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Filling in the gaps
RACHEL
CORTICALCIRCUITRY
O.L. WONG
-
Filling
in the gaps
A transiently occurring network of gap junctions synchronously activates neuronal ceils during circuit formation in the developing neocortex.
The mapping of sensory inputs to their recipient target regions in the brain is only crudely organized during the early stages of development. The initial pattern of neuronal connections is subsequently refined in a way that depends on electrical activity. However, electrical activity alone is not sufficient to promote the fine-tuning of these connections: the activities of neighboring neurons need to be temporally coordinated before highly structured pathways, characteristic of the adult pattern, arise. A recent article by Peinado, Yuste and Katz [l] elegantly demonstrates the presence of a mechanism that not only underlies the coordinated activity of neighboring neocortical cells during the period in which they synchronously undergo spontaneous depolarization [2 I, but could also be an effective means of exchanging developmentally relevant signals between cells. The Peinado et al. paper shows that the dendrites of neighboring cortical neurons in the rat neocortex are transiently inter-linked by electrical gap junctions during circuit formation. Many conditions are necessary for setting up specific connections within the central nervous system during development. One basic requirement for reiining the initially diffuse connectivity patterns is for neurons constantly to communicate with each other. Synaptic input is selected on a ‘trial and error’ basis: even when synaptic contacts first form between cells, decisions have to be made as to whether those contacts are appropriate and thus should be retained. The developing nervous system then follows cues provided by the, activity patterns of the input neurons, which eventually lead to either the retainment or rejection of the early synapses (for recent reviews see [3,4]). If several input neurons fire action potentials that simulta neously depolarize their common postsynaptic cell, then the synapses at the terminals’ of these coactive neurons tend to be strengthened and retained. Conversely, the firing of inputs from afferent cells out of synchrony with each other tends to result in the strengthening of one synapse and the weakening of the other. By undergoing this process of synaptic strengthening or weakening, the early synaptic circuitry becomes progressively refined. Because temporal coordination in the activity of large groups of neurons appears crucial for circuit formation, mere is much interest in the mechanisms that provide coordinated impulses to the target, often with the assumption that correlated activity in the target relies on the pattern of electrical activity from its afferents. ,The results of Peinado et al. [I] suggest that coordinated activity in target cortical neurons can occur, even in the absence of action potentials from their afferent input, by a mechanism that does not require synaptic release of neurotransmitters for cell-cell communication. 180
Before we explore in more detail the mechanism coordinating the activity of the developing cortical neurons, it is worth recalling that it has not been a simple task to demonstrate directly the existence of correlated activity in large groups of neurons. This was largely because of an inability to record simultaneously from more than a few cells, either in vivo or in vitro. It is only recently that new electrophysiological [ 51and imaging techniques [61 have provided a direct means for detecting the presence of synchronous activity in neurons during development e:‘ the central nervous system. With the use of techniques for imaging Ca2f in living brain slices [ 2,6],Yuste, Peinado and Katz [2] made the exciting discovery of spontaneously occurring domains of coactive cells in the rat neocortex. These cortical domains contain from 5 to 50 cells, and their spatial and electrical properties suggest that they may be involved in the development of the modular architecture of the cortex, The domains occur in the absence of synaptic innervation, disappear under halothane, and were found to persist in the presence of tetrodotoxin, a Naf channel blocker that abolishes action potentials. Taken together, these observations suggest that me coordinated activity of these domains is mediated by a non-synaptic means of intercellular communication, such as by electrical gap junctions. Gap junctions are commonly found in a variety of developing vertebrate and invertebrate systems, and have been shown to play a major role in many developmental events, including cellular differentiation [ 71. They are regions of contact between cells in which a central channel is surrounded by six protein subunits forming an annulus; when open, the channel allows the exchange of ions and small molecules. These junctions are common amongst neural glia, particularly astrocytes [S], and act as intercellular bridges conveying electrical activity from one cell to another. The presence of gap-junctional links between glial cells has often been revealed by intracellular dye-iilling of single cells with fluorescent dyes such as Lucifer yellow; however, such dyes do not always spread easily from cell to cell. Because of this, in contrast to the glial cells, the existence of gap-junctional coupling between many neuronal cell types remained unknown until the recent use of the low molecular weight tracer, neurobiotin [ 91.By injecting this tracer into single cells, Peinado et al. [ 1] found extensive coupling between large numbers of cortical cells during the time when the neuronal domains were present. Between postnatal days P7 to P9 in the rat neocortex, injection of a single cortical cell resulted in about E-25 neighboring cells being stained (Fig. l), whereas injection of Lucifer yellow only stained one or a few surrounding cells (see also [lo]).
