Potentiation of L-type calcium channels reveals nonsynaptic mechanisms that correlate spontaneous activity in the developing mammalian retina.

Although correlated neural activity is a hallmark of many regions of the developing nervous system, the neural events underlying its propagation remain largely unknown. In the developing vertebrate retina, waves of spontaneous, correlated neural activity sweep across the ganglion cell layer. Here, we demonstrate that L-type Ca(2+) channel agonists induce large, frequent, rapidly propagating waves of neural activity in the developing retina. In contrast to retinal waves that have been described previously, these L-type Ca(2+) channel agonist-potentiated waves propagate independent of fast synaptic transmission. Bath application of nicotinic acetylcholine, AMPA, NMDA, glycine, and GABA(A) receptor antagonists does not alter the velocity, frequency, or size of the potentiated waves. Additionally, these antagonists do not alter the frequency or magnitude of spontaneous depolarizations that are recorded in individual retinal ganglion cells. Like normal retinal waves, however, the area over which the potentiated waves propagate is reduced dramatically by 18alpha-glycyrrhetinic acid, a blocker of gap junctions. Additionally, like normal retinal waves, L-type Ca(2+) channel agonist-potentiated waves are abolished by adenosine deaminase, which degrades extracellular adenosine, and by aminophylline, a general adenosine receptor antagonist, indicating that they are dependent on adenosine-mediated signaling. Our study indicates that although the precise spatiotemporal properties of retinal waves are shaped by local synaptic inputs, activity may be propagated through the developing mammalian retina by nonsynaptic pathways.

Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina.

Before phototransduction, spontaneous activity in the developing mammalian retina is required for the appropriate patterning of retinothalamic connections, and there is growing evidence that this activity influences the development of circuits within the retina itself. We demonstrate here that the neural substrate that generates waves in the mouse retina develops through three distinct stages. First, between embryonic day 16 and birth [postnatal day 0 (P0)], we observed both large, propagating waves inhibited by nicotinic acetylcholine receptor (nAChR) antagonists and small clusters of cells displaying nonpropagating, correlated calcium increases that were independent of nAChR activation. Second, between P0 and P11, we observed only larger propagating waves that were abolished by toxins specific to alpha3 and beta2 subunit-containing nAChRs. Third, between P11 and P14 (eye opening) we observed propagating activity that was abolished by ionotropic glutamate receptor antagonists. The time course of this developmental shift was dramatically altered in retinas from mice lacking the beta2 nAChR subunit or the beta2 and beta4 subunits. These retinas exhibited a novel circuit at P0, no spontaneous correlated activity between P1 and P8, and the premature induction at P8 of an ionotropic glutamate receptor-based circuit. Retinas from postnatal mice lacking the alpha3 nAChR subunit exhibited spontaneous, correlated activity patterns that were similar to those observed in embryonic wild-type mice. In alpha3-/- and beta2-/- mice, the development and distribution of cholinergic neurons and processes and the density of retinal ganglion cells (RGCs) and the gross segregation of their dendrites into ON and OFF sublaminae were normal. However, the refinement of individual RGC dendrites is delayed. These results indicate that retinal waves mediated by nAChRs are involved in, but not required for, the development of neural circuits that define the ON and OFF sublamina of the inner plexiform layer.

Neurotransmitters and gap junctions in developing neural circuits.

A growing body of evidence suggests that highly correlated, spontaneous neural activity plays an important role in shaping connections in the developing nervous system prior to the maturation of sensory afferents. In this article we discuss the mechanisms involved in the generation and the regulation of spontaneous activity patterns in the developing retina and the developing neocortex. Spontaneous activity in the developing retina propagates across the ganglion cell layer as waves of action potentials and drives rhythmic increases in intracellular calcium in retinal neurons. Retinal waves are mediated by a combination of chemical synaptic transmission and gap junctions, and the circuitry responsible for generating retinal waves changes with age and between species. In the developing cortex, spontaneous calcium elevations propagate across clusters of cortical neurons called domains. Cortical domains are generated by a regenerative mechanism involving second messenger diffusion through gap junctions and subsequent calcium release from internal stores. The neocortical gap junction system is regulated by glutamate-triggered second messenger systems as well as neuromodulatory transmitters, suggesting extensive interactions between synaptic transmission and information flow through gap junctions. The interaction between gap junctions and chemical synaptic transmission observed in these developing networks represent a powerful mechanism by which activity across large groups of neurons can be correlated.

Dynamics of retinal waves are controlled by cyclic AMP.

Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.

Retinal waves are governed by collective network properties.

Propagating neural activity in the developing mammalian retina is required for the normal patterning of retinothalamic connections. This activity exhibits a complex spatiotemporal pattern of initiation, propagation, and termination. Here, we discuss the behavior of a model of the developing retina using a combination of simulation and analytic calculation. Our model produces spatially and temporally restricted waves without requiring inhibition, consistent with the early depolarizing action of neurotransmitters in the retina. We find that highly correlated, temporally regular, and spatially restricted activity occurs over a range of network parameters; this ensures that such spatiotemporal patterns can be produced robustly by immature neural networks in which synaptic transmission by individual neurons may be unreliable. Wider variation of these parameters, however, results in several different regimes of wave behavior. We also present evidence that wave properties are locally determined by a single variable, the fraction of recruitable (i.e., nonrefractory) cells within the dendritic field of a retinal neuron. From this perspective, a given local area's ability to support waves with a wide range of propagation velocities-as observed in experiment-reflects the variability in the local state of excitability of that area. This prediction is supported by whole-cell voltage-clamp recordings, which measure significant wave-to-wave variability in the amount of synaptic input a cell receives when it participates in a wave. This approach to describing the developing retina provides unique insight into how the organization of a neural circuit can lead to the generation of complex correlated activity patterns.

