influx during spontaneous activity, which is the first step in triggering activity-dependent developmental programs. For these reasons, the properties of voltage- and ligand-gated ion channels expressed by developing neurons and muscle cells often differ markedly from those of adult cells. When viewed from this perspective, the reasons for complex patterns of ion channel emergence and regression during development become much clearer.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Stimulation of transient increases in intracellular calcium (Cai2+) activates protein kinases, regulates transcription and influences motility and morphology. Developing neurons generate spontaneous Cai2+ transients, but their role in directing neuronal differentiation and the way in which they encode information are unknown. Here we image Ca2+ in spinal neurons throughout an extended period of early development, and find that two types of spontaneous events, spikes and waves, are expressed at distinct frequencies. Neuronal differentiation is altered when they are eliminated by preventing Ca2+ influx. Reimposing different frequency patterns of Ca2+ elevation demonstrates that natural spike activity is sufficient to promote normal neurotransmitter expression and channel maturation, whereas wave activity is sufficient to regulate neurite extension. Suppression of spontaneous Ca2+ elevations by BAPTA loaded intracellularly indicates that they are also necessary for differentiation. Ca2+ transients appear to encode information in their frequency, like action potentials, although they are 10(4) times longer in duration and less frequent, and implement an intrinsic development programme.
The receptor pharmacology of cholinergic ("starburst") amacrine cells was studied in a newly developed rabbit retinal slice preparation with whole-cell patch clamp. Displaced starburst cells were labeled with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), and their dendritic morphology was identified in the slice with Lucifer yellow. Under conditions in which synaptic transmission was blocked by Cd2+, starburst cells responded vigorously to the bath-applied neurotransmitters GABA, glycine, and glutamate. The response to GABA consisted of an inward current and an increase in noise, which could be mimicked by the GABAA agonists muscimol and trans-4-aminocrotonic acid (TACA), but not by the GABAB agonist baclofen or the GABAC agonist cis-4-aminocrotonic acid (CACA). The GABA-evoked currents were reversibly inhibited by bicuculline and picrotoxin and had a reversal potential close to the chloride equilibrium potential. Noise analysis of GABA-activated whole-cell currents yielded elementary conductance estimates of 12.5 pS. Glycine (30-200 microM) also activated a Cl- conductance in starburst cells, which could be completely blocked by strychnine. The non-NMDA agonists kainate (KA, 30-100 microM) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 60 microM) evoked robust responses, which were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and which reversed near the equilibrium potential for cations. NMDA coapplied with glycine in salines free of Cd2+ and Mg2+ elicited small but detectable responses. The I/V relation of the NMDA-evoked response showed a characteristic "J"-shaped region in a saline containing 1 mM Mg2+ and 0 Cd2+, indicating that NMDA receptors were present directly on starburst cells. This was consistent with our finding that whole-cell currents evoked by KA and NMDA had different noise characteristics. These results place new constraints on models of starburst cell function and suggest that GABA-mediated inhibition of the starburst cell itself may play an important role in directional selectivity in the retina.
Transmitter-like molecules are thought to influence many aspects of neuronal development, often by regulating the levels of intracellular calcium. Using the Ca2+ sensitive dye, fura-2, this study demonstrates that in the rabbit retina, ACh analogs stimulate a rise in cytosolic free Ca2+ concentration ([Ca2+]i) in many cell types, and in cells at various stages of differentiation during embryonic and neonatal development. The elevation in [Ca2+]i in cells within the ventricular zone (VZ) resulted primarily from the activation of muscarinic receptors. By contrast, the cholinergic regulation of [Ca2+]i of ganglion cells and amacrine cells, cell types which migrate to their final destinations early in fetal life, was largely mediated by nicotinic receptors. The muscarinic response of the VZ cells was mediated by the M1, rather than the M2-type of muscarinic receptor. This response was abolished in the absence of extracellular Ca2+ and in the presence of NiCl2, but it was not affected by verapamil or omega-conotoxin, thus suggesting that while Ca2+ influx occurred, it did not involve L- and N-type voltage-gated Ca2+ channels. The muscarinic response in the VZ disappeared at the end of the period of cell division in the retina, just prior to eye opening. By contrast, nicotinic-induced changes in [Ca2+]i in ganglion cells and amacrine cells persisted throughout development. Since previous studies have implicated that the precursors of ganglion cells and amacrine cells also possess muscarinic receptors (Yamashita and Fukuda, 1993), the concomitant emergence of different functional cholinergic receptor (AChR) subtypes with differentiation in vivo suggests that ACh may play diverse and temporally regulated roles in the developing retina.
