Spontaneous development of synchronous oscillatory activity during maturation of cortical networks in vitro. J Neurophysiol 88: 2196 -2206, 2002; 10.1152/jn.00316.2002. Recent studies have focused attention on mechanisms of spontaneous large-scale wavelike activity during early development of the neocortex. In this study, we describe and characterize synchronous neuronal activity that occurs in cultured cortical networks naturally without pharmacological intervention. The synchronous activity that can be detected by means of Fluo-3 fluorescence imaging starts to develop at the beginning of the second week in culture and eventually includes the entire neuronal population about 1 wk later. A synchronous increase of [Ca 2ϩ ] i in the neuronal population is associated with a burst of action potentials riding on a long-lasting depolarization recorded in a single cell. It is suggested that this depolarization results directly from synaptic current, which was comprised of at least three different components mediated by AMPA, N-methyl-D-aspartate (NMDA), and GABA A receptors. We never observed a gradually depolarizing pacemaker potential and found no evidence for a change of excitability during inter-burst periods. However, we found evidence for a period of synaptic depression after bursts. Network excitability recovers gradually over seconds from this depression that can explain the episodic nature of spontaneous network activity. Using pharmacological manipulation to investigate the propagation of activity in the network, we show that synchronous network activity depends on both glutamatergic and GABA A ergic neurotransmission during a brief period. Reversal potential of GABA A receptor-mediated current was found to be significantly more positive than resting membrane potential both at 1 and 2 wk in culture, suggesting depolarizing action of GABA. However, in cultures older than 2 wk, inhibition of GABA A receptors does not result in block of synchronous network activity but in modulation of burst width and frequency.

Neurons dissociated from embryonic cerebral rat cortex form a differentiated network of synaptic connections and develop synchronous oscillatory network activity with the beginning of the second week in culture. During an initial phase lasting 3-4 d, synchronous calcium transients can be blocked completely by either CNQX or bicuculline, showing that both glutamatergic and GABAergic neurons are required for the generation of this form of activity. By manipulating dissociation and growth conditions, cultures containing different populations of GABAergic neurons were obtained. These cultures revealed that a distinct population of large GABAergic neurons is a key element in the generation of synchronous oscillatory network activity. A minimal number of two large GABAergic neurons per square millimeter are required for the occurrence of synchronous activity. Changes in the density of all other types of GABAergic or non-GABAergic neurons has no influence on the synchronous activity. Electron microscopic analysis shows that the large GABAergic neurons form an interconnected network. Exceptionally high somatodendritic innervation and extended axonal arborization enable these neurons to collect electric network activity and to distribute it effectively throughout the neuronal network. Additional experiments indicated that most neurons developing in culture to large GABAergic neurons are derived from the primordial plexiform layer and reside in the subplate at the time of birth. We suggest that they function as an integrating element that synchronizes neuronal activity during early cortical development by collecting incoming extrinsic and intrinsic signals and distributing them effectively throughout the developing cortical plate.

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.

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.

The pontomesencephalic projection to the dorsal lateral geniculate nucleus (dLGN) of the cat was analyzed by combining retrograde transport of rhodamine-labeled latex spheres and immunohistochemistry. After injections of latex beads into the dLGN, sections of the brainstem were treated immunohistochemically for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Essentially, six regions in the brainstem contained retrogradely labeled cells: the superior colliculus, the parabigeminal nucleus, the dorsal raphe nuclei, the parabrachial area of the central tegmental field, the marginal nucleus of the brachium conjunctivum, and the nucleus coeruleus. Furthermore, isolated retrogradely labeled cells were present in the central nucleus of the raphe, in the cuneiform nucleus, and in the periaqueductal gray. Most serotoninergic double-labeled cells were found in the medial and lateral divisions of the dorsal raphe nuclei, but a few were also present in the central nucleus of the raphe. In the sections immunostained for ChAT, double-labeled cells were located in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In the sections treated for TH and DBH, double-labeled cells showed a similar distribution, and like the ChAT(+) cells, they were located mainly in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In these regions the cholinergic and noradrenergic cells that projected to the lateral geniculate nucleus were intermingled, the former predominating rostrally and the latter caudally. The majority of retrogradely labeled cells were located in the region of the central tegmental field in the vicinity of the brachium conjunctivum, and most of these cells were also ChAT-immunoreactive. We, therefore, conclude that the cholinergic projection is the most important of the central core projections ascending to the dLGN.