GABAergic system and chloride cotransporters as potential therapeutic targets to mitigate cell death in ischemia.

Gamma aminobutyric acid (GABA) is a critical inhibitory neurotransmitter in the central nervous system that plays a vital role in modulating neuronal excitability. Dysregulation of GABAergic signaling, particularly involving the cotransporters NKCC1 and KCC2, has been implicated in various pathologies, including epilepsy, schizophrenia, autism spectrum disorder, Down syndrome, and ischemia. NKCC1 facilitates chloride influx, whereas KCC2 mediates chloride efflux via potassium gradient. Altered expression and function of these cotransporters have been associated with excitotoxicity, inflammation, and cellular death in ischemic events characterized by reduced cerebral blood flow, leading to compromised tissue metabolism and subsequent cell death. NKCC1 inhibition has emerged as a potential therapeutic approach to attenuate intracellular chloride accumulation and mitigate neuronal damage during ischemic events. Similarly, targeting KCC2, which regulates chloride efflux, holds promise for improving outcomes and reducing neuronal damage under ischemic conditions. This review emphasizes the critical roles of GABA, NKCC1, and KCC2 in ischemic pathologies and their potential as therapeutic targets. Inhibiting or modulating the activity of these cotransporters represents a promising strategy for reducing neuronal damage, preventing excitotoxicity, and improving neurological outcomes following ischemic events. Furthermore, exploring the interactions between natural compounds and NKCC1/KCC2 provides additional avenues for potential therapeutic interventions for ischemic injury.

Caffeine and Its Neuroprotective Role in Ischemic Events: A Mechanism Dependent on Adenosine Receptors.

Ischemia is characterized by a transient, insufficient, or permanent interruption of blood flow to a tissue, which leads to an inadequate glucose and oxygen supply. The nervous tissue is highly active, and it closely depends on glucose and oxygen to satisfy its metabolic demand. Therefore, ischemic conditions promote cell death and lead to a secondary wave of cell damage that progressively spreads to the neighborhood areas, called penumbra. Brain ischemia is one of the main causes of deaths and summed with retinal ischemia comprises one of the principal reasons of disability. Although several studies have been performed to investigate the mechanisms of damage to find protective/preventive interventions, an effective treatment does not exist yet. Adenosine is a well-described neuromodulator in the central nervous system (CNS), and acts through four subtypes of G-protein-coupled receptors. Adenosine receptors, especially A1 and A2A receptors, are the main targets of caffeine in daily consumption doses. Accordingly, caffeine has been greatly studied in the context of CNS pathologies. In fact, adenosine system, as well as caffeine, is involved in neuroprotection effects in different pathological situations. Therefore, the present review focuses on the role of adenosine/caffeine in CNS, brain and retina, ischemic events.

Towards diversity in science - a glance at gender disparity in the Brazilian Society of Neuroscience and Behavior (SBNeC).

Gender equity is far from being achieved in most academic institutions worldwide. Women representation in scientific leadership faces multiple obstacles. Implicit bias and stereotype threat are considered important driving forces concerning gender disparities. Negative cultural stereotypes of weak scientific performance, unrelated to true capacity, are implicitly associated with women and other social groups, influencing, without awareness, attitudes and judgments towards them. Meetings of scientific societies are the forum in which members from all stages of scientific careers are brought together. Visibility in the scientific community stems partly from presenting research as a speaker. Here, we investigated gender disparities in the Brazilian Society of Neuroscience and Behavior (SBNeC). Across the 15 mandates (1978-2020), women occupied 30% of the directory board posts, and only twice was a woman president. We evaluated six meetings held between 2010 and 2019. During this period, the membership of women outnumbered that of men in all categories. A total of 57.50% of faculty members, representing the potential pool of speakers and chairs, were female. Compared to this expected value, female speakers across the six meetings were scarce in full conferences (χ2(5)=173.54, P<0.001) and low in symposia (χ2(5)=36.92, P<0.001). Additionally, women chaired fewer symposia (χ2(5)=47.83, P<0.001). Furthermore, men-chaired symposia had significantly fewer women speakers than women-chaired symposia (χ2(1)=56.44, P<0.001). The gender disparities observed here are similar to those in other scientific societies worldwide, urging them to lead actions to pursue gender balance and diversity. Diversity leads not only to fairness but also to higher-quality science.

