Ana D. de Lima

Neuritic differentiation and synaptogenesis in serum‐free neuronal cultures of the rat cerebral cortex

To better understand the dynamics of the cellular processes involved in early neocortical development, we studied the neuritic differentiation and synaptogenesis of dispersed neurons grown in serum‐free cultures under a wide variety of culture conditions. Microtubule‐associated protein (MAP2), phosphorylated neurofilament (SMI 31) and synaptophysin immunocytochemistry was complemented with time‐lapse studies. During the first week in vitro dissociated cortical neurons developed from roundish cells without processes to neurons with axons and differentiated dendrites, going through five distinct phases. The sequence of these phases was unaltered in a wide range of culturing methods, but the timing of the steps varied among cultures started with different cell densities. Synaptic terminals were first observed after 3‐4 days in vitro, coincident with the beginning of dendritic differentiation. Synaptogenesis progressed at least until the end of the third week in vitro, despite a decline in cell density during the second week in vitro. The process of cellular differentiation of cerebral cortical neurons in vitro resembled the development of these cells in the intact tissue, suggesting that organized cell migration is not a prerequisite for the differentiation of single cortical neurons. J. Comp. Neurol. 382:230‐246, 1997.

Activation of Early Silent Synapses by Spontaneous Synchronous Network Activity Limits the Range of Neocortical Connections

During the early development of neocortical networks, many glutamatergic synapses lack AMPA receptors and are physiologically silent. We show in neocortical cultures that spontaneous synchronous network activity is able to convert silent synapses to active synapses by the incorporation of AMPA receptors into synaptic complexes throughout the network within a few minutes. To test the effect of synaptic activation on the connectivity of neuronal populations, we created separated neuronal networks that could innervate each other. We allowed outgrowing axons to invade the neighboring network either before or after the onset of synchronous network activity. In the first case, both subnetworks connected to each other and synchronized their activity, whereas in the second case, axonal connections failed to form and network activity did not synchronize between compartments. We conclude that early spontaneous synchronous network activity triggers a global AMPAfication of immature synapses, which in turn prevents later-arriving axons from forming afferent connections. This activity-dependent process may set the range of corticocortical connections during early network development before experience-dependent mechanisms begin elaborating the mature layout of the neocortical connections and modules.

Synchronization of Neuronal Activity Promotes Survival of Individual Rat Neocortical Neurons in Early Development

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co‐activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.

Irreversible loss of a subpopulation of cortical interneurons in the absence of glutamatergic network activity

In the cerebral cortex of mammals, γ‐aminobutyric acid (GABA)ergic neurons represent 15–25% of all neurons, depending on the species and area being examined. Because converging evidence suggests that activity may play an important role in the neuritic maturation and synaptic function of GABAergic neurons, it is feasible that activity plays a role in the regulation of the proportion of GABAergic neurons. Here we provide direct evidence that early in cortical development activity blockade may deplete the network of a subpopulation of GABA immunoreactive neurons characterized by their small size and late generation in vitro. In a period of time coinciding with the emergence of synchronous network activity, the survival and morphological differentiation of GABAergic neurons was influenced by long‐term blockade of synaptic activity. While GABAA receptor antagonists had a minor promoting effect on interneuronal survival during the second week in vitro, antagonists of ionotropic glutamate receptors strongly impaired survival and differentiation of immature GABAergic interneurons. Interneuronal loss was more severe when N‐methyl‐d‐aspartate receptors were blocked than after blockade of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionic acid (AMPA)/kainate receptors. The decrease in the density of GABAergic neurons was irreversible, but could be prevented by the simultaneous addition of brain‐derived neurotrophic factor (BDNF). These results suggest that there is a narrow time window during neocortical development when glutamatergic activity, and specially NMDA receptor stimulation, is crucial to assure survival and maturation of a subpopulation of late developing GABAergic interneurons.