During the first 3 weeks of postnatal development in the ferret retina, cells in the ganglion cell layer spontaneously generate waves of electrical activity that travel across the retina in the absence of mature photoreceptors (Meister et al., 1991; Wong et al., 1993). Since few chemical synapses are present at the earliest stages when waves are present, we have explored whether gap junctions could act to correlate the activity of cells in the immature ganglion cell layer. Retinal ganglion cells in a living in vitro preparation from postnatal day 1 (P1) to P45 were intracellularly injected with the tracer Neurobiotin and the fluorescent dye Lucifer yellow, molecules that are known to pass through gap junctions. Lucifer yellow consistently filled only the injected cell, whereas Neurobiotin filled not only the injected cell but also passed to a constellation of neighboring cells. Coupling revealed by Neurobiotin is seen as early as P1, but, at this stage, it was not possible to identify the various morphological types of cells that were coupled. Thereafter, alpha ganglion cells showed homologous coupling to other alpha cells and to both conventionally placed and displaced amacrine cells. Likewise, gamma ganglion cells appeared coupled to other gamma cells and to amacrine cells. However, beta ganglion cells never showed tracer coupling in the neonatal or in adult retinas. The percentage of alpha and gamma cells that were coupled to other cells increased progressively with age. By the end of the third postnatal week, the pattern of Neurobiotin coupling in the ferret retina was adult-like, with virtually every injected alpha cell showing tracer coupling. Our observations suggest that intercellular junctions able to pass Neurobiotin are present in the inner plexiform layer during the period when the firing of retinal ganglion cells is highly correlated. Such junctions could contribute to synchronization of the activity of subsets of neighboring ganglion cells during development, but it cannot be the sole mediator of this activity because beta cells, which also participate in the correlated activity, showed no coupling at any stage. In addition, the continued presence of coupling in the adult retina implies that other changes in retinal circuitry are likely to contribute to the disappearance of the waves.
Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.
Even before birth and the onset of sensory experience, neural activity plays an important role in shaping the vertebrate nervous system. In the embryonic chick visual system, activity in the retina before vision has been implicated in the refinement of retinotopic maps, the elimination of transient projections, and the survival of a full complement of neurons. In this study, we report the detection of a physiological substrate for these phenomena: waves of spontaneous activity in the ganglion cell layer of the embryonic chick retina. The activity is robust and highly patterned, taking the form of large amplitude, rhythmic, and wide-ranging waves of excitation that propagate across the retina. Activity waves are most prominent and organized between embryonic days 13-18, coinciding with the developmental period during which retinal axons refine their connections in their targets. The spatial and temporal features of the patterns observed are consistent with the role of activity patterns in shaping eye-specific projections and retinotopic maps but inconsistent with the hypothesis that they specify lamina-specific projections in the tectum. Antagonists of glutamatergic and glycinergic transmission and of gap junctional communication suppress spontaneous activity, whereas antagonists to GABAergic transmission potentiate it. Based on these results, we propose that spontaneous activity in the ganglion cells is regulated by chemical inputs from both bipolar and amacrine cells and by gap junctional coupling involving ganglion cells.
Spontaneous neuronal firing during development has the potential to shape many aspects of neuronal wiring throughout the brain. Bursts of electrical activity coordinated among large numbers of neurons, occurring during a brief developmental window, have been described in many regions of the CNS, including retina, hippocampus, and spinal cord, but evidence for this type of activity in developing neocortex has so far been lacking. To identify conditions that may give rise to patterned spontaneous electrical activity in developing neocortex, cholinergic agonists were applied to immature rat cortical slices while large-scale activity was imaged optically with fura-2 AM. Here I show that activation of muscarinic acetylcholine receptors results in waves of correlated neural activity. Waves recruit large numbers of neurons, are slowly propagating, regenerative events involving depolarization and associated calcium transients, and advance for many millimeters as a sharp wave front perpendicular to the pial surface, at speeds ranging between 50 and 300 m/sec. The expression of waves is restricted temporally to a brief period in postnatal development, until postnatal day 6, and spatially to some neocortical areas. The ability of isolated neocortical networks to generate large-scale patterned activity endogenously during a period of massive neurite extension and synaptogenesis raises the possibility that at least in some cortical areas these processes might be influenced by patterned neuronal firing generated independently of thalamocortical input.