Towards diversity in science - a glance at gender disparity in the Brazilian Society of Neuroscience and Behavior (SBNeC)

Gender equity is far from being achieved in most academic institutions worldwide. Women representation in scientific leadership faces multiple obstacles. Implicit bias and stereotype threat are considered important driving forces concerning gender disparities. Negative cultural stereotypes of weak scientific performance, unrelated to true capacity, are implicitly associated with women and other social groups, influencing, without awareness, attitudes and judgments towards them. Meetings of scientific societies are the forum in which members from all stages of scientific careers are brought together. Visibility in the scientific community stems partly from presenting research as a speaker. Here, we investigated gender disparities in the Brazilian Society of Neuroscience and Behavior (SBNeC). Across the 15 mandates (1978-2020), women occupied 30% of the directory board posts, and only twice was a woman president. We evaluated six meetings held between 2010 and 2019. During this period, the membership of women outnumbered that of men in all categories. A total of 57.50% of faculty members, representing the potential pool of speakers and chairs, were female. Compared to this expected value, female speakers across the six meetings were scarce in full conferences (χ2(5)=173.54, P<0.001) and low in symposia (χ2(5)=36.92, P<0.001). Additionally, women chaired fewer symposia (χ2(5)=47.83, P<0.001). Furthermore, men-chaired symposia had significantly fewer women speakers than women-chaired symposia (χ2(1)=56.44, P<0.001). The gender disparities observed here are similar to those in other scientific societies worldwide, urging them to lead actions to pursue gender balance and diversity. Diversity leads not only to fairness but also to higher-quality science.

Caffeine exposure ameliorates acute ischemic cell death in avian developing retina.

In infants, the main cause of blindness is retinopathy of prematurity that stems in a hypoxic-ischemic condition. Caffeine is a psychoactive compound that at low to moderate concentrations, selectively inhibits adenosine A1 and A2A receptors. Caffeine exerts beneficial effects in central nervous system of adult animal models and humans, whereas it seems to have malefic effect on the developing tissue. We observed that 48-h exposure (during synaptogenesis) to a moderate dose of caffeine (30 mg/kg of egg) activated pro-survival signaling pathways, including ERK, CREB, and Akt phosphorylation, alongside BDNF production, and reduced retinal cell death promoted by oxygen glucose deprivation in the chick retina. Blockade of TrkB receptors and inhibition of CREB prevented caffeine protection effect. Similar signaling pathways were described in previously reported data concerning chemical preconditioning mechanism triggered by NMDA receptors activation, with low concentrations of agonist. In agreement to these data, caffeine increased NMDA receptor activity. Caffeine decreased the levels of the chloride co-transporter KCC2 and delayed the developmental shift on GABAA receptor response from depolarizing to hyperpolarizing. These results suggest that the caffeine-induced delaying in depolarizing effect of GABA could be facilitating NMDA receptor activity. DPCPX, an A1 adenosine receptor antagonist, but not A2A receptor inhibitor, mimicked the effect of caffeine, suggesting that the effect of caffeine occurs through A1 receptor blockade. In summary, an in vivo caffeine exposure could increase the resistance of the retina to ischemia-induced cell death, by triggering survival pathways involving CREB phosphorylation and BDNF production/TrkB activation.

Retinal exposure to high glucose condition modifies the GABAergic system: Regulation by nitric oxide.

Diabetic retinopathy is a severe retinal complication that diabetic patients are susceptible to present. Although this disease is currently characterized as a microvascular disease, there is growing evidence that neural changes occur and maybe precede vascular impairments. Using chicken retina, an avascular tissue with no direct contact with blood vessels and neural retina, this study aimed to evaluate the influence of acute exposure to high glucose concentration in the retinal GABAergic system, and the role of nitric oxide (NO) in this modulation. Therefore, in ex vivo experiments, retinas were incubated in control (10 mM glucose) or high glucose condition (35 mM) for 30 min. By using DAF-FM to evaluate NO production, it was possible to show that high glucose (HG) significantly increased NO levels in the outer nuclear layer, inner nuclear layer (outer and inner portion), and inner plexiform layer. It was also observed that HG increased GABA immunoreactivity (IR) in amacrine and horizontal cells. HG did not change glutamic acid decarboxylase-IR, whereas it decreased GABA Transporter (GAT) 1-IR and increased GAT-3-IR. The co-treatment with 7-NI, an inhibitor of neuronal nitric oxide synthase (nNOS), blocked all changes stimulated by HG exposure. The concomitant exposure with SNAP-5114, a GAT-2/3 inhibitor, blocked the increase in GABA-IR caused by HG incubation. Therefore, our data suggest that hyperglycemia induces GABA accumulation in the cytosol by modulating GABA transporters. This response is dependent on NO production and signaling.

