![The cell cycle in the chick retina. A , Progenitor cells ( PC ) undergo interkinetic nuclear migration. In this process, the nucleus moves between the VZ, which is permissive for mitosis ( M ), and the GCL. The nucleus moves toward the GCL in G 1 , replicates its DNA (S phase), and returns toward the VZ in G 2 . Throughout this period, the cell retains contact with both surfaces of the retina by means of thin cytoplasmic processes. During the transition from G 2 to M phase, the cell retracts its vitreal process before dividing. PCs can either undergo symmetrical division, in which both daughter cells continue in the cell cycle, or divide asymmetrically to give rise to a PC and a newly differentiated cell ( NDC ) that then migrates to its fi nal location. B , Combined phase contrast and fl uorescence image of a section through an E6 chick retina, stained with propidium iodide. Mitotic cells are con fi ned to the VZ and contact the RPE. Mitotic cells can be identi fi ed by the presence of strongly staining and highly condensed chromatin that contrasts with the weakly stained and dispersed chromatin of the interphase cells. C , Single PC injected with FITC and dextran to show the processes that extend to the ventricular and vitreal surfaces. The nucleus of the cell is located in the bulge within the cell below the VZ. D, E , VZ cells can be labeled with both Fluo-4 AM, to measure their [Ca 2 ϩ ] i responses, and Hoechst 33342, to determine their mitotic status. D , In Fluo-4-labeled preparations, two distinct populations of cells can be identi fi ed within the VZ. Large, dark cells ( arrows ) with an average diameter of 4.6 Ϯ 0.2 m ( n ϭ 80; N ϭ 3) and smaller, more brightly labeled ones ( arrowheads ) of 2.7 Ϯ 0.2 m ( n ϭ 80; N ϭ 3) in diameter. Cells were viewed at resting [Ca 2 ϩ ] i . E , Same cells shown in A labeled with Hoechst 33342 (2 M ). The large pro fi les contain condensed chromatin and are mitotic ( arrows ). The chromatin within the nuclei of the brightly labeled and smaller cells is typical of cells in interphase ( arrowheads ). F , VZ of an E6 retina labeled with TuJ-1 ( red ), an antibody for neuron-speci fi c tubulin, and Hoechst 33342 ( green ). Approximately one-third of the interphase cells of the VZ are TuJ-1 positive (see Results). Scale bars, 10 m.](https://www.researchgate.net/profile/David-Becker-6/publication/11190913/figure/fig1/AS:282236353040392@1444301798896/The-cell-cycle-in-the-chick-retina-A-Progenitor-cells-PC-undergo-interkinetic_Q320.jpg)
![Spontaneous [Ca 2 ϩ ] i transients in E6 VZ cells. A , Examples of the spontaneous changes in [Ca 2 ϩ ] i ( ⌬ F/F ) seen in individual cells in the VZ when the temperature is raised to 37 ° C. In any given cell, events occurred approximately once every 250 sec ( n ϭ 99; N ϭ 3) and had durations of 13.8 Ϯ 1.3 sec. B , The frequency of spontaneous events is not reduced by the Na ϩ channel blocker TTX (10 M ; N ϭ 3). C , Paired event](https://www.researchgate.net/profile/David-Becker-6/publication/11190913/figure/fig2/AS:282236357234690@1444301799139/Spontaneous-Ca-2-i-transients-in-E6-VZ-cells-A-Examples-of-the-spontaneous_Q320.jpg)
![The mitotic and interphase VZ cell populations respond to different agonists. A-C, Changes in [Ca 2 ] i can be correlated with the mitotic status of individual cells. A, Confocal image of flat-mount E6 retina labeled with Fluo-4 AM. Four cells are indicated by numbered boxes and show increased fluorescence in the presence of carbachol (center panel ). Left, center, and right panels correspond to 0, 70, and 300 sec, respectively. B, Hoechst 33342 image of the same region shown in A reveals that cells 1 and 2 are mitotic, and 3 and 4 are in interphase. Scale bar, 5 m. C, Changes in [Ca 2 ] i (F/F ) in cells 1-4 during drug application. D, At E6, most cells respond to carbachol, and smaller numbers respond to UTP, GABA, and glutamate. Responses to UTP (100 M) and carbachol (100 M) arose equally from the mitotic and the interphase populations, whereas those to GABA (100 M) and glutamate (100 M) were predominantly from the interphase cells. Values plotted are mean SEM; N 4.](https://www.researchgate.net/profile/David-Becker-6/publication/11190913/figure/fig8/AS:668339827388425@1536356040959/The-mitotic-and-interphase-VZ-cell-populations-respond-to-different-agonists-A-C_Q320.jpg)
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Purinergic and Muscarinic Modulation of the Cell Cycle and Calcium Signaling in the Chick Retinal Ventricular Zone
Abstract and Figures
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.