Before phototransduction, spontaneous activity in the developing mammalian retina is required for the appropriate patterning of retinothalamic connections, and there is growing evidence that this activity influences the development of circuits within the retina itself. We demonstrate here that the neural substrate that generates waves in the mouse retina develops through three distinct stages. First, between embryonic day 16 and birth [post-natal day 0 (P0)], we observed both large, propagating waves inhibited by nicotinic acetylcholine receptor (nAChR) antagonists and small clusters of cells displaying nonpropagating, correlated calcium increases that were independent of nAChR activation. Second, between P0 and P11, we observed only larger propagating waves that were abolished by toxins specific to α3 and β2 subunit-containing nAChRs. Third, between P11 and P14 (eye opening) we observed propagating activity that was abolished by ionotropic glutamate receptor antagonists. The time course of this developmental shift was dramatically altered in retinas from mice lacking the β2 nAChR subunit or the β2 and β4 subunits. These retinas exhibited a novel circuit at P0, no spontaneous correlated activity between P1 and P8, and the premature induction at P8 of an ionotropic glutamate receptor-based circuit. Retinas from postnatal mice lacking the α3 nAChR subunit exhibited spontaneous, correlated activity patterns that were similar to those observed in embryonic wild-type mice. In α3-/- and β2-/- mice, the development and distribution of cholinergic neurons and processes and the density of retinal ganglion cells (RGCs) and the gross segregation of their dendrites into ON and OFF sublaminae were normal. However, the refinement of individual RGC dendrites is delayed. These results indicate that retinal waves mediated by nAChRs are involved in, but not required for, the development of neural circuits that define the ON and OFF sublamina of the inner plexiform layer.
In the developing vertebrate retina, spontaneous electric activity occurs rhythmically in the form of propagating waves and is believed to play a critical role in activity-dependent visual system development, including the establishment of precise retinal and geniculate circuitry. To elucidate how spontaneous retinal waves encode specific developmental cues at various developmental stages, it is necessary to understand how the waves are generated and regulated. Using Ca(2+) imaging and patch clamp in a flat-mount perinatal rabbit retinal preparation, this study demonstrates that, in addition to the cholinergic system, a strychnine-sensitive system in the inner retina plays an obligatory and developmentally regulated role in the initiation and propagation of spontaneous retinal waves. This system, which is believed to be the glycinergic network, provided an excitatory drive during early retinal development. It then became inhibitory after postnatal day 1 (P1) to P2, an age when a number of coordinated transitions in neurotransmitter systems occurred concomitantly, and finally contributed to the complete inhibition and disappearance of spontaneous waves after P7-P9. This glycinergic contribution was notably distinct from that of the ionotropic GABAergic system, which was found to exert an inhibitory but nonessential influence on the early wave formation. Blocking glycine- and GABA-gated anion currents had opposing effects on spontaneous retinal waves between embryonic day 29 and P0, suggesting that Cl(-) transporters, particularly R(+)-butylindazone-sensitive K-Cl cotransporters, may have a synapse- and/or cell type-specific distribution pattern, in addition to an age-dependent expression pattern in the inner retina. Overall, the results revealed an important reliance of spontaneous retinal waves on dynamic and coordinated interactions among multiple, nonredundant neurotransmitter systems.
Although correlated neural activity is a hallmark of many regions of the developing nervous system, the neural events underlying its propagation remain largely unknown. In the developing vertebrate retina, waves of spontaneous, correlated neural activity sweep across the ganglion cell layer. Here, we demonstrate that L-type Ca(2+) channel agonists induce large, frequent, rapidly propagating waves of neural activity in the developing retina. In contrast to retinal waves that have been described previously, these L-type Ca(2+) channel agonist-potentiated waves propagate independent of fast synaptic transmission. Bath application of nicotinic acetylcholine, AMPA, NMDA, glycine, and GABA(A) receptor antagonists does not alter the velocity, frequency, or size of the potentiated waves. Additionally, these antagonists do not alter the frequency or magnitude of spontaneous depolarizations that are recorded in individual retinal ganglion cells. Like normal retinal waves, however, the area over which the potentiated waves propagate is reduced dramatically by 18alpha-glycyrrhetinic acid, a blocker of gap junctions. Additionally, like normal retinal waves, L-type Ca(2+) channel agonist-potentiated waves are abolished by adenosine deaminase, which degrades extracellular adenosine, and by aminophylline, a general adenosine receptor antagonist, indicating that they are dependent on adenosine-mediated signaling. Our study indicates that although the precise spatiotemporal properties of retinal waves are shaped by local synaptic inputs, activity may be propagated through the developing mammalian retina by nonsynaptic pathways.