Cannabinoid receptors and TRPA1 on neuroprotection in a model of retinal ischemia.

Retinal ischemia is a pathological event present in several retinopathies such as diabetic retinopathy and glaucoma, leading to partial or full blindness with no effective treatment available. Since synthetic and endogenous cannabinoids have been studied as modulators of ischemic events in the central nervous system (CNS), the present study aimed to investigate the involvement of cannabinoid system in the cell death induced by ischemia in an avascular (chick) retina. We observed that chick retinal treatment with a combination of WIN 55212-2 and cannabinoid receptor antagonists (either AM251/O-2050 or AM630) decreased the release of lactate dehydrogenase (LDH) induced by retinal ischemia in an oxygen and glucose deprivation (OGD) model. Further, the increased availability of endocannabinoids together with cannabinoid receptor antagonists also had a neuroprotective effect. Surprisingly, retinal exposure to any of these drugs alone did not prevent the release of LDH stimulated by OGD. Since cannabinoids may also activate transient receptor potential (TRP) channels, we investigated the involvement of TRPA1 receptors (TRPA1) in retinal cell death induced by ischemic events. We demonstrated the presence of TRPA1 in the chick retina, and observed an increase in TRPA1 content after OGD, both by western blot and immunohistochemistry. In addition, the selective activation of TRPA1 by mustard oil (MO) did not worsen retinal LDH release induced by OGD, whereas the blockage of TRPA1 completely prevented the extravasation of cellular LDH in ischemic condition. Hence, these results show that during the ischemic event there is an augment of TRPA1, and activation of this receptor is important in cell death induction. The data also indicate that metabotropic cannabinoid receptors, both type 1 and 2, are not involved with the cell death found in the early stages of ischemia. Therefore, the study points to a potential role of TRPA1 as a target for neuroprotective approaches in retinal ischemia.

Fragile X Mental Retardation Protein expression in the retina is regulated by light.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick.

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation.

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.

Selective activation of group III metabotropic glutamate receptor subtypes produces different patterns of γ-aminobutyric acid immunoreactivity and glutamate release in the retina.

Glutamate, the major excitatory neurotransmitter in the retina, functions by activation of both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. Group III mGluRs, except for mGluR6, are mostly found in the inner plexiform layer (IPL), and their retinal functions are not well known. Therefore, we decided to investigate the effect of mGluRIII on glutamate release and GABAergic amacrine cells in the chick retina. The nonselective mGluRIII agonist L-SOP promoted a decrease in the number of γ-aminobutyric acid (GABA)-positive cells and in the GABA immunoreactivity in all sublayers of the IPL. This effect was prevented by the antagonist MAP-4, by GAT-1 inhibitor, and by antagonists of iGluR. Under the conditions used, L-SOP did not alter endogenous glutamate release. VU0155041, an mGluR4-positive allosteric modulator, reduced GABA immunoreactivity in amacrine cells and in sublayers 2 and 4 of the IPL but evoked an increase in the glutamate released. VU0155041's effect was inhibited by the absence of calcium. AMN082, a selective mGluR7-positive allosteric modulator, also decreased GABA immunoreactivity in amacrine cells and sublayers 1, 2, and 3 and increased glutamate release, and this effect was also inhibited by calcium absence. DCPG, an mGluR8-selective agonist, did not significantly alter GABA immunoreactivity in amacrine cells or glutamate release. However, it did significantly increase GABA immunoreactivity in sublayers 4 and 5. The results suggest that mGluRIIIs are involved in the modulation of glutamate and GABA release in the retina, possibly participating in distinct visual pathways: mGluR4 might be involved with cholinergic circuitry, whereas mGluR7 and mGluR8 might participate, respectively, in the OFF and the ON pathways.

A calcium-dependent glutamate release induced by metabotropic glutamate receptors I/II promotes GABA efflux from amacrine cells via a transporter-mediated process.