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... As a whole, the entire set of neurotransmitters inherent in a certain type of cell determines the nature of interactions between neurons and their morphological features. There are several mechanisms in retinal cells that ensure the formation of neurotransmitter communication (Pearson et al., 2002). The signaling systems of neurotransmitters and neuromodulators, including ATP, acetylcholine, GABA (gammaaminobutyric acid), and glutamate, begin to participate in the control of the processes of proliferation, differentiation, and maintenance of cell viability long before the formation of synaptic connections, using various mechanisms of release from cells, by both vesicular and nonvesicular transport (Attwell et al., 1993;Voigt et al., 2015). ...
... Changes in the intracellular concentration of Ca 2+ ions regulate the successful passage of proliferating cells through all stages of the cell cycle, including the transition from G 1 to S-phase, all key phases of mitosis, the transition from metaphase to anaphase, and the induction of cytokinesis (Arredouani et al., 2010). A wave-like increase in the transport of Ca 2+ ions is observed during the peak of neurogenesis and is mainly associated with the formation of intercellular synaptic connections between differentiating retinal cells (Pearson et al., 2002). The processes of transport of Ca 2+ ions, characterized by a certain frequency, amplitude, and duration, also affect the synthesis of neurotransmitters, the expression of specific receptors in maturing neurons, and the growth rate of their neurites (Rosenberg and Spitzer, 2011). ...
The purinergic signaling system (PSS) occupies an exceptional position in the regulation of cellular
processes, maintaining the homeostasis of the retina of vertebrates. In addition to general regularities, taxonspecific
differences, genetically and metabolically determined, are manifested in the functioning of PSS in
the retina. Signal cascades with the participation of PSS components can perform a dual role in the retina:
they have both a damaging and protective effect, which is largely determined by the conditions of the cellular
microenvironment and the molecular context. The identification of key components in the functioning of the
PSS, linking it with other endogenous regulatory systems, creates prerequisites for the selection of cellular
and molecular targets for neuroprotection in human retinal pathologies.
... Melanin in the RPE has calcium buffering capacity and has been associated with the tight regulation of calcium levels available to the neural retina (Ambrosio et al., 2016;Bellono and Oancea, 2014;Bush and Simon, 2007;Drager, 1985;Salceda and Sanchez-Chavez, 2000). In turn, calcium transients have been shown to regulate retinal development by inducing progenitor proliferation (Pearson et al., 2002). Here, stimulation of L-type calcium channels with BayK-8644 to pregnant dams, led to markedly increased CyclinD2 expression, a larger Zic2 + RGC population in the albino retina, and ultimately improved function of the binocular circuit. ...
In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. We focus here on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ has the competence to generate predominantly ipsilaterally-projecting RGCs, but this competence is altered in the albino visual system due to CyclinD2 downregulation and disturbed temporal control of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit a diminished ipsilateral retinogeniculate projection and compromised depth perception. Pharmacological stimulation of calcium channels in albino mice, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs, and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.
Highlights
The mouse ventral CMZ produces predominantly ipsilateral RGCs.
In the albino visual system, CyclinD2 downregulation leads to delayed G1/S transition toward mitotic exit of CMZ progenitors.
Perturbations in the temporal control of cell cycle by CyclinD2 lead to reduced Zic2 ⁺ RGCs and consequently, a diminished ipsilateral retinogeniculate projection and compromised depth perception.
Calcium channel modulation during embryogenesis normalizes the levels of CyclinD2 and restores binocular vision in albino mice.
... Pearson и соавт. выяснили, что развитие куриных эмбрионов в присутствии уридинтрифосфата значительно увеличивает диаметр глаза [27]. ...