Spontaneous calcium transients occur in the ventricular zone of the chick retina and result from the endogenous release of neurotransmitters in the absence of action potentials. Calcium transients resulting from the activation of purinergic and muscarinic receptors occur in a mixed population of interphase and mitotic cells, whereas those produced by ionotropic GABA and glutamate receptors are mostly restricted to the interphase population, the GABA responses primarily coming from cells that express the neuronal marker TuJ-1. Muscarinic and purinergic receptors can act respectively as a brake and an accelerator on mitosis, whereas GABA and glutamate receptors are without effect. Our results suggest that the balance between muscarinic and purinergic activation acts to control the rate of retinal proliferation in early development.
In the immature retina, correlated spontaneous activity in the form of propagating waves is thought to be necessary for the refinement of connections between the retina and its targets. The continued presence of this activity in the mature retina would interfere with the transmission of information about the visual scene. The mechanisms responsible for the disappearance of retinal waves are not well understood, but one hypothesis is that visual experience is important. To test this hypothesis, we monitored the developmental changes in spontaneous retinal activity of both normal mice and mice reared in the dark. Using multi-electrode array recordings, we found that retinal waves in normally reared mice are present at postnatal day (P) 9 and begin to break down shortly after eye opening, around P15. By P21, waves have disappeared, and synchronous firing is comparable with that observed in the adult (6 weeks). In mice raised in the dark, we found a similar time course for the disappearance of waves. However, at P15, dark-reared retinas occasionally showed abnormally long periods of relative inactivity, not seen in controls. Apart from this quiescence, we found no striking differences between the patterns of spontaneous retinal activity from normal and dark-reared mice. We therefore suggest that visual experience is not required for the loss of synchronous spontaneous activity.
Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.
The development of cholinergic cells in the rat retina has been examined with immunocytochemistry by using antisera against choline acetyltransferase (ChAT). ChAT-immunoreactive (IR) cells were first detected at embryonic day 17 (E17) in the transitional zone between the neuroblastic layer (NBL) and ganglion cell layer (GCL). At E20, ChAT-IR cells are located exclusively in the GCL. At postnatal day 0 (P0), ChAT immunoreactivity appeared for the first time in cells at the distal margin of the NBL. Two prominent bands of labeled processes were first visible at P3, and by P15, these two bands resembled those of the adult retina. In addition, ChAT immunoreactivity appeared transiently in horizontal cells from P5 to P10. The number of ChAT-IR cells increased steadily up to P15. This resulted in a 93.8-fold increase between E17 and P15 (680–63,800 cells). However, after P15, the number declined by 19% from 63,800 cells at P15 to 51,800 in the adult. At all ages, the spatial density of each ChAT-IR cell population in the central retina was higher than in the periphery. In both central and peripheral regions, the peak density of ChAT-IR cells in the GCL was attained at E20. However, in the INL, the peak densities occurred at P3 in the central region and at P5 in the peripheral region. Up to P15, the soma diameter of ChAT-IR cells in the INL and GCL in each region increased continuously, reaching peak values at P15. Our results demonstrate that ChAT immunoreactivity is expressed in early developmental stages in the rat retina, as in other mammals, and that acetylcholine released from ChAT-IR cells may have neurotrophic functions in retinal maturation. J. Comp. Neurol. 427:604–616, 2000. © 2000 Wiley-Liss, Inc.
When contacts are first forming in the developing nervous system, many neurons generate spontaneous activity that has been hypothesized to shape appropriately patterned connections. In Mustela putorius furo, monocular intraocular blockade of spontaneous retinal waves of action potentials by cholinergic agents altered the subsequent eye-specific lamination pattern of the lateral geniculate nucleus (LGN). The projection from the active retina was greatly expanded into territory normally belonging to the other eye, and the projection from the inactive retina was substantially reduced. Thus, interocular competition driven by endogenous retinal activity determines the pattern of eye-specific connections from retina to LGN, demonstrating that spontaneous activity can produce highly stereotyped patterns of connections before the onset of visual experience.
The development of the central nervous system is dependent on spontaneous action potentials and changes in [Ca2+]i occurring in neurons [1-4]. In the mammalian retina, waves of spontaneous electrical activity spread between retinal neurons, raising [Ca2+]i as they pass [5-7]. In the ferret retina, the first spontaneous Ca2+ waves have been reported at postnatal day 2 and are thought to result from the Ca2+ influx associated with bursts of action potentials seen in ganglion cells at this time [5-7]. These waves depend on depolarisation produced by voltage-gated sodium channels, but their initiation and/or propagation also depends upon nicotinic cholinergic synaptic transmission between amacrine cells and ganglion cells [8]. Here, we report contrasting results for the chick retina where Ca2+ transients are seen at times before retinal synapse formation but when there are extensive networks of gap junctions. These Ca2+ transients do not require nicotinic cholinergic transmission but are modulated by acetylcholine (ACh), dopamine and glycine. Furthermore, they propagate into the depth of the retina, suggesting that they are not restricted to ganglion and amacrine cells. The transients are abolished by the gap-junctional blocker octanol. Thus, the Ca2+ transients seen early in chick retinal development are triggered and propagate in the absence of synapses by a mechanism that involves several neurotransmitters and gap junctions.