Competition in retinogeniculate patterning driven by spontaneous activity.

When contacts are first forming in the developing nervous system, many neurons generate spontaneous activity that has been hypothesized to shape appropriately patterned connections. In Mustela putorius furo, monocular intraocular blockade of spontaneous retinal waves of action potentials by cholinergic agents altered the subsequent eye-specific lamination pattern of the lateral geniculate nucleus (LGN). The projection from the active retina was greatly expanded into territory normally belonging to the other eye, and the projection from the inactive retina was substantially reduced. Thus, interocular competition driven by endogenous retinal activity determines the pattern of eye-specific connections from retina to LGN, demonstrating that spontaneous activity can produce highly stereotyped patterns of connections before the onset of visual experience.

Dynamic processes shape spatiotemporal properties of retinal waves.

In the developing mammalian retina, spontaneous waves of action potentials are present in the ganglion cell layer weeks before vision. These waves are known to be generated by a synaptically connected network of amacrine cells and retinal ganglion cells, and exhibit complex spatiotemporal patterns, characterized by shifting domains of coactivation. Here, we present a novel dynamical model consisting of two coupled populations of cells that quantitatively reproduces the experimentally observed domain sizes, interwave intervals, and wavefront velocity profiles. Model and experiment together show that the highly correlated activity generated by retinal waves can be explained by a combination of random spontaneous activation of cells and the past history of local retinal activity.

Migration of neocortical neurons in the absence of functional NMDA receptors.

A variety of factors, from cell adhesion to changes in intracellular calcium, are thought to influence neuronal migration. Here we examine the possibility that calcium influx mediated via NMDA receptors regulates migration of neocortical neurons. We have examined the cytoarchitecture of the cortex in transgenic mice lacking functional NMDA receptors. Using cell birthdating techniques we found that cells in the developing neocortex of NMDAR-1 mutant mice have a distribution indistinguishable from that in animals with functional NMDA receptors, implying normal rates and routes of migration. These observations contrast with previous in vitro pharmacological studies of cerebellar granule cell migration, in which a role for NMDA receptors has been demonstrated. Thus, either different mechanisms are responsible for controlling neuronal migration in neocortex versus cerebellum or, more likely, neocortical neurons in NMDAR-1 mutant mice have acquired compensatory mechanisms for cell migration.

Presynaptic calcium dynamics at the frog retinotectal synapse.

1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipiens with the use of microfluorimetry. Isolated frog brains were incubated with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2+ indicator fura-2. An optic nerve shock caused a transient decrease in the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of seconds. 2. In normal saline, the amplitude of the fluorescence transients was dependent on stimulus intensity and at all levels it was directly correlated with the amplitude of postsynaptic field potentials produced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluorescence transients remained essentially unchanged while postsynaptic field potential amplitude was greatly reduced. Replacing extracellular Ca2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approximately linearly with extracellular [Ca2+], whereas the amplitude the corresponding field potential was nonlinearly related to the fluorescent transient amplitude (approximately 2.5 power). In thin sections of labeled tecta, fluorescence labeling was localized to 1-micron puncta in the termination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve stimulation, the amplitude of fluorescence transients to succeeding action potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buffering capacity of fura-2-labeled terminals was increased by incubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced response was produced by saturation of the fura-2. We provide a mathematical analysis of this saturation effect and use it to estimate the Ca2+ change per action potential. 4. Both BAPTA-AM and EGTA-AM reduced the overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qualitative difference in their effects on the shape of the transient. Incubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subsequent phase observed with BAPTA. We demonstrate that these results are consistent with numerical simulations of Ca2+ dynamics in a single-compartment model where the fast initial decay is produced by the forward rate of Ca2+ binding to EGTA. 5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic nerve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affected (approximately 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz stimulus trains, synaptic enhancement of unmyelinated fibers decayed with a characteristic time similar to fura-2 fluorescence decays. Incubation with EDTA-AM or EGTA-AM produced little effect on evoked release but reduced both the amplitude of the fura-2-measured Ca2+ transient and the amplitude of short-term synaptic enhancement.

Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves.

Highly correlated neural activity in the form of spontaneous waves of action potentials is present in the developing retina weeks before vision. Optical imaging revealed that these waves consist of spatially restricted domains of activity that form a mosaic pattern over the entire retinal ganglion cell layer. Whole-cell recordings indicate that wave generation requires synaptic activation of neuronal nicotinic acetylcholine receptors on ganglion cells. The only cholinergic cells in these immature retinas are a uniformly distributed bistratified population of amacrine cells, as assessed by antibodies to choline acetyltransferase. The results indicate that the major source of synaptic input to retinal ganglion cells is a system of cholinergic amacrine cells, whose activity is required for wave propagation in the developing retina.