Competition driven by retinal waves promotes morphological and functional synaptic development of neurons in the superior colliculus

Abstract

Prior to eye opening, waves of spontaneous activity sweep across the developing retina. These "retinal waves," together with genetically encoded molecular mechanisms, mediate the formation of visual maps in the brain. However, the specific role of wave activity in synapse development in retino-recipient brain regions is unclear. Here we compare the functional development of synapses and the morphological development of neurons in the superior colliculus (SC) of wild-type (WT) and transgenic (β2-TG) mice in which retinal wave propagation is spatially truncated (Xu HP, Furman M, Mineur YS, Chen H, King SL, Zenisek D, Zhou ZJ, Butts DA, Tian N, Picciotto MR, Crair MC. Neuron 70: 1115-1127, 2011). We use two recently developed brain slice preparations to examine neurons and synapses in the binocular vs. mainly monocular SC. We find that retinocollicular synaptic strength is reduced whereas the number of retinal inputs is increased in the binocular SC of β2-TG mice compared with WT mice. In contrast, in the mainly monocular SC the number of retinal inputs is normal in β2-TG mice, but, transiently, synapses are abnormally strong, possibly because of enhanced activity-dependent competition between local, "small" retinal wave domains. These findings demonstrate that retinal wave size plays an instructive role in the synaptic and morphological development of SC neurons, possibly through a competitive process among retinofugal axons.

Keywords: morphological development; nicotine; nicotinic acetylcholine receptor; retinal waves; superior colliculus; synapse maturation; visual map development.

Fig. 1.

Examining synapse development in the medial and lateral superior colliculus (SC) of wild-type (WT) and transgenic (β2-TG) mice. A: example spontaneous retinal waves recorded with a multielectrode array in WT (top) and β2-TG (bottom) mice at postnatal day (P)4 (from Xu et al. 2011). The size and color of the circles correspond to the activity level recorded from the underlying electrodes, which were spaced 100 μm apart, with 500 ms between frames from left to right. Waves in WT mice encompass the entire electrode array, whereas in β2-TG mice waves are much more circumscribed and encompass a smaller portion of the retina. B: schematic of mapping phenotype in β2-TG mice. At P7, focal dye injections into the retina (left, different colors) result in dense target zones in corresponding locations of the SC of WT mice (center, dorsal view). In contrast, in β2-TG mice (right), target zones are enlarged for ventro-temporal retinal injections, indicating poor retinotopic refinement in this part of the map but not in the remaining parts of the map. The ventro-temporal retina and its corresponding antero-medial crescent of the SC (shaded) represent the binocular visual field. To record selectively from the medial (binocular) vs. lateral (mainly monocular) SC, we designed 2 brain slice preparations that differ in their cutting angles. C: schematic of retinocollicular axon refinement. A and B were adapted with permission from Xu et al. (2011), and C was adapted with permission from Dhande et al. (2011).

Fig. 2.

Development of AMPA miniature currents (“AMPA-minis”) in β2-TG and WT mice. A and B: examples of evoked AMPA-minis recorded from medial (A) and lateral (B) slices of β2-TG and WT mice at different ages. In these experiments, synchronous evoked AMPA responses were first recorded under a Ca²⁺-ACSF bath (gray, average trace over 10–20 sweeps). The extracellular Ca²⁺ was then replaced by Sr²⁺ to desynchronize synaptic release and evoke “miniature” AMPA currents (black). C and D: quantification of AMPA-mini amplitudes in medial (C) and lateral (D) slices. Number of cells per group is indicated. WT data include recordings from Furman and Crair (2012) (see materials and methods; 9/10, 11/14, and 8/10 cells for P3–4, P6–7, and P12–13, respectively, in C and 7/9, 7/11, 5/7 in D). Also, traces WT-P7 (A) and WT-P4 and WT-P12 (B) are reproduced from Furman and Crair (2012) with permission. In all recordings only 1 cell per slice and 1 slice per animal were used. Effect of age was tested by 1-way ANOVA with Tukey's post hoc test; differences between WT and TG mice were tested with 2-tailed Student's t-test with false discovery rate procedure for multiple comparisons. *P < 0.05, ***P < 0.001.

Fig. 3.

