Retinal waves in mice lacking the ␤2 subunit of the nicotinic receptor

Chao Sun*, David K. Warland*, Jose M. Ballesteros*, Deborah van der List*, and Leo M. Chalupa*†‡

Departments of *Neurobiology, Physiology, and Behavior, and †Ophthalmology and Vision Science, University of California, Davis, CA 95616

Communicated by Edward G. Jones, University of California, Davis, CA, July 24, 2008 (received for review June 9, 2008)

The structural and functional properties of the are disrupted in mutant animals lacking the ␤2 subunit of the nicotinic acetylcholine receptor. In particular, eye-specific retinogeniculate projections do not develop normally in these mutants. It is widely thought that the developing of ␤2؊/؊ mutants do not manifest correlated activity, leading to the notion that retinal waves play an instructional role in the formation of eye-specific retinogeniculate projections. By multielectrode array recordings, we show here that the ␤2؊/؊ mutants have robust retinal waves during the formation of eye-specific projections. Unlike in WT animals, however, the mutant retinal waves are propagated by gap junctions rather than cholinergic circuitry. These results indi- cate that lack of retinal waves cannot account for the abnormalities that have been documented in the retinogeniculate pathway of the ␤2؊/؊ mutants and suggest that other factors must contribute Fig. 1. Confocal images of and LGN using antibodies against Chrnb2 (M270). WT retina (A) stains (red) in the (IPL) with two to the deficits in the visual system that have been noted in these distinguishable bands. In contrast, the sections from the Picciotto (B) and Xu animals. (C) mutant mice show no M270 binding. INL, inner nuclear layer; GCL, gan- glion cell layer. (D–F) Confocal images showing M270 staining (red) in the lateral geniculate nucleus ͉ multielectrode array ͉ retinal ganglion cells ͉ dLGN (outlined) of the P4 WT mouse (D) and a lack of staining with this retinogeniculate segregation ͉ gap junction antibody for the Picciotto (E) and Xu (F) mutant mice at comparable ages. D, dorsal; V, ventral; M, medial; L, lateral. DAPI-stained nuclei are pseudocolored green. (Scale bars: A–C,50␮m 50; D–F, 200 ␮m.) he precise connections that characterize the mature nervous Tsystem often arise from an early exuberant pattern that becomes refined through a combination of molecular and and ␤2Ϫ/Ϫ mutants have been reported to lack retinal waves during activity-dependent cues. In the case of the visual system, the this developmental period (15, 17, 19). Other visual system defects projections of the two eyes to the dorsal lateral geniculate have been reported including: (i) the projections of the two eyes nucleus (dLGN) are initially intermingled before gradually remain intermingled within both the dLGN and the superior becoming segregated into separate layers in animals with a colliculus at an age when they are fully segregated in WT mice laminated dLGN such as ferret, cat, and monkey or to different (15–17), (ii) retinotopic organization is less precise than in WT regions of the geniculate as in the mouse and rat (1–3). During animals (19, 21, 26), and (iii) receptive field properties