This literature review focuses on the role of purinergic P2-receptors in the physiology and pathophysiology of the eye, as well as on the possibilities of pharmacologic stimulation of these receptors. The most important studies from the clinical point of view on the involvement of purinergic neuronal transmission in the physiological processes have been analyzed, ranging from normal embryogenesis to cell apoptosis in age-related degenerative diseases of the eye. Data on the effect of agonists and antagonists of P2 receptors on corneal wound healing, lacrimal fluid production, regulation of intraocular pressure, neurotransmission, proliferation of glial tissue in the retina has been presented along with data on the possibilities of modulating these processes by using potential drugs acting via P2 receptors.
... In the postnatal rat retina, activation of P2Y1 receptors regulates the transition from G1 to S phase of the cell cycle [18], and P2Y12 receptors are required for the successful exit from the cell cycle [19]. In the chick retina, while UTP induces the proliferation of ganglion, amacrine, photoreceptor, and horizontal cell precursors through activation of P2Y2/4 receptors [20], ADP induces the proliferation of bipolar and Müller glial cell precursors through P2Y1/13 receptors, PLC, MAPK, CREB, and PI3K/ Akt pathways [21][22][23][24][25]. In addition to cell proliferation, nucleotides also control cell death in the chicken retina. ...
Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [³H]-thymidine incorporation was observed when cultures were incubated with 0.5–1.0 μM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5–5.0 μM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50 mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1 mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.
The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.
Objective:
The aim: This study was carried out to examine the presence of P2Y4 receptors in rat epidermal tissue and how their in vivo activation leads to histological and genetic changes.
Patients and methods:
Materials and methods: Thirty-six Wistar rats were separated into six groups each of six rats, the control group and five injected groups with increasing concentrations of ATP intradermally (0.1, 5.0, 10.0, 50.0, 100.0 μg/ml). The histological and genetic examination was performed from excised tissues.
Results:
Results: Noticeable histological thickening of the epidermal layer in rats injected with high concentrations of ATP. No apparent histological damage was seen in all injected groups. The genetic expression seems to also increase following exposure to variable concentrations of ATP.
Conclusion:
Conclusions: Purinergic receptors activated by ATP molecules are highly involved in the development of adult tissues. Their precise location within the epidermal layer indicated their importance in cellular proliferation and differentiation of epidermal cells. Excessive exposure to ATP results in their robust genetic ectopic over expression indicative of increased cellular activity.
In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.
Cyclophosphamide is a common anticancer drug that belongs to the nitrogen mustard group of alkylating agents, their acts by adding an alkyl group to DNA. Due to this cytotoxic property it is used extensively in a variety of carcinomas singly or in combination with other drugs to treat a variety of cancers. Purinergic nucleotides targets a group of receptors known as purinergic receptors, which fall into two classes P2Y and P2X receptors, the P2Y receptors are membrane bound G protein coupled receptors. Purinergic signaling has been shown to play a significant role in the regulation of proliferation in many animal and human tissues. Therefore, the present study was conducted to compare and correlate histomorphological alterations in different anatomical sites of adult wistar albino rat induced by a gradual increase in cyclophosphoamide dose administered intraperitoneally and subcutaneously and correlate the effect of cyclophosphamide with purinergic receptor gene expression. The aim of this study is to examine the effect of cyclophosphoamide on histological and cellular changes when administered in two different methods as well as examine purinergic receptor gene expression post cyclophosphoamide injection. Material and methods: Laboratory wistar albino rats were injected with different bolus doses of cyclophosphoamide intraperitoneally and subcutaneously and were sacrificed after seven days. liver, kidney, testes and subcutaneous injection sites were examined histologically and compared to control animals. Purinergic receptors P2Y1, P2Y2 and P2Y4 Gene expression changes were assessed using Quantitative reverse transcriptase polymerase chain reaction. Results: histological examination of all tissues in both groups shows gradual cytotoxic effect of cyclophosphoamide reaching extreme histological damage with increased bolus dose. Tissue damage included hemorrhaging and lymphocytic collection. Gene fold change of purinergic receptors showed an increase in genetic expression of P2Y1, P2Y2 and P2Y4 receptors in all examined site which indicates ectopic overexpression of purinergic receptors with increases cyclophosphamide specific dose. Increased gene fold change is an indication to cellular response to inflammatory histological damage caused by cyclophosphamide administration. The results of this study show that purinergic receptors are highly involved in cellular damage restoration through ectopic gene overexpression in tissues damaged by cyclophosphamide. Conclusion: This study concludes that cyclophosphoamide has a strong cytotoxic effect on certain organs if injected at a high dose intraperitoneally of subcutaneously. However histological damage is more prominent in intraperitoneal injected animals compared to subcutaneously injected animals. Cytotoxic effect and histological damage is confirmed by the gradual increase in purinergic receptors gene expression in both intraperitoneally and subcutaneously injected animals indicating an inflammatory response by increased gene fold change post administration of cyclophosphoamide and with gradual increase in cyclophosphoamide dose. It is a safer and less damaging to administer cyclophosphoamide subcutaneously.