A recent highlight in the study of the retina has been the publication of evidence that the response of the ON bipolar cells is generated by a cGMP-mediated second messenger system. This GTP-binding protein mechanism is activated by the binding of glutamate, the photoreceptor neurotransmitter, to the 2-amino-4-phosphonobutyrate (APB) class of receptor.
1.
Long before onset of synaptogenesis in the chicken neural tube, the closely related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed in a mutually exclusive manner. Accordingly, neuroblasts on the ventricular side of the neural tube transiently express BChE before they abruptly accumulate AChE while approaching the outer brain surface.
2.
By exploiting AChE as a sensitive and early histochemical differentiation marker, we have demonstrated complex polycentric waves of differentiation spreading upon the cranial part of the chicken neural tube but a smooth rostrocaudal wave along the spinal cord. Shortly after expression of AChE, these cells extend long projecting neurites. In particular, segmented spinal motor axons originate from AChE-positive motoneurones; they navigate through a BChEactive zone within the rostral half of the sclerotomes before contacting BChE/AChE-positive myotome cells. At synaptogenetic stages, cholinesterases additionally are detectable in neurofibrillar laminae foreshadowing the establishment of cholinergic synapses.
3.
In order to elucidate the functional significance of cholinesterases at early stages, we have investigated specific cholinesterase molecules and their mechanism of actionin vivo andin vitro. A developmental shift from the low molecular weight forms to the tetramers of both enzymes has been determined.In vitro, the addition of a selective BChE inhibitor leads to a reduction of AChE gene expression. Thus,in vivo andin vitro data suggest roles of cholinesterases in the regulation of cell proliferation and neurite growth.
4.
Future research has to show whether neurogenetic functioning of cholinesterases can help to understand their reported alterations in neural tube defects, mental retardations, dementias and in some tumours.
The development of orderly connections in the mammalian visual system depends on action potentials in the optic nerve fibers,
even before the retina receives visual input. In particular, it has been suggested that correlated firing of retinal ganglion
cells in the same eye directs the segregation of their synaptic terminals into eye-specific layers within the lateral geniculate
nucleus. Such correlations in electrical activity were found by simultaneous recording of the extracellular action potentials
of up to 100 ganglion cells in the isolated retina of the newborn ferret and the fetal cat. These neurons fired spikes in
nearly synchronous bursts lasting a few seconds and separated by 1 to 2 minutes of silence. Individual bursts consisted of
a wave of excitation, several hundred micrometers wide, sweeping across the retina at about 100 micrometers per second. These
concerted firing patterns have the appropriate spatial and temporal properties to guide the refinement of connections between
the retina and the lateral geniculate nucleus.
Methods are described for isolating adult rabbit retinal and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, nd electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue.
In the adult mammalian retina, the principal direction of information flow is along a vertical pathway from photoreceptors to retinal interneurons to ganglion cells, the output neurons of the retina. We report here, however, that initially in development, at a time when the photoreceptors are not yet even present, there are already functionally defined networks within the retina. These networks are spontaneously active rather than visually driven, and they involve horizontal rather than vertical pathways. By means of optical recording using the calcium-sensitive dye Fura-2, we have found that sets of retinal ganglion cells and amacrine cells, a type of retinal interneuron, undergo synchronized oscillations in intracellular calcium concentration. These oscillations are highly correlated among subgroups of neighbouring cells, and spread in a wave-like fashion tangentially across the retina. Thus, in development of retinal circuitry, the initial patterning of neuronal function occurs in the horizontal domain before the adult pattern of vertical information transfer emerges.