Glutamate and GABA are, respectively, the major excitatory and inhibitory neurotransmitters in the retina, participating in the two pathways through which the retina processes light information. It has already been shown that glutamate induces GABA release from amacrine cells through a transporter-mediated mechanism, and that this process is mediated by ionotropic glutamate receptors. It is well established that glutamate can also activate metabotropic glutamate receptors, which are widely distributed in the retina, and can be detected in amacrine cell bodies and synaptic contacts. Thus, we decided to investigate the role of the activation of groups I and II metabotropic glutamate receptors in GABA release from amacrine cells in the chicken retina. Group I/II agonist trans-ACPD promoted a 40% decrease in the number of GABA-positive cells in relation to the control, effect that was prevented by antagonists of both groups. Also, the trans-ACPD effect was blocked by GAT-1 inhibitor or by antagonists of ionotropic glutamate receptors. Trans-ACPD induced release of GABA was abolished when the experiment was conducted in absence of calcium ions. Under the superfusing conditions used, trans-ACPD promoted an increase in endogenous glutamate release that was prevented when calcium was omitted from the bathing medium. The results suggest that mGluRI/II regulate the release of glutamate, likely from bipolar cells, that in turn activates GABA release from amacrine cells via a transporter mediated process.

Modulation of GABA release by nitric oxide in the chick retina: different effects of nitric oxide depending on the cell population.

gamma-Aminobutyric acid (GABA) is considered to be the most important inhibitory neurotransmitter in the central nervous system, including the retina. It has been shown that nitric oxide (NO) can influence the physiology of all retinal neuronal types, by mechanisms including modulation of GABA release. However, until now, there have been no data concerning the effects on endogenous GABA release of NO produced by cells in the intact retina. In the present study, we have investigated how NO production induced by drugs influences the release of endogenous GABA in cells of the intact retina of mature chicken. Retinas were exposed to different drugs that affect NO production, and GABA remaining in the tissue was detected by immunohistochemical procedures. A specific nNOS inhibitor (7-NI) reduced the number of GABA+amacrine cells and cells in the ganglion cell layer (GCL) by 33% and 41%, respectively. A GABA transporter inhibitor blocked this effect. L-arginine (100 microM), the precursor of NO, induced increases of 62% and 34% in the number of GABA+amacrine cells and GCL cells, respectively. A sodium (Na(+))-free solution, 7-NI and a PKG inhibitor prevented the effect of L-arginine (100 microM). However, a higher concentration of L-arginine (1mM) induced a 35% reduction in the number of GABA+cells by a Na(+)-dependent mechanism that was restricted to the GCL population. NMDA, which stimulates NO production, increased GABA release as indicated by 53% and 38% reductions in the number of GABA+amacrine cells and GCL cells, respectively. This effect was blocked by 7-NI only in GCL cells. We conclude that basal NO production and moderate NO production (possibly induced by L-arginine; 100 microM) inhibit basal GABA release from amacrine cells and GCL cells. However, NMDA or L-arginine (1mM) induce a NO-dependent increase in GABA release in GCL cells, possibly by stimulating higher NO production.

Transporter mediated GABA release in the retina: role of excitatory amino acids and dopamine.

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.

GABAergic circuitry in the opossum retina: a GABA release induced by L-aspartate.

Glutamate and gamma-amino butyric acid (GABA) are the major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system (CNS), including the retina. Although in a number of studies the retinal source of GABA was identified, in several species, as horizontal, amacrine cells and cells in the ganglion cell layer, nothing was described for the opossum retina. Thus, the first goal of this study was to determine the pattern of GABAergic cell expression in the South America opossum retina by using an immunohistochemical approach for GABA and for its synthetic enzyme, glutamic acid decarboxylase (GAD). GABA and GAD immunoreactivity showed a similar cellular pattern by appearing in a few faint horizontal cells, topic and displaced amacrine cells. In an effort to extend the knowledge of the opossum retinal circuitry, the possible influence of glutamatergic inputs in GABAergic cells was also studied. Retinas were stimulated with different glutamatergic agonists and aspartate (Asp), and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to NMDA and kainate resulted the reduction of the number of GABA immunoreactive topic and displaced amacrine cells. The Asp treatment also resulted in reduction of the number of GABA immunoreactive amacrine cells but, in contrast, the displaced amacrine cells were not affected. Finally, the Asp effect was totally blocked by MK-801. This result suggests that Asp could be indeed a putative neurotransmitter in this non-placental animal by acting on an amacrine cell sub-population of GABA-positive NMDA-sensitive cells.