This thesis investigates the roles of neurotransmitters and their receptors, changes in intracellular calcium ([Ca2+]i), and the retinal pigment epithelium (RPE) in the regulation of cell proliferation and migration in the developing chick retina. Confocal imaging of retinae is used to show that before synapses are formed, cells in the ventricular zone (VZ) display intermittent spontaneous [Ca2+]i transients that depend upon the endogenous release of neurotransmitters. Purinergic and muscarinic receptor- evoked transients occur in a mixed population of interphase and mitotic cells. Those produced by GABAergic and glutamatergic receptors are mostly restricted to the interphase population. Muscarinic receptor activation is shown to slow down, and purinergic activation to speed up, mitosis. These actions may result from the [Ca2+]i transients these agonists evoke. GABA and glutamate receptor activation are without effect on mitosis. The nuclei of retinal progenitor cells (PCs) migrate back-and-forth across the retina in a process called interkinetic nuclear migration (INM). To study the possible influence of neurotransmitter receptor activation on INM, a 'gene gun' technique was used to label cells in the VZ; the speed of movement of some cells is influenced by neurotransmitters. [Ca2+]i transients occur in cells during INM, which may be important in its regulation. Gap junctional communication between the RPE and the neural retina was investigated. Ca2+ -imaging experiments show that gap junctions support the spread of spontaneous Ca2+ signals between neighbouring cells. Whole-cell patch clamp recording was used to fill VZ cells with a combination of gap junction-permeable and impermeable dyes. These injections show that gap junctions couple PCs into clusters that largely exclude differentiated neurons. Coupling was also observed between cells in the RPE and the neural retina. These pathways may be important in the regulation of proliferation. The RPE is shown to express both purinergic and muscarinic receptors and to have a profound influence on the rate of cell proliferation in the neural retina. The RPE may speed mitosis in the retina through the release of ATP and other factors.
The albino visual system is characterised by a range of abnormalities including a severe rod deficit, an underdevelopment of the central retina and a misprojection of a portion of the temporal ganglion cells. During development of the retina levels of proliferation and apoptosis are elevated, and the maturational gradient is delayed. There is evidence from in vitro studies to suggest that L-DOPA, a precursor in both the melanin and catecholamine synthesis pathways, is able to regulate these abnormal patterns of cell proliferation in a manner which is consistent with its role in the regulation of retinal development. The aims of this thesis were; (1) to further characterise the developmental abnormalities associated with albinism, (2) to examine the effects of L-DOPA on patterns of cell division and neurogenesis within an in vivo context, and (3) to determine the mechanisms by which L-DOPA mediates its effects. Using both in vivo and in vitro methods, patterns of cell division and retinal differentiation were examined in pigmented and albino rat tissue. Results demonstrate that coincidental with abnormal levels of proliferation, the early postnatal albino retina is characterised by; a thickening of the neuroblastic layer, an excess of cells in the cell cycle, defects in the distribution of cleavage orientations within the apical-basal plane and elevations in gap junction expression. Furthermore, the vast majority of these parameters could be regulated by the introduction of L-DOPA to the retina and -preliminary experiments would suggest- through the addition of dopamine. These findings are consistent with a model in which reduced levels of L-DOPA in the albino retina lead to spatiotemporal defects in patterns of cell division, potentially as a result of secondary consequences on concentrations of retinal dopamine.