@ Current Biology 1993, Vol 3 No 3
181
Peinado et al went on to show that the tracer-c&pling of cells, like the neuronal domains. is reversibly blocked by halothane, a gap-junctional blocker, thus strengthening their conclusion that the transfer of neurobiotin occurs via intercellular junctions.
i
Fig. 1. Injection of the tracer neurobiotin into one cell in layers 2/3 of the rat neocortex results in the staining of many surrounding ceils by passage of the tracer via gap junctions.
Several properties of the cortical gap-junctional network appear useful for the activity-dependent modulation of responses in cortical cells, For example, the degree of coactivation between cells can be modulated according to how open 01: closed the junctions are at a particular time. In addition to correlating the electrical activity of cells within the cluster, the gap-junctional network could also provide a novel means by which these cells can communicate during subthreshold activity or exchange signals, such as second messengers like CAMP and IP3, which trigger developmental events. Another significant result is that the pattern of neurobiotin-coupling in the neocortex appears to be cell-type specific. For example, injection of neurons did not result in the staining of glial cells, and vice versa. Furthermore, there are variations in the pattern of coupling between different neuronal cell types: smooth non-pyramidal cells form only small clusters, whereas pyramidal cells form clusters that are about 4-5 times as large and comprise mainly cells of the same type, Thus, gap junctions specifically coupling members of the same cell class may be the means by which further segregation of afferent inputs can take place in an otherwise continuous syncytium of a mixed population of cortical cells. Only like cells should theiefore be directly affected by activation of a member of each cluster. Although we are aware on a single cell of the transient nature of some initial synaptic contacts, it is astounding to discover that entire networks of cells could be temporanily set up to promote the formation of the adult pattern of neuronal circuitry. For example, large numbers of neurons in the subplate, located below the cortical plate, could form a transient synaptic scafYold to direct the appropriate ingrowth and patterning of connections in the thalamocortical pathway [ 111. The observation of Peinado et al. [II that fewer and fewer cells in the neocortex
appear coupled with increasing exampie that transient neuronal non-synaptic, may be important connections within a target.
age is another elegant networks, in this case, for the tial pattern of
In summary, intercellular communication between cells during development is necessary for many aspects of development of the nervous system. Often, the means of cell-cell communication during the early stages of development cannot be mediated by chemical synapses alone, because the synaptic circuitry has to develop and mature further. The importance of spontaneously generated electrical activity, in particular activity that is synchronized between neighbours, appears to be crucial for the refinement of many neuronal pathways. The finding by Peinado et al. 111, that synchrony in spontaneous electrical activity of cortical cells can be mediated by a transient network of gap junctions prior to synaptogenesis, highlights a mechanism in the target neurons, rather than the input neurons, that could further coordinate and modulate the responses of the cortical cells. This observation draws our attention to the presence of a mechanism that can either compliment the developmental cues provided by synaptic transmission or work earlier and independently of synaptic activation. It also underscores the important contribution of gap-junctional communication in helping to shape the modular appearance of various functional units in the cortex and, perhaps, other parts of the brain.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
PELWO A,
YUSE R, KATZ LC: Extensive dye-coupling between rat neocortical neurons during the period of circuit formation. Neuron 1993, 10~1-12. YUSTER, PEINADOA, IWE LC: Neuronal domains in developing neocortex. Science 1992, 257665-669. SHATZCl: Impulse activity and the patterning of connections during CNS development. Neuron 1990, 5:745-756. CONSTANTINE.PATON M, CLINEHT, DEBSKI E: Patterned activity. synaptic convergence, and the NMDA receptor in developing visual pathways. Annu Rev Neurrxci 1990, 13~129-154. MEISTER M, WONGROL, BAYLOR DA, SHA’IZCJ: Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 1991, 252:939-943. YUSTER, KATZ LC: Control of postsynaptic Caz+ influx in developing neocortex by excitatory and inhibitory neurotrans. mitters. Neuron 1991, 6:333-344. GUTHUESC, GIUJLANB: Gap-junctional communication and development. Trends Neurosci 1989, 12:12-15. CORNELL.BELLAH, FINKBEINERSM, COOPER MS, SMITH SJ: Glutamate induces calcium waves in cultured astrocytes: long-range glial signailing. Science 1990, 247:47ti73. VANEY DI: Many diverse types of retinal neurons show tracer coupling when injected with biocytin or neurobiotin- Neuro sci Let& 1991, 125:187-190. LOTURCOJJ, KRIEGSTEINAR: Clusters of coupled neuroblasts in embryo& neocortex. Science 1991, 252~563-566. GHOSH A ANTONINIA, MCCONNELL SK, SHATZCJ: Requirement for subplate neurons in the formation of thalamocortical connections. Nature 1990, 347~179-181.
Rachel O.L. Wong, Vision, Touch and Hearing Research Centre, University of Queensland, QLD 4072, Australia.