The function of acetylcholinesterase (AChE) is to terminate the action of acetylcholine at the cholinergic synapse. Recent evidence suggests additional roles for acetylcholinesterase as a peptidase and/or a protease which is expressed by growing neurites as part of their invasion of developing neural structures. We report the localization of acetylcholinesterase in developing ferret retina. AChE histochemical staining is seen in the developing inner plexiform layer (IPL) of ferret retina at birth (post-natal day zero, PO), the earliest developmental stage examined. Transient expression is seen at the border between the ganglion cell layer and the nerve fiber layer at P14 and P21. A small amount of transient expression is seen in the outer plexiform layer (OPL) at this age as well. By P28, the transient expression in the OPL is at its peak, and is found at photoreceptor terminals and associated with apparent horizontal cell axons. Labeling is also seen intracellularly in the inner nuclear layer (INL), at the OPL/INL border, suggesting that horizontal cells are the source of the transient AChE expression in the OPL. Overt synaptic profiles also appear in the inner plexiform layer (IPL) at P21 and P28. About 2 days layer, the eyes open and the photoreceptor outer segments are fully developed. By 2 weeks later, at P42, the AChE staining pattern in the retina has taken on its adult appearance: no reaction product in the outer retina; intracellular reaction product in the Golgi apparatus of a subset of amacrine and displaced amacrine cells which manufacture AChE; and extracellular reaction product at both synaptic and non-synaptic sites in the IPL. These data are consistent with a role for AChE as a peptidase early in development, and as an enzyme essential in the termination of synaptic action at mature synapses.
Waves of spontaneous electrical activity and calcium transients occur in the retina during its development. Recent work raises the question of how these waves are produced and propagated.
Highly correlated neural activity in the form of spontaneous waves of action potentials is present in the developing retina weeks before vision. Optical imaging revealed that these waves consist of spatially restricted domains of activity that form a mosaic pattern over the entire retinal ganglion cell layer. Whole-cell recordings indicate that wave generation requires synaptic activation of neuronal nicotinic acetylcholine receptors on ganglion cells. The only cholinergic cells in these immature retinas are a uniformly distributed bistratified population of amacrine cells, as assessed by antibodies to choline acetyltransferase. The results indicate that the major source of synaptic input to retinal ganglion cells is a system of cholinergic amacrine cells, whose activity is required for wave propagation in the developing retina.
Vision is critical for the functional and structural maturation of connections in the mammalian visual system. Visual experience, however, is a subset of a more general requirement for neural activity in transforming immature circuits into the organized connections that subserve adult brain function. Early in development, internally generated spontaneous activity sculpts circuits on the basis of the brain's "best guess" at the initial configuration of connections necessary for function and survival. With maturation of the sense organs, the developing brain relies less on spontaneous activity and increasingly on sensory experience. The sequential combination of spontaneously generated and experience-dependent neural activity endows the brain with an ongoing ability to accommodate to dynamically changing inputs during development and throughout life.
In the developing mammalian retina, spontaneous waves of action potentials are present in the ganglion cell layer weeks before vision. These waves are known to be generated by a synaptically connected network of amacrine cells and retinal ganglion cells, and exhibit complex spatiotemporal patterns, characterized by shifting domains of coactivation. Here, we present a novel dynamical model consisting of two coupled populations of cells that quantitatively reproduces the experimentally observed domain sizes, interwave intervals, and wavefront velocity profiles. Model and experiment together show that the highly correlated activity generated by retinal waves can be explained by a combination of random spontaneous activation of cells and the past history of local retinal activity.
In the embryonic chicken neuroepithelium, butyrylcholinesterase (BChE) as a proliferation marker and then acetylcholinesterase (AChE) as a differentiation marker are expressed in a mutually exclusive manner. These and other data indicate a coregulation of cholinesterase expression, and also possible roles of cholinesterases during neurogenesis. Here, both aspects are investigated by two independent transfection protocols of dissociated retina cells of the 6-day-old chick embryo in reaggregation culture, both protocols leading to efficient overexpression of AChE protein. The effect of the overexpressed AChE protein on the re-establishment of retina-like three-dimensional networks (so-called retinospheroids) was studied. In a first approach, we transfected retinospheroids with a pSVK3 expression vector into which a cDNA construct encoding the entire rabbit AChE gene had been inserted in sense orientation. As detected at the mRNA level, rabbit AChE was heterologously overexpressed in chicken retinospheroids. Remarkably, this was accompanied by a strong increase in endogenous chicken AChE protein, while the total AChE activity was only slightly increased. This increase was due to chicken enzyme, as shown by species-specific inhibition studies using fasciculin. Clearly, total AChE activity is regulated post-translationally. As an alternative method of AChE overexpression, transfection of spheroids was performed with an antisense-5'-BChE vector, which not only resulted in the down-regulation of BChE expression, but also strongly increased chicken AChE transcripts, protein and enzyme activity. Histologically, a higher concentration of AChE protein (as a consequence of either AChE overexpression or BChE suppression) was associated with an advanced degree of tissue differentiation, as detected by immunostaining for the cytoskeletal protein vimentin.
Spontaneous neuronal activity has been detected in many parts of the developing vertebrate nervous system. Recent studies suggest that this activity depends on properties that are probably shared by all developing networks. Of particular importance is the high excitability of recurrently connected, developing networks and the presence of activity-induced transient depression of network excitability. In the spinal cord, it has been proposed that the interaction of these properties gives rise to spontaneous, periodic activity.