GABA release induced by aspartate-mediated activation of NMDA receptors is modulated by dopamine in a selective subpopulation of amacrine cells.

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS, including the retina. In the chick retina, GABA is located in horizontal and amacrine cells and in some cells in the ganglion cell layer. It has been shown that glutamate and its agonists, NMDA, kainate, and aspartate, promote the release of GABA from isolated retina and from cultured retinal cells. Dopamine, the major catecholamine in the retina, inhibits the induction of GABA release by NMDA. Two to seven-day-old intact chicken retinas were stimulated with different glutamatergic agonists and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to 100 microM NMDA for 30 minutes resulted in 50% reduction in the number of GABA-immunoreactive amacrine cells. Aspartate (100 microM) treatment also resulted in 60% decrease in the number of GABA-immunoreactive amacrine cells. The number of GABA-immunoreactive horizontal cells was not affected by either NMDA or aspartate. In addition, dopamine reversed by 50% the reduction of the number of GABA-immunoreactive amacrine cells exposed to NMDA or aspartate. Kainate stimulation promoted a 50% reduction in the number of both GABA-immunoreactive amacrine and horizontal cells. Dopamine did not interfere with the kainate effect. While in control and in non-stimulated retinas a continuous and homogeneous immunolabeling was observed throughout the inner plexiform layer, retinas exposed to NMDA, kainate and aspartate displayed only a faint punctate labeling in the inner plexiform layer. It is concluded that, under our experimental conditions, both NMDA and aspartate induce the release of GABA exclusively from amacrine cells, and that the release is modulated by dopamine. On the other hand, kainate stimulates GABA release from both amacrine and horizontal cells with no interference of dopamine.

Evidence of muscarinic acetylcholine receptors in the retinal centrifugal system of the chick.

In this study we characterize the presence of muscarinic acetylcholine receptors (mAChR) in the isthmo-optic nucleus (ION) of chicks by immunohistochemistry with the M35 antibody. Some M35-immunoreactive fibers were observed emerging from the retinal optic nerve insertion, suggesting that they could be centrifugal fibers. Indeed, intraocular injections of cholera toxin B (CTb), a retrograde tracer, and double-labeling with M35 and CTb in the ION confirmed this hypothesis. The presence of M35-immunoreactive cells and the possible mAChR expression in ION and ectopic neuron cells in the chick brain strongly suggest the existence of such a cholinergic system in this nucleus and that acetylcholine release from amacrine cells may mediate interactions between retinal cells and ION terminals.

Evidence of muscarinic acetylcholine receptors in the retinal centrifugal system of the chick

In this study we characterize the presence of muscarinic acetylcholine receptors (mAChR) in the isthmo-optic nucleus (ION) of chicks by immunohistochemistry with the M35 antibody. Some M35-immunoreactive fibers were observed emerging from the retinal optic nerve insertion, suggesting that they could be centrifugal fibers. Indeed, intraocular injections of cholera toxin B (CTb), a retrograde tracer, and double-labeling with M35 and CTb in the ION confirmed this hypothesis. The presence of M35-immunoreactive cells and the possible mAChR expression in ION and ectopic neuron cells in the chick brain strongly suggest the existence of such a cholinergic system in this nucleus and that acetylcholine release from amacrine cells may mediate interactions between retinal cells and ION terminals

Neurogenesis of cholinoceptive neurons in the chick retina.

Immunocytochemistry and [3H]thymidine autoradiography were combined in this study to determine the neurogenesis of cholinoceptive cells in the chick retina. After injections of [3H]thymidine between embryonic days 1 and 11, the time of birth of retinal neurons containing either the alpha 3 or the alpha 8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the alpha 3-positive neurons in the ganglion cell layer leave the cell cycle from E2 through E7, and those in the inner nuclear layer (amacrine and displaced ganglion cells) from E2 through E9. The alpha 8-positive cells in the ganglion cell layer were born from E1 through E7, and those in the inner nuclear layer (amacrine and bipolar cells) from E2 through E11. These data suggest that the time of birth of cholinoceptive neurons in the chick retina follows the general pattern of cell generation in the chick retina, and that alpha 8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the alpha 3-positive cells in the same layer.