In the central nervous system, dendritic arborizations of neurons undergo dynamic structural remodelling during development. Processes are elaborated, maintained or eliminated to attain the adult pattern of synaptic connections. Although neuronal activity influences this remodelling, it is not known how activity exerts its effects. Here we show that neurotransmission-evoked calcium (Ca(2+)) release from intracellular stores stabilizes dendrites during the period of synapse formation. Using a ballistic labelling method to load cells with Ca(2+) indicator dyes, we simultaneously monitored dendritic activity and structure in the intact retina. Two distinct patterns of spontaneous Ca(2+) increases occurred in developing retinal ganglion cells--global increases throughout the arborization, and local 'flashes' of activity restricted to small dendritic segments. Blockade of local, but not global, activity caused rapid retraction of dendrites. This retraction was prevented locally by focal uncaging of caged Ca(2+) that triggered Ca(2+) release from internal stores. Thus, local Ca(2+) release is a mechanism by which afferent activity can selectively and differentially regulate dendritic structure across the developing arborization.

Synchronized spontaneous rhythmic activity is a feature common to many parts of the developing nervous system. In the early visual system, before vision, developing circuits in the retina generate synchronized patterns of bursting activity that contain information useful for patterning connections between retinal ganglion cells and their central targets. However, how developing retinal circuits generate and regulate these spontaneous activity patterns is still incompletely understood. Here we show that in developing retinal circuits, the nature of excitatory neurotransmission driving correlated bursting activity in ganglion cells is not fixed but undergoes a developmental shift from cholinergic to glutamatergic transmission. In addition, we show that this shift occurs as presynaptic glutamatergic bipolar cells form functional connections onto the ganglion cells, implicating the role of bipolar cells in providing endogenous drive to bursting activity later in development. This transition coincides with the period when subsets of ganglion cells (On and Off cells) develop distinct activity patterns that are thought to underlie the refinement of their connectivity with their central targets. Here, our results suggest that the differences in activity patterns of On and Off ganglion cells may be conferred by differential synaptic drive from On and Off bipolar cells, respectively. Taken together, our results suggest that the regulation of patterned spontaneous activity by neurotransmitters undergoes systematic change as new cellular elements are added to developing circuits and also that these new elements can help specify distinct activity patterns appropriate for shaping connectivity patterns at later ages.Key words: retinal development; ferret retina; spontaneous activity; retinal waves; activity dependent; APB; glutamate Electrical activity in the developing nervous system is characterized by spontaneous periodic bursts of action potentials that are synchronized among neighboring cells (Feller, 1999;O'Donovan, 1999;Wong, 1999). Such activity occurs in the immature nervous systems of different species (Masland, 1977;Galli and Maffei, 1988;Meister et al., 1991;Gummer and Mark, 1994; Sernagor and Grzywacz, 1996;Wong et al., 1998;Z hou, 1998) and has been implicated in the development and refinement of neuronal connectivity (Katz and Shatz, 1996;Wong, 1999). Because of this functional importance, recent work has focused on how coordinated network oscillations are produced and regulated across development.Spontaneous rhythmic activity in structures from the spinal cord to the hippocampus and retina often requires excitatory neurotransmission (O'Donovan, 1999). Intriguingly, spontaneous rhythmic activity occurs before and throughout the period when synaptic networks are assembled (Wong et al., 1993;Spitzer et al., 1995; C atsicas et al., 1998;Milner and Landmesser, 1999), raising the question of whether unique or even transient mechanisms are required for its production. Additionally, as new synaptic elements are incorp...

Patterned neuronal activity is implicated in the refinement of connectivity during development. Calcium-imaging studies of the immature ferret visual system demonstrated previously that functionally separate ON and OFF retinal ganglion cells (RGCs) develop distinct temporal patterns of spontaneous activity as their axonal projections undergo refinement. OFF RGCs become spontaneously more active compared with ON cells, resulting in a decrease in synchronous activity between these cell types. This change in ON and OFF activity patterns is suitable for driving the activity-dependent refinement of their axonal projections. Here, we used whole-cell and perforatedpatch recording techniques to elucidate the mechanisms that underlie the developmental alteration in the ON and OFF RGC activity patterns. First, we show that before the refinement period, ON and OFF RGCs have similar spike patterns; however, during the period of segregation, OFF RGCs demonstrate significantly higher spike rates relative to ON cells. With increasing age, OFF cells require less depolarization to reach their action potential threshold and fire more spikes in response to current injection compared with ON cells. In addition, spontaneous postsynaptic currents and potentials are greater in magnitude in OFF cells than ON cells. In contrast, before axonal refinement, there are no differences in the intrinsic excitability or synaptic drive onto ON and OFF cells. Together, our results show that developmental changes in ON and OFF RGC excitability and in the strength of their synaptic drives act together to reshape the spike patterns of these cells in a manner appropriate for the refinement of their connectivity.