Many pathways in the developing visual system are restructured and become highly organized even before vision occurs. Yet the developmental processes underlying the remodeling of visual connectivity are crucially dependent on retinal activity. Surprisingly, the immature and light-insensitive retina spontaneously generates a pattern of rhythmic bursting activity during the period when the connectivity patterns of retinal ganglion cells are shaped. Spatially, the activity is seen to spread across the retina in the form of waves that bring into synchrony the bursts of neighboring cells. Waves are present in the developing retina of higher and lower vertebrates, which suggests that this form of activity may be a common and fundamental mechanism employed in the activity-dependent refinement of early patterns of visual connections. Unraveling the cues encoded by the waves promises to provide important insights into how interactions driven by specific patterns of activity could lead to the modification of connectivity during development.
The localization of nitric oxide synthase (NOS) was investigated by immunocytochemistry and immunoblotting using an antiserum against neuronal NOS in the rat, mouse, guinea pig, rabbit and cat retinae. Western blot analysis of retinal tissue extracts showed that the NOS-immunoreactive band of 155 kDa was present in all species. In the rat, mouse, guinea pig and rabbit retinae, two types of amacrine cells and a class of displaced amacrine cells were consistently NOS-labeled. In the cat retina, unlike other mammals, one type of amacrine cells and two types of displaced amacrine cells showed NOS immunoreactivity. NOS immunoreactivity was further found in some bipolar cells of the rat and guinea pig, some interplexiform cells of the mouse, some photoreceptor cells of the rabbit and some Müller cells of the cat.
Johnson PT, Williams RR, Cusato K, Reese BE. 1999. Rods and cones project to the inner plexiform layer during development. J Comp Neurol 414:1–12.
Numerous errors were introduced during the production of the above cited article after receipt of the corrected proofs. The correct text is as follows:
The citation “Bowes, 1988” on page 2 should read “Bowes et al., 1988”.
The subsection under Materials and Methods entitled “DI labelling” should read “DiI labeling”. The reference to “(Fig. 3, arrows)” at the top of page 9 should read “(Fig. 3i, arrows)”.
The final sentence on page 10 should read: “Many photoreceptors are generated long before an OPL has formed, and so it might be expected that the terminals of these cells would overshoot their future target stratum.”
Lastly, the final sentence of the text on page 11 should read: “The source of this environmental signal is unclear but since the retraction is coincident with the maturation of bipolar and horizontal cells, processes within the OPL are promising candidates.”
The Publisher regrets these errors.
Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.
Cell-cell signaling within the neocortical ventricular zone (VZ) has been shown to influence the proliferation of VZ precursor cells and the subsequent differentiation and fate of postmitotic neurons. Calcium (Ca(2+)), a ubiquitous second messenger implicated in the regulation of many aspects of development, may play a role in these signaling events. Accordingly, we have examined the spatiotemporal patterns of spontaneous intracellular free Ca(2+) ([Ca(2+)](i)) fluctuations of cells within the intact neocortical VZ. Previous observations have demonstrated that similar patterns of spontaneous [Ca(2+)](i) increase occur in both proliferative and postmitotic cortical cells, suggesting that they may be mechanistically similar. Our results suggest that the changes in [Ca(2+)](i) in VZ cells and cortical plate neurons are likely triggered by different mechansims, and imply that similar changes in [Ca(2+)](i) may underlie different signaling events during distinct phases of neocortical development.
Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.
Spontaneous waves of excitation in the developing mammalian retina are mediated, to a large extent, by neurotransmission. However, it is unclear how the underlying neurotransmitter systems interact with each other to play specific roles in the formation of retinal waves at various developmental stages. In particular, it is puzzling why the waves maintain a similar propagation pattern even after underlying neurotransmitter systems have undergone drastic developmental changes. Using Ca(2+) imaging and patch clamp in a whole-mount preparation of the developing rabbit retina, we discovered two dramatic and coordinated transitions in the excitatory drive for retinal waves: one from a nicotinic to a muscarinic system, and the other from a fast cholinergic to a fast glutamatergic input. Retinal waves before the age of postnatal day 1 (P1) were blocked by nicotinic antagonists, but not by muscarinic or glutamatergic antagonists. After P3, however, the spontaneous wave, whose basic spatiotemporal pattern remained similar, was completely inhibited by muscarinic or glutamate antagonists, but not by nicotinic antagonists. We also found that the muscarinic drive, mediated primarily by M1 and M3 receptors, was particularly important for wave propagation, whereas the glutamatergic drive seemed more important for local excitation. Our results suggest (1) a novel mechanism by which a neurotransmitter system changes its functional role via a switch between two completely different classes of receptors for the same transmitter, (2) the cholinergic system plays a critical role in not only early but also late spontaneous waves, and (3) the continued participation of the cholinergic system may provide a network basis for the consistency in the overall propagation pattern of spontaneous retinal waves.