CORTICALCIRCUITRY
O.L. WONG
-
Filling
in the gaps
A transiently occurring network of gap junctions synchronously activates neuronal ceils during circuit formation in the developing neocortex.
The mapping of sensory inputs to their recipient target regions in the brain is only crudely organized during the early stages of development. The initial pattern of neuronal connections is subsequently refined in a way that depends on electrical activity. However, electrical activity alone is not sufficient to promote the fine-tuning of these connections: the activities of neighboring neurons need to be temporally coordinated before highly structured pathways, characteristic of the adult pattern, arise. A recent article by Peinado, Yuste and Katz [l] elegantly demonstrates the presence of a mechanism that not only underlies the coordinated activity of neighboring neocortical cells during the period in which they synchronously undergo spontaneous depolarization [2 I, but could also be an effective means of exchanging developmentally relevant signals between cells. The Peinado et al. paper shows that the dendrites of neighboring cortical neurons in the rat neocortex are transiently inter-linked by electrical gap junctions during circuit formation. Many conditions are necessary for setting up specific connections within the central nervous system during development. One basic requirement for reiining the initially diffuse connectivity patterns is for neurons constantly to communicate with each other. Synaptic input is selected on a ‘trial and error’ basis: even when synaptic contacts first form between cells, decisions have to be made as to whether those contacts are appropriate and thus should be retained. The developing nervous system then follows cues provided by the, activity patterns of the input neurons, which eventually lead to either the retainment or rejection of the early synapses (for recent reviews see [3,4]). If several input neurons fire action potentials that simulta neously depolarize their common postsynaptic cell, then the synapses at the terminals’ of these coactive neurons tend to be strengthened and retained. Conversely, the firing of inputs from afferent cells out of synchrony with each other tends to result in the strengthening of one synapse and the weakening of the other. By undergoing this process of synaptic strengthening or weakening, the early synaptic circuitry becomes progressively refined. Because temporal coordination in the activity of large groups of neurons appears crucial for circuit formation, mere is much interest in the mechanisms that provide coordinated impulses to the target, often with the assumption that correlated activity in the target relies on the pattern of electrical activity from its afferents. ,The results of Peinado et al. [I] suggest that coordinated activity in target cortical neurons can occur, even in the absence of action potentials from their afferent input, by a mechanism that does not require synaptic release of neurotransmitters for cell-cell communication. 180
Before we explore in more detail the mechanism coordinating the activity of the developing cortical neurons, it is worth recalling that it has not been a simple task to demonstrate directly the existence of correlated activity in large groups of neurons. This was largely because of an inability to record simultaneously from more than a few cells, either in vivo or in vitro. It is only recently that new electrophysiological [ 51and imaging techniques [61 have provided a direct means for detecting the presence of synchronous activity in neurons during development e:‘ the central nervous system. With the use of techniques for imaging Ca2f in living brain slices [ 2,6],Yuste, Peinado and Katz [2] made the exciting discovery of spontaneously occurring domains of coactive cells in the rat neocortex. These cortical domains contain from 5 to 50 cells, and their spatial and electrical properties suggest that they may be involved in the development of the modular architecture of the cortex, The domains occur in the absence of synaptic innervation, disappear under halothane, and were found to persist in the presence of tetrodotoxin, a Naf channel blocker that abolishes action potentials. Taken together, these observations suggest that me coordinated activity of these domains is mediated by a non-synaptic means of intercellular communication, such as by electrical gap junctions. Gap junctions are commonly found in a variety of developing vertebrate and invertebrate systems, and have been shown to play a major role in many developmental events, including cellular differentiation [ 71. They are regions of contact between cells in which a central channel is surrounded by six protein subunits forming an annulus; when open, the channel allows the exchange of ions and small molecules. These junctions are common amongst neural glia, particularly astrocytes [S], and act as intercellular bridges conveying electrical activity from one cell to another. The presence of gap-junctional links between glial cells has often been revealed by intracellular dye-iilling of single cells with fluorescent dyes such as Lucifer yellow; however, such dyes do not always spread easily from cell to cell. Because of this, in contrast to the glial cells, the existence of gap-junctional coupling between many neuronal cell types remained unknown until the recent use of the low molecular weight tracer, neurobiotin [ 91.By injecting this tracer into single cells, Peinado et al. [ 1] found extensive coupling between large numbers of cortical cells during the time when the neuronal domains were present. Between postnatal days P7 to P9 in the rat neocortex, injection of a single cortical cell resulted in about E-25 neighboring cells being stained (Fig. l), whereas injection of Lucifer yellow only stained one or a few surrounding cells (see also [lo]).
@ Current Biology 1993, Vol 3 No 3
181
Peinado et al went on to show that the tracer-c&pling of cells, like the neuronal domains. is reversibly blocked by halothane, a gap-junctional blocker, thus strengthening their conclusion that the transfer of neurobiotin occurs via intercellular junctions.
i
Fig. 1. Injection of the tracer neurobiotin into one cell in layers 2/3 of the rat neocortex results in the staining of many surrounding ceils by passage of the tracer via gap junctions.
Several properties of the cortical gap-junctional network appear useful for the activity-dependent modulation of responses in cortical cells, For example, the degree of coactivation between cells can be modulated according to how open 01: closed the junctions are at a particular time. In addition to correlating the electrical activity of cells within the cluster, the gap-junctional network could also provide a novel means by which these cells can communicate during subthreshold activity or exchange signals, such as second messengers like CAMP and IP3, which trigger developmental events. Another significant result is that the pattern of neurobiotin-coupling in the neocortex appears to be cell-type specific. For example, injection of neurons did not result in the staining of glial cells, and vice versa. Furthermore, there are variations in the pattern of coupling between different neuronal cell types: smooth non-pyramidal cells form only small clusters, whereas pyramidal cells form clusters that are about 4-5 times as large and comprise mainly cells of the same type, Thus, gap junctions specifically coupling members of the same cell class may be the means by which further segregation of afferent inputs can take place in an otherwise continuous syncytium of a mixed population of cortical cells. Only like cells should theiefore be directly affected by activation of a member of each cluster. Although we are aware on a single cell of the transient nature of some initial synaptic contacts, it is astounding to discover that entire networks of cells could be temporanily set up to promote the formation of the adult pattern of neuronal circuitry. For example, large numbers of neurons in the subplate, located below the cortical plate, could form a transient synaptic scafYold to direct the appropriate ingrowth and patterning of connections in the thalamocortical pathway [ 111. The observation of Peinado et al. [II that fewer and fewer cells in the neocortex
appear coupled with increasing exampie that transient neuronal non-synaptic, may be important connections within a target.
age is another elegant networks, in this case, for the tial pattern of
In summary, intercellular communication between cells during development is necessary for many aspects of development of the nervous system. Often, the means of cell-cell communication during the early stages of development cannot be mediated by chemical synapses alone, because the synaptic circuitry has to develop and mature further. The importance of spontaneously generated electrical activity, in particular activity that is synchronized between neighbours, appears to be crucial for the refinement of many neuronal pathways. The finding by Peinado et al. 111, that synchrony in spontaneous electrical activity of cortical cells can be mediated by a transient network of gap junctions prior to synaptogenesis, highlights a mechanism in the target neurons, rather than the input neurons, that could further coordinate and modulate the responses of the cortical cells. This observation draws our attention to the presence of a mechanism that can either compliment the developmental cues provided by synaptic transmission or work earlier and independently of synaptic activation. It also underscores the important contribution of gap-junctional communication in helping to shape the modular appearance of various functional units in the cortex and, perhaps, other parts of the brain.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
PELWO A,
YUSE R, KATZ LC: Extensive dye-coupling between rat neocortical neurons during the period of circuit formation. Neuron 1993, 10~1-12. YUSTER, PEINADOA, IWE LC: Neuronal domains in developing neocortex. Science 1992, 257665-669. SHATZCl: Impulse activity and the patterning of connections during CNS development. Neuron 1990, 5:745-756. CONSTANTINE.PATON M, CLINEHT, DEBSKI E: Patterned activity. synaptic convergence, and the NMDA receptor in developing visual pathways. Annu Rev Neurrxci 1990, 13~129-154. MEISTER M, WONGROL, BAYLOR DA, SHA’IZCJ: Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 1991, 252:939-943. YUSTER, KATZ LC: Control of postsynaptic Caz+ influx in developing neocortex by excitatory and inhibitory neurotrans. mitters. Neuron 1991, 6:333-344. GUTHUESC, GIUJLANB: Gap-junctional communication and development. Trends Neurosci 1989, 12:12-15. CORNELL.BELLAH, FINKBEINERSM, COOPER MS, SMITH SJ: Glutamate induces calcium waves in cultured astrocytes: long-range glial signailing. Science 1990, 247:47ti73. VANEY DI: Many diverse types of retinal neurons show tracer coupling when injected with biocytin or neurobiotin- Neuro sci Let& 1991, 125:187-190. LOTURCOJJ, KRIEGSTEINAR: Clusters of coupled neuroblasts in embryo& neocortex. Science 1991, 252~563-566. GHOSH A ANTONINIA, MCCONNELL SK, SHATZCJ: Requirement for subplate neurons in the formation of thalamocortical connections. Nature 1990, 347~179-181.
Rachel O.L. Wong, Vision, Touch and Hearing Research Centre, University of Queensland, QLD 4072, Australia.