Activity-dependent refinement of synaptic connections occurs throughout the developing nervous system, including the visual system. Retinal ganglion cells (RGCs) overproduce synapses then refine them in an activity-dependent manner that segregates RGC connections into multicellular patterns, such as eye-specific regions and retinotopic maps. Ferrets additionally segregate ON and OFF retinogeniculate pathways in an activity-dependent manner. It was unknown whether differences in ON versus OFF intrinsic and spontaneous activity occur in postnatal mouse. The work reported here measured the intrinsic properties and spontaneous activity of morphologically identified postnatal mouse RGCs, and tested the hypothesis that mouse ON and OFF RGCs develop differences in spontaneous activity. We found developmental changes in resting potential, action potential threshold, depolarization to threshold, action potential width, action potential patterns, and maximal firing rates. These results are consistent with the maturation of the intrinsic properties of RGCs extending through the first three postnatal weeks. However, there were no differences among mouse ON, OFF, and multistratified RGCs in intrinsic excitability, spontaneous synaptic drive or spontaneous action potential patterns. The absence of differences between ON and OFF activity patterns is unlike the differences that arise in ferrets. In contrast to the ferret, the ON and OFF target neurons in the mouse are organized in a random pattern, not layers. This supports the hypothesis that the absence of systematic differences in activity results in the nonlayered distribution of retinogeniculate connections.

The effects of cholinergic agonists and antagonists on electrical coupling between horizontal cells were studied in dark-adapted mudpuppy retinas. Carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) uncoupled horizontal cells, but the muscarinic agonist oxotremorine did not. The uncoupling effects of carbachol and DMPP were blocked by the nicotinic antagonist D-tubocurarine and by the dopamine antagonist fluphenazine, indicating that carbachol uncoupled horizontal cells by stimulating dopamine release via nicotinic receptors. Carbachol also caused an increase in release of [3H]dopamine from retinas. D-Tubocurarine increased horizontal cell coupling, indicating that tonic cholinergic input was present in dark-adapted retinas. D-Tubocurarine did not reduce light-evoked uncoupling of horizontal cells, suggesting that cholinergic neurons are not an essential part of the direct pathway by which light causes an immediate increase in dopamine release.

The retinal ganglion cells (RGCs) have diverse morphology and physiology. Although some studies show that correlations between morphological properties and physiological properties exist in cat RGCs, these properties are much less distinct and their correlations are unknown in mouse RGCs. In this study, using three-dimensional digital neuron reconstruction, we systematically analyzed twelve morphological parameters of mouse RGCs as they developed in the first four postnatal weeks. The development of these parameters fell into three different patterns and suggested that contact from bipolar cells and eye opening might play important roles in RGC morphological development. Although there has been a general impression that the morphological parameters are not independent, such as RGCs with larger dendritic fields usually have longer but sparser dendrites, there was not systematic study and statistical analysis proving it. We used Pearson's correlation coefficients to determine the relationship among these morphological parameters and demonstrated that many morphological parameters showed high statistical correlation. In the same cells we also measured seven physiological parameters using whole-cell patch-clamp recording, focusing on intrinsic excitability. We previously reported the increase in intrinsic excitability in mouse RGCs during early postnatal development. Here we showed that strong correlations also existed among many physiological parameters that measure the intrinsic excitability. However, Pearson's correlation coefficient revealed very limited correlation across morphological and physiological parameters. In addition, principle component analysis failed to separate RGCs into clusters using combined morphological and physiological parameters. Therefore, despite strong correlations within the morphological parameters and within the physiological parameters, postnatal mouse RGCs had only limited correlation between morphology and physiology. This may be due to developmental immaturity, or to selection of parameters.

This paper describes a novel technique to identify and study neurons that express specific mRNAs. The method is called Live FISH because it is like fluorescent in situ hybridization (FISH) but can be applied to living tissues. In Live FISH, molecular beacons, which fluoresce only in the presence of the complementary mRNA, are delivered by gene gun into living neurons. One of its many benefits over existing gene expression assays is that the neurons are alive. Because identified neurons are living, they can be targeted for subsequent single-cell assays, such as electrophysiology. In addition, dye delivered with the molecular beacons illuminates dendritic morphology. Live FISH is cheaper than generating transgenic animals, and the endogenous mRNA is assayed without covalent modification or genetic manipulation. This report demonstrates the feasibility of Live FISH with yellow fluorescent protein (YFP) mRNA in living mouse retinas, although molecular beacons can be rationally designed to any mRNA of interest.

1. The relative effectiveness of adapting lights of different wave-lengths on uncoupling of horizontal cells was measured in dark-adapted mudpuppy retinas. Diffuse blue (470 nm) or red (620 nm) adapting stimuli were adjusted in intensity to be equally effective for rods or for cones. Uncoupling of horizontal cells was measured by intracellular recording of changes in their responses to spot and annulus stimuli. The intensities of the adapting light pairs were varied over 3 log units. The responses of the horizontal cells indicated that both rods and cones were stimulated by the adapting lights. 2. Relatively dim adapting lights did not produce detectable changes in horizontal cell coupling. Brighter adapting lights caused uncoupling of horizontal cells. When the brighter adapting lights were rod matched, the uncoupling effect of the 620-nm light was significantly greater than that of the 470 nm light, indicating that cones contribute to the uncoupling effect. 3. When the adapting lights were cone matched, the effects of the two wavelengths were not significantly different, but this did not rule out a rod contribution because the effective adapting lights probably produced maximal or nearly maximal, and hence equal or nearly equal, responses in rods. 4. The results indicate that cones contribute to the light-evoked uncoupling of horizontal cells in mudpuppy, although a contribution from rods could not be ruled out. Because it was shown previously that light-evoked uncoupling of horizontal cells in mudpuppy is mediated by dopamine, the results also suggest that cones contribute to the light-evoked release of dopamine.