The development of cholinergic cells in the rat retina has been examined with immunocytochemistry by using antisera against choline acetyltransferase (ChAT). ChAT-immunoreactive (IR) cells were first detected at embryonic day 17 (E17) in the transitional zone between the neuroblastic layer (NBL) and ganglion cell layer (GCL). At E20, ChAT-IR cells are located exclusively in the GCL. At postnatal day 0 (P0), ChAT immunoreactivity appeared for the first time in cells at the distal margin of the NBL. Two prominent bands of labeled processes were first visible at P3, and by P15, these two bands resembled those of the adult retina. In addition, ChAT immunoreactivity appeared transiently in horizontal cells from P5 to P10. The number of ChAT-IR cells increased steadily up to P15. This resulted in a 93.8-fold increase between E17 and P15 (680-63,800 cells). However, after P15, the number declined by 19% from 63,800 cells at P15 to 51,800 in the adult. At all ages, the spatial density of each ChAT-IR cell population in the central retina was higher than in the periphery. In both central and peripheral regions, the peak density of ChAT-IR cells in the GCL was attained at E20. However, in the INL, the peak densities occurred at P3 in the central region and at P5 in the peripheral region. Up to P15, the soma diameter of ChAT-IR cells in the INL and GCL in each region increased continuously, reaching peak values at P15. Our results demonstrate that ChAT immunoreactivity is expressed in early developmental stages in the rat retina, as in other mammals, and that acetylcholine released from ChAT-IR cells may have neurotrophic functions in retinal maturation.
Immunoreactivity for m2 and m4 muscarinic acetylcholine receptors (mAChRs) was demonstrated in the adult newt retina. The m2 mAChR was localized to somata on either side of the inner plexiform layer (IPL), especially ganglion cells, and also distributed into two bands within the IPL. The distal band at a depth of 0-15% IPL co-localized with one of two choline acetyltransferase (ChAT) immunoreactive bands, while the proximal band at 85-100% depth did not overlap with either of the ChAT-ir bands. The m4 mAChR was localized to somata closely apposed to either side of the IPL, probably amacrine cell somata, and no immunoreactivity was detectable throughout the IPL. The time course of appearance of the m2 and m4 mAChRs was examined in both developing and regenerating retinas. Like acetylcholinesterase (AChE), the m2 was first detected in somata located at the most proximal level of the retina well before ChAT-ir cholinergic neurons appeared, while the m4 was detected at the time of appearance of ChAT, in both developing and regenerating retinas. When the outer plexiform layer (OPL) began to form, somata in the horizontal cell layer became transiently immunoreactive to the m2. The discrepancy in distribution of the m2 and ChAT in the IPL suggests that mAChR may play a role other than cholinergic neurotransmission. Furthermore, the similarity in time course of appearance of the m2 and m4, as well as other cholinergic system components [4], in both developing and regenerating retinas would suggest that the mechanisms that control neuronal differentiation during retinal development and regeneration are similar.
Photoreceptors in the ferret's retina have been shown to project transiently to the inner plexiform layer (IPL) prior to their differentiation of an outer segment. On postnatal day 15 (P-15), when this projection achieves maximal density, the photoreceptors projecting into the IPL extend primarily to one of two depths, coincident with the processes of cholinergic amacrine cells. The present study has used an excitotoxic approach employing subcutaneous injections of L-glutamate to ablate these cholinergic amacrine cells on P-7, in order to see whether their elimination alters this targeting of photoreceptor terminals within the IPL. The near-complete elimination of cholinergic amacrine cells at P-15 was confirmed, although the population of retinal ganglion cells was also affected, being depleted by roughly 50%. The rod opsin-immunopositive terminals in such treated ferrets no longer showed a stratified distribution, being found throughout the depth of the IPL, as well as extending into the ganglion cell layer. This effect should not be due to the partial loss of retinal ganglion cells, however, since optic nerve transection at P-2, which eliminates the ganglion cells entirely while leaving the cholinergic amacrine cell population intact, was shown not to affect the stratification pattern of the photoreceptors within the IPL. These results strongly suggest that the targeting of the photoreceptor terminals to discrete strata within the IPL is dependent upon the cholinergic amacrine cell processes.
The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1 alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1 alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals.