Cells In Optic Tectum Research Articles

Loss of Stim2 in zebrafish induces glaucoma-like phenotype.

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology.

Unveiling the neurotoxicity of methylmercury in fish (Diplodus sargus) through a regional morphometric analysis of brain and swimming behavior assessment

The current study aims to shed light on the neurotoxicity of MeHg in fish (white seabream – Diplodus sargus) by the combined assessment of: (i) MeHg toxicokinetics in the brain, (ii) brain morphometry (volume and number of neurons plus glial cells in specific brain regions) and (iii) fish swimming behavior (endpoints associated with the motor performance and the fear/anxiety-like status). Fish were surveyed for all the components after 7 (E7) and 14 (E14) days of dietary exposure to MeHg (8.7μgg−1), as well as after a post-exposure period of 28days (PE28). MeHg was accumulated in the brain of D. sargus after a short time (E7) and reached a maximum at the end of the exposure period (E14), suggesting an efficient transport of this toxicant into fish brain. Divalent inorganic Hg was also detected in fish brain along the experiment (indicating demethylation reactions), although levels were 100–200 times lower than MeHg, which pinpoints the organic counterpart as the great liable for the recorded effects. In this regard, a decreased number of cells in medial pallium and optic tectum, as well as an increased hypothalamic volume, occurred at E7. Such morphometric alterations were followed by an impairment of fish motor condition as evidenced by a decrease in the total swimming time, while the fear/anxiety-like status was not altered. Moreover, at E14 fish swam a greater distance, although no morphometric alterations were found in any of the brain areas, probably due to compensatory mechanisms. Additionally, although MeHg decreased almost two-fold in the brain during post-exposure, the levels were still high and led to a loss of cells in the optic tectum at PE28. This is an interesting result that highlights the optic tectum as particularly vulnerable to MeHg exposure in fish. Despite the morphometric alterations reported in the optic tectum at PE28, no significant changes were found in fish behavior. Globally, the effects of MeHg followed a multiphasic profile, where homeostatic mechanisms prevented circumstantially morphometric alterations in the brain and behavioral shifts. Although it has become clear the complexity of matching brain morphometric changes and behavioral shifts, motor-related alterations induced by MeHg seem to depend on a combination of disruptions in different brain regions.

Developmental and hypoxia-induced cell death share common ultrastructural and biochemical apoptotic features in the central nervous system

Hypoxic insults during the perinatal period lead to motor and cognitive impairments that later appear during childhood. In the adult brain, hypoxic events often lead to necrotic neuronal death, depending on the region and intensity of the event. During development an active apoptotic cell death occurs and could be an important variable affecting the hypoxic insult outcome.In the present work we performed a comparative study, in a chick embryo model, of the phenotypes and molecular markers exhibited during developmental and hypoxic cell death (HxCD). Ultrastructural analysis of optic tectum cells of embryos subjected to hypoxia (8% O2, 60min) revealed a clear apoptotic morphology that did not differ from the one exhibited during developmental cell death. Integrity of plasma membrane, condensation of chromatin in round well-defined bodies, and gradual shrinkage of the cell are all hallmarks of the apoptotic process and were present in both control and hypoxic cells. To elucidate if hypoxic and developmental cell deaths share a common mechanism we evaluated the activation of both intrinsic and extrinsic apoptotic pathways. A basal cleavage of caspase-9 and cytochrome c release was observed by co-immunofluorescence in control embryos, but hypoxic insult significantly increased the incidence of this colocalization. Caspase-8 cleavage remained unchanged after the hypoxic insult, suggesting that the extrinsic pathway would not be involved in hypoxic death. We also observed a significant decrease of Akt activation immediately after hypoxia, possibly facilitating the later release of cytochrome c. In addition we analyzed the influence of retinal ganglion cells (RGC) in neuronal survival. Transection of RGC fibers at embryonic day (ED) 3 did not induce any change in developmental and HxCD at ED12. In conclusion, our findings demonstrate that a hypoxic insult in the developing brain triggers the same apoptotic pathway as the active developmental death.

Visual receptive field properties of cells in the optic tectum of the archer fish

The archer fish is well known for its extreme visual behavior in shooting water jets at prey hanging on vegetation above water. This fish is a promising model in the study of visual system function because it can be trained to respond to artificial targets and thus to provide valuable psychophysical data. Although much behavioral data have indeed been collected over the past two decades, little is known about the functional organization of the main visual area supporting this visual behavior, namely, the fish optic tectum. In this article we focus on a fundamental aspect of this functional organization and provide a detailed analysis of receptive field properties of cells in the archer fish optic tectum. Using extracellular measurements to record activities of single cells, we first measure their retinotectal mapping. We then determine their receptive field properties such as size, selectivity for stimulus direction and orientation, tuning for spatial frequency, and tuning for temporal frequency. Finally, on the basis of all these measurements, we demonstrate that optic tectum cells can be classified into three categories: orientation-tuned cells, direction-tuned cells, and direction-agnostic cells. Our results provide an essential basis for future investigations of information processing in the archer fish visual system.