Transient elevations of intracellular Ca2+ levels play critical roles in neuronal development, but such elevations have not been demonstrated in migrating neurons. Here, we show that the amplitude and frequency components of Ca2+ fluctuations are correlated positively with the rate of granule cell movement in cerebellar microexplant cultures. Moreover, depression of the amplitude and frequency components of Ca2+ fluctuations by blockade of Ca2+ influx across the plasma membrane results in a reversible retardation of cell movement. These results indicate that the combination of amplitude and frequency components of intracellular Ca2+ fluctuations may provide an intracellular signal controlling the rate of neuronal cell migration.
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 regulation of cell cycle progression is a complex process which involves kinase cascades, protease action, production of second messengers and other operations. Increasing evidence now compellingly suggests that changes in the intracellular Ca2+ concentration may also have a crucial role. Ca2+ transients occur at the awakening from quiescence, at the G/S transition, during S-phase, and at the exit from mitosis. They may lead to the activation of Ca2+ binding proteins like S-100, but the key decoder of the Ca2+ signals in the cycle is calmodulin. Activation of calmodulin leads to the stimulation of protein kinases, i.e., CaM-kinase II, and of the CaM-dependent protein phosphatase calcineurin. Ample evidence now indicates the G/S transition, the progression from G2 to M, and the metaphase/anaphase transition as specific points of intervention of CaM-kinase II. Another attractive possibility for the role of Ca2+ in the cycle is through the activation of the Ca(2+)-dependent protease calpain: other proteases (e.g., the proteasome) have been suggested to be responsible for the degradation of some of cyclins, which is essential to the progression of the cycle. One of the cyclins, however, (D1) is instead degraded by calpain, which has been shown to promote both mitosis and meiosis when injected into somatic cells or oocytes.
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.
Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.
Patch-clamp recordings of granule cells in thin slices of developing rat cerebellum maintained in vitro displayed spontaneous single-channel activity mediated via activation of N-methyl-D-aspartate (NMDA) receptors. The frequency of tonic single-channel activity was reversibly inhibited by the NMDA receptor/channel antagonists D-2-amino-5-phosphonovalerate (D-AP5), 7-chloro-kynurenate (7-Cl-Kynu) and MgCl2, potentiated by glycine, and unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or tetrodotoxin (TTX). Tonic channel activity was also reversibly inhibited by enzymatic degradation of endogenous glutamate by glutamate pyruvate transaminase, which did not affect the NMDA sensitivity of granule cells. Both the frequency of spontaneous channel activity and the NMDA sensitivity were low in premigratory cells of the external germinal layer (EGL), with large increases observed in migrating cells in the molecular layer (ML) and in postmigratory cells within the internal granule cell layer (GCL). Tonic channel activity was enhanced by the glutamate uptake inhibitor L-alpha-aminoadipate (L-alpha-AA), the degree of enhancement being greater in the EGL than the GCL. The results demonstrate that a dramatic increase in the tonic NMDA receptor-channel activity occurs during the stages of granule cell differentiation, migration and synaptogenesis, which is driven by endogenous glutamate release and regulated by NMDA receptor density and local glutamate uptake.
Growth curves of the retinal cell population of embryonic chicks were fitted by a branching-process model of cell population growth, thereby estimating the proliferative ratios and mean cell-cycle times of the generations of cell cycles that underlie retinal growth. The proliferative ratio determines the proportion of cells that divides in the next generation, so the numbers of proliferative and non-proliferative cells in each generation of cell cycles were obtained. The mean cell-cycle times determine the times over which the generations are extant. Assuming growth starts from one cell in generation 0, the proliferative cells reach 3.6 x 10(6) and the non-proliferative cells reach 1.1 x 10(6) by generation 23. The next four generations increase the proliferative cell numbers to 13.9 x 10(6) and produce 20.1 x 10(6) non-proliferative cells. In the next five generations in the end phase of growth, non-proliferative cells are produced in large numbers at an average of 13.9 x 10(6) cells per generation as the retinal lineages are completed. The retinal cell population reaches a maximum estimated here at 98.2 x 10(6) cells. The mean cell-cycle time estimates range between 6.8 and 10.1 h in generations before the end phase of growth and between 10.6 and 17.2 h in generations in the end phase. The retinal cell population growth is limited by the depletion of the proliferative cell population that the production of non-proliferative cells entails. The proliferative ratios and the cell-cycle-time distribution parameters are the likely determinants of retinal growth rates. The results are discussed in relation to other results of spatial and temporal patterns of the cessation of cell cycling in the embryonic chick retina.