of dLGN and the developmental period when eye-specific retinogeniculate visual cortical neurons are abnormal (18, 20, 26). projections are being established the retina manifests a remark- To probe further into the relationship between neuronal able pattern of activity. Immature retinal ganglion cells (RGCs) activity and developing retinal projections, we sought to docu- discharge periodic bursts of action potentials, with adjacent cells ment in greater detail the spontaneous discharge patterns of Ϫ Ϫ firing in a temporally correlated manner, resulting in waves of RGCs in the ␤2 / mutant retinas during the developmental activity sweeping across the retinal surface (4–7). These retinal period when the projections of the two eyes become segregated. ␤ Ϫ/Ϫ waves have been considered to be essential for the formation of Multielectrode array (MEA) recordings from 2 mutants eye-specific retinogeniculate projections through a Hebbian- unexpectedly revealed retinal waves and correlated discharges in type mechanism where the connections stemming from one eye all cases. These surprising results contradict previous lack-of- are strengthened and maintained, whereas those of the other eye waves reports (15, 17, 19). We conclude that the segregation defects and other structural and functional abnormalities that become eliminated based on which set of inputs is more capable ␤ Ϫ/Ϫ of activating dLGN neurons (8, 9). Evidence in support of this have been documented in the 2 mutants are not caused by the absence of retinal waves. prevalent notion has been provided by studies that have relied on pharmacological agents to alter the normal activity patterns of Results the developing retina. Several studies have shown that blocking ؊ ؊ Characterization of the Two Independent ␤2 / Mouse Lines. Based or perturbing retinal activity prevents the formation of segre- on published information, the Picciotto mutant allele deletes only gated retinogeniculate projections (9–11). These observations have led to the conclusion that retinal waves instruct the formation of eye-specific domains (8, 12–14). Author contributions: C.S., D.K.W., D.v.d.L., and L.M.C. designed research; C.S., J.M.B., and Studies that have used mutant mice have also supported a linkage D.v.d.L. performed research; C.S., D.K.W., J.M.B., and D.v.d.L. analyzed data; and C.S., between retinal waves and development of segregated retino- D.K.W., J.M.B., D.v.d.L., and L.M.C. wrote the paper. geniculate projections. In particular, animals in which the ␤2 The authors declare no conflict of interest. subunit of the nicotinic acetylcholine receptor gene (Chrnb2) has Freely available online through the PNAS open access option. been deleted (throughout the text, these mice are designated as ‡To whom correspondence should be addressed. E-mail: [email protected]. Ϫ/Ϫ ␤2 mutants) have served as a popular model in this arena This article contains supporting information online at www.pnas.org/cgi/content/full/ (15–22). When eye-specific projections are being formed, the 0807178105/DCSupplemental. correlated discharges of RGCs reflect cholinergic activity (23–25), © 2008 by The National Academy of Sciences of the USA

13638–13643 ͉ PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807178105 Downloaded by guest on October 2, 2021 NEUROSCIENCE

Fig. 2. Retinal waves in WT and ␤2Ϫ/Ϫ mutants by MEA recording. (A) Raster plots of individual RGC spike activity obtained from 18 P4–5 WT and mutant retinas via MEA recording. Each row is the spike train from a single RGC. Within each row, vertical lines represent spikes from the corresponding cell. (Scale bar ϭ 2 min.) (B) The retinal wave frequency (waves/min) in P4–5 WT and mutant mice. The WT mice showed significantly lower retinal wave frequencies than the two mutants. There was no difference between Picciotto and Xu mice. Numbers reflect retinas recorded. Data are given as mean Ϯ SEM. *,P Ͻ 0.05; Student’s t test. (C) The mean firing frequency (spikes/s) of RGCs in P4–5 WT and mutant mice were significantly different from each other. The WT mice had the lowest firing frequency, and the Picciotto mice had the highest firing frequency. Numbers reflect RGCs recorded. Data are given as mean Ϯ SEM. *, P Ͻ 0.05; Kruskal-Wallis test.

part of the exon 1, which results in an mRNA abnormally truncated through adulthood [supporting information (SI) Fig. S1A]. At at the 5Ј end. Although a truncated peptide might exist, Picciotto P1, there was considerable overlap of the contralateral and ipsilat- et al. (27) have shown that the Chrnb2 protein is undetectable in the eral input in the WT mouse and the two ␤2Ϫ/Ϫ mutants. In WT homozygous mutant mice. In contrast, the Xu mutant allele deletes animals, the ipsilateral projection decreased with age, becoming five exons. The entire coding region for the Chrnb2 mRNA is confined to the gap within the contralateral input, a pattern clearly essentially deleted in the Xu allele. This allele should be a null allele evident at P5. By contrast, in the two mutants, the ipsilateral because no Chrnb2 protein can be generated. Both the Picciotto projection remained relatively widespread at P5. At P12 as in the and Xu mutant alleles were generated by inserting a neo (neomycin- adult, the ipsilateral projection became patchy, with distinct clumps resistant) cassette into the genomic sequence. We also examined of label interspersed within the contralateral projections (Fig. S2). the presence of Chrnb2 proteins in the two mutant mice by using Quantitative analysis revealed that in the WT mouse the segrega- antibodies against Chrnb2 (Fig. 1). As may be seen, there is clear tion of the retinogeniculate projections was essentially complete by staining in the retina and dLGN of the WT mice, with no detectable P8, whereas significant binocular overlap was still present in the two staining in either the Picciotto or Xu mutants. These analyses mutants at P12 (Fig. S1B) the retinogeniculate projection remains indicate that we can unequivocally identify the homozygous mu- abnormal in the adult ␤2Ϫ/Ϫ mutants. tants for both mutant lines and that these two mutant lines are essentially null. Retinal Waves Are Present in Both ␤2؊/؊ Mutant Lines. We next assessed retinal activity by MEA recordings. Such recordings Retinogeniculate Projections Are Abnormally Segregated in Both from developing WT retinas at P4 and P5 revealed typical retinal ;2؊/؊ Mutant Lines. To assess the retinogeniculate pathway, waves with periodic correlated discharges (Fig. 2A; n ϭ 3 for P4␤ intraocular injections of different tracers were made into each n ϭ 4 for P5). We also performed MEA recordings on ␤2Ϫ/Ϫ eye of developing mice ranging in age from postnatal day 1 (P1) mutant retinas from both Picciotto mutants (Fig. 2A; n ϭ 4 for

Sun et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13639 Downloaded by guest on October 2, 2021 Fig. 3. CI of pairs of RGCs in WT and ␤2Ϫ/Ϫ mutants. (A–C) Three typical MEA recordings are shown for each P4 mouse type: WT (A), Picciotto (B), and Xu (C). RGCs in both WT and mutant mice exhibit highly correlated spontaneous activity as a function of intercell distance. Note that because the interelectrode spacing used in these recordings was 200 microns, the signals of a given cell can appear only on one electrode. As a result, quantization of data points around particular intercell distances can be seen. For each pair of recorded cells, the CI was plotted logarithmically against the intercell distance. The red regression line represents an exponential fit to the data CI ϭ A exp (ϪX/L), where A is the maximal CI and L is the correlation length. Note that the trend lines are similar in all three groups of animals (red lines). Vertical lines denote quintile boundaries, and the horizontal thin line denotes the 99% limit of a control shuffle analysis. The y-intercepts (maximum CI) from the scatter plots are a measure of the strength of the overall correlations recorded from cell pairs. (D) The mean CIs as a function of intercell distance for the WT and mutant mice are plotted with SEM (error bars). *, P Ͻ 0.05; Mann–Whitney U test.