Visual Activity Regulates Neural Progenitor Cells in Developing Xenopus CNS through Musashi1

Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis invivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity.

Evidence for neural stem cells in the medaka optic tectum proliferation zones

Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [1999]: J Comp Neurol 413:385-404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid-binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency-associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio-temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.

Live Imaging of Radiation-Induced Apoptosis by Yolk Injection of Acridine Orange in the Developing Optic Tectum of Medaka

To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) solution into the yolk of embryo and visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT, were irradiated with 5 Gy X-rays which is a non-lethal dose for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase up to 24 h after irradiation and completely disappeared up to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.

The Effects of Cell Surface NgCAM Expression on Developing Chick Brain Axons In Vitro

During embryonic brain development, molecular guidance cues work in concert to transduce signals to guide axons to their appropriate targets. Of these cues are proteins of the Ig superfamily, including L1 and its chick homologue, neuron‐glia cell adhesion molecule (NgCAM). L1 and NgCAM are neuronal cell surface membrane proteins shown by others to accelerate axonogenesis and increase axon elongation. In vitro bioassays were performed in order to test the hypothesis that NgCAM expression on the cell monolayer will increase rate of outgrowth, axon length, and number of axon processes. Embryonic Day 7 optic tectum (OT) explants were first used to study outgrowth, yet failed to be an efficient and reproducible model. Aggregates were then made from dissociated OT cells and plated onto NIH3T3 monolayers that did or did not express uniform NgCAM and onto monolayers exhibiting patterned NgCAM expression. The aggregates were visualized using static images and time‐lapse microscopy by fluorescent membrane dye. Current data has shown an increase in both axon length and number in the presence of NgCAM, suggesting its crucial role as a positive axon guidance cue. Supported in part by HHMI and NIH.

Radial glia produce and align the ligand fibronectin during neuronal migration in the developing chick brain.

We demonstrated previously that alpha8beta1 integrin regulates the migration and survival of immature neurons during development of the chicken optic tectum; however, the potential extracellular ligand was unknown. We used immunohistochemistry to determine if several potential ligands (fibronectin, tenascin, vitronectin, and osteopontin) were expressed during neuronal migration along radial glia (RG). Fibronectin was localized in a pattern relevant to radial migration and survival of neurons; it was present before and during neuronal migration and appeared oriented along RG fibers by conventional fluorescence microscopy. Confocal microscopy confirmed that fibronectin was localized along RG cells during radial migration. It was more concentrated in some superficial laminae, which might support directional movement. Fibronectin was present after formation of definitive tectal laminae, but was diffuse and not aligned along RG, which persist. Flow cytometry analysis of dissociated optic tectum cells revealed that almost all RG were positive for fibronectin. Short-term cell culture experiments using an exocytosis inhibitor revealed that fibronectin accumulated in most RG cells. Thus, fibronectin is produced by RG and is aligned along their surfaces before and during migration. Fibronectin, therefore, is a potential ligand for general radial neuronal migration in the chick optic tectum. Its predominant source appears to be RG, in contrast with developing mammalian cortex, where fibronectin was not found in a pattern that could guide widespread radial migration and where neurons are the predominant producers of fibronectin during migration.

Early variations of the disialoganglioside GD3 in chicken embryonic brain support its role in cell migration

In the present study a primary culture system of chicken embryo brain neurons was used in the early period of chicken brain development from day 6 until day 8, which was shown to be a suitable model of neuritogenesis, cell migration and reaggregation. Dissociated chicken optic tectum cells from embryonic stage 31 were cultured on polylysine-coated dishes under serum-free conditions up to 3 days. Freshly dissociated neurons developed short processes, which contacted one another and formed fasciculated bundles. Cell somata migrated along the neurite bundles, similar to migrating neurons in vivo, forming three-dimensional tissue-like clusters. This system was used to study the possible functions of the disialoganglioside GD3 for these neuronal differentiation steps. GD3 represents the predominant ganglioside of embryonic neurons before neuritogenesis in vitro and in vivo. Its biosynthesis is followed during day 6 until day 8 of embryonic brain development. Incubation of dissociated neurons with the monoclonal antibody R-24, recognising the GD3 on the cell surface, led to a total blocking of neurite outgrowth. Accordingly, neither cell migration nor reaggregation could be found. These results indicate that the disialoganglioside GD3 plays a central role in neuronal differentiation and development in the embryonic chicken brain.