Contribution of GABAergic Interneurons to the Development of Spontaneous Activity Patterns in Cultured Neocortical Networks

Periodic synchronized events are a hallmark feature of developing neuronal networks and are assumed to be crucial for the maturation of the neuronal circuitry. In the developing neocortex, the early network oscillations coincide with an excitatory action of the neurotransmitter gamma-aminobutyric acid (GABA). A relationship between the emerging inhibitory action of GABA and the gradual disappearance of early synchronized network activity has been previously suggested. Therefore we investigate the interplay between the action of GABA and spontaneous activity in cultured networks of the lateral or dorsal embryonic rat neocortex, which show considerable difference in the content of GABAergic neurons. Here we present the results of long-term monitoring of spontaneous electrical activity of cultured networks growing on microelectrode arrays and the time course of changes in GABA action using calcium imaging. All cultures studied displayed stereotyped synchronized burst events at the end of the first week in vitro. As the GABAA depolarizing action decreases, naturally or after bumetanide treatment, network activity in lateral cortex cultures changed from stereotypic bursting to more clustered and asynchronous activity patterns. Dorsal cortex cultures and cultures lacking GABAA-receptor mediated synaptic transmission, retained an immature synchronous firing pattern, but developed prominent intraburst oscillations (∼3–10 Hz). Large, mostly parvalbumin positive, GABAergic neurons dominate the GABAergic population in lateral cortex cultures. These large interneurons were virtually absent in dorsal cortex cultures. Based on these results, we suggest that the richly interconnected large GABAergic neurons contribute to desynchronize and temporally differentiate the spontaneous activity of cultured cortical networks.

Spike-timing-dependent plasticity in small-world networks

Biologically plausible excitatory neural networks develop a persistent synchronized pattern of activity depending on spontaneous activity and synaptic refractoriness (short term depression). By fixed synaptic weights synchronous bursts of oscillatory activity are stable and involve the whole network. In our modeling study we investigate the effect of a dynamic Hebbian-like learning mechanism, spike-timing-dependent plasticity (STDP), on the changes of synaptic weights depending on synchronous activity and network connection strategies (small-world topology). We show that STDP modifies the weights of synaptic connections in such a way that synchronization of neuronal activity is considerably weakened. Networks with a higher proportion of long connections can sustain a higher level of synchronization in spite of STDP influence. The resulting distribution of the synaptic weights in single neurons depends both on the global statistics of firing dynamics and on the number of incoming and outgoing connections.

Relationship between GABAergic interneurons migration and early neocortical network activity.

Available evidence converges to suggest that during the early development of the cerebral cortex, the emergence of the spontaneous network activity chronologically overlap with the end of the cell migration period in the developing cortex. We approached the functional regulation of neuronal migration in a culture model of neocortical networks, using time lapses to detect migratory movements, calcium‐imaging to assess the activity of migratory neurons, and immunocytochemical methods to identify the migratory cells retrospectively. In cell cultures, early physiological development and cell migration are reproduced at a local network level, thus allowing the study of the interrelationships between cell migration and network development independent of the topographical complexity. Neurons migrate at least until 12 days in vitro and GABAergic neurons migrate faster compared with non‐GABAergic neurons. A decline of migratory activity was coincident with the development of spontaneous synchronous network activity. Migrating interneurons did not participate in synchronous network activity, but interneurons that ended cell migration during observation time frequently engaged in synchronous activity within less than an hour. Application of GABAA and ionotropic glutamate receptor antagonists significantly increased the number of migrating GABAergic neurons without changing the dynamics of the migratory movements. Thus, neurotransmitters released by early network activity might favor the termination of neuronal migration. These results reinforce the idea that network activity plays an important role in the development of late‐born GABAergic cells.

Identification of two distinct populations of γ‐aminobutyric acidergic neurons in cultures of the rat cerebral cortex

Two types of neurons containing γ‐aminobutyric acid (GABA) were identified in cultures of embryonic rat neocortex. Large GABAergic neurons were already present 4 hours after plating, whereas small ones appeared later. Both types were shown to be neurons by double labeling with GABA and microtubule‐associated protein 2 (MAP2) immunocytochemistry. The large GABAergic neurons represented less than 5% of the adherent cells, developed neurites rapidly, and progressed synchronously through the polarization and differentiation steps characteristic of the whole neuronal population. During the second week in culture, these GABA‐immunoreactive cells developed into large, stellate neurons with fairly homogeneous morphology and poorly ramified, straight dendrites. At the same time, the GABAergic neuropil increased greatly, and neurites of GABAergic neurons showed advancing maturity and smoothness. The axon of each cell covered extensive areas of the culture, frequently encircling the somata of unlabeled neurons in a basket‐like fashion.

Astroglia inhibit the proliferation of neocortical cells and prevent the generation of small GABAergic neurons in vitro

We quantitatively studied the dynamics of rat neocortical precursor proliferation in vitro, and additionally examined the effects of neuron–glia interactions on the proliferation and differentiation of neurons, and particularly of γ‐aminobutyric acid (GABA)‐containing cells. In cultures grown on glia‐free substrate, cellular proliferation was detected at least until the end of the second week in vitro, but most neurons which expressed detectable amounts of microtubule‐associated protein at 12 days in vitro were generated early during the first week. Further double‐labelling experiments, combining 5′‐bromo‐2′‐deoxyuridine with GABA or β‐tubulin III immunohistochemistry, provided direct evidence that neuronal proliferation continued through the second week in vitro, and that a population of small GABAergic neurons was generated between 3 and 12 days in vitro. Culturing cells on a glial substrate significantly reduced the generation of small GABAergic cells and strongly inhibited the total cell proliferation. Inhibition also occurred if astrocytes were added to the culture after 6 days in vitro, but was significantly decreased if cells were grown on a fixed glial substrate, suggesting that the effect might be at least partially mediated by active interactions between neurons and glia. In conclusion, our results show that the sustained proliferation of precursor cells in neocortical cultures is necessary for the differentiation of small GABAergic neurons, and that mature astroglia effectively inhibit the proliferation of neocortical precursors thereby affecting the appearance of a population of GABAergic cells.

Developmental downregulation of GABAergic drive parallels formation of functional synapses in cultured mouse neocortical networks

Networks of cortical neurons in vitro spontaneously develop synchronous oscillatory electrical activity at around the second week in culture. However, the underlying mechanisms and in particular the role of GABAergic interneurons in initiation and synchronization of oscillatory activity in developing cortical networks remain elusive. Here, we examined the intrinsic properties and the development of GABAergic and glutamatergic input onto presumed projection neurons (PNs) and large interneurons (L‐INs) in cortical cultures of GAD67‐GFP mice. Cultures developed spontaneous synchronous activity already at 5–7 days in vitro (DIV), as revealed by imaging transient changes in Fluo‐3 fluorescence. Concurrently, spontaneous glutamate‐mediated and GABAA‐mediated postsynaptic currents (sPSCs) occured at 5 DIV. For both types of neurons the frequency of glutamatergic and GABAergic sPSCs increased with DIV, whereas the charge transfer of glutamatergic sPSCs increased and the charge transfer of GABAergic sPSCs decreased with cultivation time. The ratio between GABAergic and the overall charge transfer was significantly reduced with DIV for L‐INs and PNs, indicating an overall reduction in GABAergic synaptic drive with maturation of the network. In contrast, analysis of miniature PSCs (mPSCs) revealed no significant changes of charge transfer with DIV for both types of neurons, indicating that the reduction in GABAergic drive was not due to a decreased number of functional synapses. Our data suggest that the global reduction in GABAergic synaptic drive together with more synaptic input to PNs and L‐INs during maturation may enhance rhythmogenesis of the network and increase the synchronization at the level of population bursts.