Development of AMPA-to-NMDA ratios and NMDA decay time constant (τ_NMDA). A and B: examples of AMPA- and NMDA-mediated currents recorded from medial (A) and lateral (B) slices of β2-TG and WT mice at different ages. Each trace represents average response of 10–20 sweeps. C–F: quantification of τ_NMDA (C and D) and AMPA-to-NMDA ratios (E and F) (1-way ANOVA with Tukey's post hoc test for the effect of age; Student's t-test with false discovery rate procedure for differences between WT and β2-TG mice at each age). WT data include recordings from Furman and Crair (2012) as follows (see materials and methods): 7/8, 14/15, and 7/9 cells for P3–4, P6–7, and P12–13, respectively, in C and E and 5/6, 11/13, and 5/8 cells in D and F. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Number-of-inputs analysis. A–D: examples of graded-stimulation experiments at P6–7 in medial and lateral slices from β2-TG and WT mice. In these experiments, we measured NMDA-mediated responses at different stimulation strengths. The lower stimulation strength was adjusted to obtain a mixture of no-response trials (“failures”) and responses (“successes”). The mean peak amplitude of the success responses at the minimal stimulation strength is an estimate of the single-fiber response. Stimulation intensity was then gradually increased until saturation response amplitude was obtained. The saturation response divided by the single-fiber response is an estimate of the number of retinal inputs to the neuron. EPSC, excitatory postsynaptic current. E–G: quantification of the number of inputs (E), single-fiber response (F), and saturation response (G) for the 4 experimental groups, color coded as indicated in the schematic. Differences were tested by one-way ANOVA with Tukey's post hoc test. The WT traces in A and B are reproduced from Furman and Crair (2012) with permission. Also, part of the WT data in E–G (medial slice: 10/12 cells; lateral slice: 8/10 cells) is from Furman and Crair (2012). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Morphological analysis of SC neurons. A: in some of the experiments, we labeled neurons during recording and reconstructed their morphology. Shown are examples of reconstructed neurons from medial and lateral slices of β2-TG and WT mice at P6–7. All cells are oriented so that the pial surface is facing upward. Scale bar, 100 μm in all panels. B–D: dendritic complexity of reconstructed neurons was quantified with the number of dendritic nodes (B), total dendritic length (C), and fractal dimension of the dendritic tree (D). Differences were tested by 1-way ANOVA with Tukey's post hoc test. One of the reconstructed cells in A (WT P6–7, lateral slice, bottom) and part of the WT data in B–D (medial slice: 10/12, lateral slice: 9/11 cells) are from Furman and Crair (2012). *P < 0.05, **P < 0.01.

Fig. 6.

Results summary and conceptual model. Top: retinocollicular synapse development was examined with brain slice preparations that cut through the medial (binocular) or lateral (mainly monocular) SC (left and right, respectively). Middle and bottom: schematic of synaptic and anatomical phenotype at P6–7 in WT and β2-TG mice, respectively. In WT mice, retinal waves propagate over relatively long distances. Thus, within each eye, retinal ganglion cells (RGCs) at different locations are activated consecutively (when participating in the same wave). These within-eye correlations support segregation of retinal axon branches into eye-specific layers in the binocularly innervated, medial SC. Large green circles represent formation of strong synaptic contacts to the postsynaptic neuron. In both medial and lateral SC, WT waves support retinotopic refinement of the map, so that RGC axons branch in the corresponding retinotopic location of the SC. SC neurons are shown as receiving monocular input, although in reality the possibility exists of binocular input in the medial SC. In β2-TG mice, retinal wave propagation is truncated, resulting in “small waves” that are insufficient for supporting eye-specific segregation and retinotopic refinement in the binocular SC. Physiologically, SC neurons receive a larger number of retinal inputs. Some of these inputs could potentially arise from the ipsilateral eye (as shown) or, alternatively, from the contralateral eye only. In the lateral SC, where binocular interactions are less prominent, the local spatial information contained in small waves is sufficient for retinotopic refinement of the map. Furthermore, synaptic strength is enhanced in the lateral SC of β2-TG mice because of large temporal offsets in the bursting activity of nearby regions in the retina. These temporal offsets are nearly absent during normal development and, according to a recently proposed model, may promote synaptic strengthening through enhanced axonal competition.