Spatiotemporal gradient of oligodendrocyte differentiation in chick optic tectum requires brain integrity and cell‐cell interactions

The development of oligodendrocytes in the chicken optic tectum (OT) was studied in vivo and in vitro by analyzing expression of myelin-associated glycoprotein (MAG) with a monoclonal antibody. MAG(+) cells first appeared in the anterior OT on embryonic day (E) 12, were present throughout the anterior half on E15, and eventually filled the tectum on E17. This spatiotemporal appearance of MAG(+) oligodendrocytes resembled two streams of cells entering the OT along the afferent and efferent axonal layers. However, experiments determined that this appearance of MAG immunoreactivity was the result of a gradient of oligodendrocyte differentiation and was not cell migration. First, retroviral vector labeling of OT progenitors in vivo on E3 resulted in labeled oligodendrocytes in late embryos. In addition, pieces of OT from as early as E3 kept in culture for a week developed numerous MAG(+) oligodendrocytes. Pieces of both anterior and posterior E7 OT developed MAG(+) oligodendrocytes after 3 days in culture, well ahead of their normal schedule in vivo. BrdU incorporation studies revealed that these cells were not born in culture, but merely differentiated. Monolayer cultures made from dissociated E10 or later OT cells developed MAG(+) oligodendrocytes, but monolayers made from E7 OT cells did not. These experiments demonstrate that oligodendrocyte progenitors were present in the OT as early as E3, that they could differentiate precociously, and that their normal progressive differentiation in situ must be due to removal of inhibitory constraints rather than the onset of inductive factors. Also, certain cell-cell interactions occur between E7 and E10, which cannot be disrupted if oligodendrocyte differentiation is to occur.

Retroviral Transfer of Antisense Integrin α6 or α8 Sequences Results in Laminar Redistribution or Clonal Cell Death in Developing Brain

To assess the roles of two integrin alpha subunits (alpha6 and alpha8) in the developing chicken optic tectum, progenitors were infected with retroviral vectors that contained the marker gene lacZ plus antisense sequences from either the alpha6 or alpha8 integrin subunit cDNAs. On embryonic day 3 (E3), the vector was injected into tectal ventricles of chicken embryos. On E6, E7.5, E9, or later, chicken embryos were killed, and optic tecta were dissected and processed for histochemical detection of lacZ-positive cells. The antisense-bearing cell clones (descendants of a single infected progenitor) were analyzed for proliferation and migration patterns and were compared with lacZ-only vector-infected control clones. At E6, both alpha6 and alpha8 integrin antisense-containing cell clones were similar to controls. At E7.5, integrin alpha8 antisense-containing clones exhibited a cell number reduction in upper laminae (intermediate zone and tectal plate), and at E9, they exhibited a reduction in the ventricular zone as well. Integrin alpha6 antisense-containing cell clones exhibited no difference in total cell number at E9 but had a net laminar redistribution of more cells in the ventricular zone and less cells in the tectal plate. Our data show that different integrins play different roles during brain development: alpha6 integrin is essential for migration of tectal cells into specific laminae, and alpha8 integrin is essential for the survival of optic tectum cells. Also alpha8 integrin-substrate interactions may suppress early programmed cell death in premigratory and migratory neuroblasts.

Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions.

MN9D is an immortalized dopamine-containing neuronal hybrid cell line. When MN9D cells were coaggregated with primary embryonic cells of optic tectum, a brain region that does not receive a dopaminergic innervation, there was a marked reduction in their dopamine content, tyrosine hydroxylase immunoreactivity, and tyrosine hydroxylase mRNA. Similar reductions in dopamine content were produced by coaggregation with cells from embryonic thalamus, another brain region devoid of dopaminergic innervation. Coaggregation of MN9D cells with dopaminoceptive cells from the corpus striatum or the cortex did not have a demonstrable stimulatory effect on the dopamine content of MN9D cells. The decrease in MN9D dopamine content produced by optic tectum cells was not reversed by addition of corpus striatum cells. Thus, the MN9D hybrid cells are able to respond to an inhibitory factor(s) from cells derived from brain areas that are not targets for dopaminergic neurons. Catecholamine-producing PC12 cells did not respond in a similar manner, suggesting that the response of MN9D cells is a function of their mesencephalic origin. Given the selective response of MN9D cells to different brain cell populations, this hybrid cell line should facilitate investigations of cell-cell interactions in the central nervous system that may be involved in the expression of neurotransmitter phenotype and establishment of specific neuronal connections.

Immunomagnetic removal of neurons from developing chick optic tectum results in glial phenotypic instability.

The ability of embryonic day 12 and 13 optic tectum cells to replace depleted A2B5(+) cells and neurons was tested by immunomagnetic cell separation. Nearly all purified surface A2B5(-) cells were identified as glia by immunoreactivity for either glutamine synthetase of galactocerebroside. Most (approximately 80%) of the purified A2B5(-) cells became A2B5(+) after 1 day in culture, although no increase in the percentage of A2B5(+) cells (from 45%) was observed in control cultures of unpurified cells. Long-term monolayer cultures from purified cells contained A2B5(+) cells with mostly flattened glial-like or round process-free morphology, whereas those from unpurified cells contained many A2B5(+) neurons. The non-neuronal A2B5(+) cells frequently reacted with antibodies against glial fibrillary acidic protein and another marker expressed by embryonic brain glia, 5A11. Additionally, some flattened glia-like cells exhibited elaborate networks of anti-neurofilament-M-reactive filaments. We believe these unusual phenotypes, which appeared only in cultures of purified A2B5(-) cells, arose in response to the immunomagnetic removal of neurons. In conjunction with previous findings, we conclude that the abnormal phenotypes in purified cell cultures represent glia that were unsuccessful in attempting to replenish the depleted neuronal population. This may reflect restricted developmental potentials that arise during brain ontogeny.

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(−) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(−) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (≈50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(−) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (−) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.

Radial arrangement of clonally related cells in the chicken optic tectum: lineage analysis with a recombinant retrovirus.

A recently described method for lineage analysis in rodents uses a recombinant retrovirus to insert a foreign gene into the genome of a precursor cell; the gene's product is later detected histochemically in the infected cell's progeny. We have adapted this method for use in chicken embryos and used it to study the disposition of clonally related cells in the optic tectum. We report that descendants of a single precursor form narrow, radially oriented columns that span the thickness of the developing tectum. Analysis of embryos injected with virus at different stages suggests a developmental scheme in which early-born progeny are displaced laterally, late-born progeny are displaced radially, and cell mixing is limited. Many clones remain radially arrayed as laminae form and contribute neurons of diverse types of several laminae. In light of previous studies showing physiological relationships among vertically arrayed neurons, our results suggest that neurons descended from a single precursor are richly interconnected and functionally related.

Neuroanatomy and electrophysiology of the lacertilian nucleus isthmi

Horseradish peroxidase (HRP) tracings showed a bidirectional connection between the optic tectum (OT) and the ipsilateral nucleus isthmi (NI) pars magnocellularis (Imc) in nocturnal Gekko gekko and diurnal Shinisaurus crocodilurus. We found that, in addition to a direct neuronal pathway, there is an indirect pathway from OT to Imc via the nucleus profundus mesencephali (NPM). The morphology of cells in OT, NPM and NI was studied. Visual units extracellularly recorded from NI were located within Imc based on cobalt sulphide markings. They responded to moving contrast targets, without reacting to tactile and auditory stimulation. HRP and Golgi-Cox studies showed that Imc is a nucleus independent of its parvocellular partner, Ipc.

Two chick embryonic adhesion systems: molecular vs tissue specificity.

We have investigated the adhesive properties of cells from several neural and nonneural chick embryonic tissues dissociated using modifications of the standard dissociation procedures employed routinely in this laboratory to obtain retinal cells. Each of these tissues (7-day optic tectum, retina, and heart, and 3.75-day limb bud) displayed both Ca++-dependent (CD) and Ca++-independent (CI) aggregation, the relative rates of which differed from tissue to tissue. In every case, cells prepared so as to display one mode of aggregation or the other cross-adhered readily to cells--regardless of tissue origin--displaying the same mode of aggregation. Cross adhesion was negligible between cells--even from the same tissue--prepared so as to display different modes of aggregation. Anti-retinal Fab molecules which inhibit selectively either the CI or CD aggregation of retina cells strongly inhibited the corresponding aggregation of optic tectum cells, but had no effect upon the aggregation (CI or CD) of heart cells. These results demonstrate the existence in the tissues examined of dual adhesion mechanisms similar in Ca++ dependence and recognition properties to those of the retina, but showing certain immunological distinctions from the latter. The immunological relationship among the adhesion mechanisms from the various tissues is under continuing investigation.

Temporal changes in embryonic nerve cell recognition: Correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.