P4; n ϭ 5 for P5) and Xu mutants (Fig. 2A; n ϭ 6 for P4; n ϭ waves during the developmental period when eye-specific pro- 6 for P5). Comparison of these recordings clearly demon- jections are being formed. At the same time, the characteristics strated that retinal waves are present in the two ␤2Ϫ/Ϫ mutant of the retinal activity in the ␤2Ϫ/Ϫ mutants differ significantly retinas. Although there was variability in the firing patterns from the retinal waves in the WT animals, suggesting that the among the studied retinas from each of the three groups, the retinal circuitry that generates the waves may be different electrical discharges were highly correlated and clearly wave- between WT and ␤2Ϫ/Ϫ mutants. like in every recorded retina whether from WT or mutant mice. Moreover, the wave and spike frequencies were found to be higher in ␤2Ϫ/Ϫ mutants than in the retinas isolated from WT animals. These conclusions were corroborated by quan- titative analyses (Fig. 2 B and C). Recordings from younger animals showed that retinal waves are present in the mutants as early as P2 (data not shown). To compare the spontaneous firing patterns of RGCs in the two ␤2Ϫ/Ϫ mutant lines with those of cells in the WT animals, we calculated the correlation index (CI) of pairs of spiking cells in P4 retinas and plotted these values against the estimated intercell distance (Fig. 3 A–C). In addition, we compared the CI as a function of intercell distance for the three strains of mice (Fig. 3D), which showed that the CI of closely adjacent cells was essentially equivalent, but at greater intercell distances it was higher in the two mutant lines compared with the WT animals. To further characterize the retinal waves in ␤2Ϫ/Ϫ mutants, we compared the discharge properties of single cells from mutants with those from WT animals. Ten different parameters were used to quantify the spiking activity (Fig. 4). The results showed Fig. 4. Normalized single RGC activity analysis. WT-normalized single RGC that retinal activity in both mutant lines had higher firing firings show that the mutants have higher mean firing frequency, burst fre- frequencies, higher burst frequencies, and higher interspike quency, and interspike interval in bursts, but lower percentage of total spikes in intervals in a burst. On the other hand, the percentage of total bursts, peak firing frequency in burst, and interburst interval. The WT absolute values from top to bottom are (mean Ϯ SE): 0.62 Ϯ 0.03 spikes/s, 2.48 Ϯ 0.05, spikes in a burst, the peak firing frequency in a burst, the 0.53 Ϯ 0.01 bursts/min, 86 Ϯ 1, 4.8 Ϯ 0.1 s, 64 Ϯ 3 spikes/burst, 0.11 Ϯ 0.01 s, 11.8 Ϯ interburst intervals, and the percent of time that cells fired at 0.3 spikes/s, 122 Ϯ 3 s. The Poisson surprise method of Legendy and Salcman (39), frequencies Ͼ10 Hz were all lower in the mutants. Collectively, as implemented in NeuroExplorer was used to detect RGC burst activity. Data are Ϫ/Ϫ these results indicate that the ␤2 mutants manifest robust given as mean Ϯ SEM. *, P Ͻ 0.05; Kruskal-Wallis test.

13640 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807178105 Sun et al. Downloaded by guest on October 2, 2021 another pharmacological agent, 18 ␤-glycyrrhetinic acid (18 ␤-GA), a gap junction blocker (29). In WT retina, 75 ␮M18 ␤-GA had no effect on wave activities. By contrast, the retinal waves were severely disrupted by this gap junction blocker in ␤2Ϫ/Ϫ mutants (Fig. 5A). Fig. 5B compares and contrasts the effects of curare and 18 ␤-GA on wave activity in the WT animals with the two mutants. As seen, curare decreased wave frequency only in the WT retinas. By contrast, the gap junction blocker caused a pronounced decrease in wave frequency only in the two mutants, whereas curare had no appreciate effect in these retinas. Taken together, these results indicate that gap junction activities are essential for the highly correlated electrical dis- charges in ␤2Ϫ/Ϫ mutants and suggest that gap junctions are abnormally up-regulated in these mice. Discussion The ␤2Ϫ/Ϫ mutant has been used in many studies dealing with the development of mammalian visual pathways, reflecting the asser- tion that the mutant lacks retinal waves during the time when segregated eye-specific and retinotopic projections are formed (30). The functional and structural aberrations that have been discovered in the visual system of this animal have been attributed to the lack of retinal waves, and by extension it has been argued that such activity is required to instill these features in the WT animal. Two different strategies have been used to generate the ␤2Ϫ/Ϫ mutants (27, 31), and although both types have been used in studies of visual development, to our knowledge retinal recordings have been made only in the Xu mutants previously (17, 19).

Retinal Waves Are Present in ␤2؊/؊ Mutants. Here, we show by means of MEA recordings that the Xu and Picciotto ␤2Ϫ/Ϫ mutants both manifest robust retinal waves, which was the case in every mutant retina from which recordings were made. Importantly, each animal was genotyped to confirm mutation of Chrnb2. At the same time, we confirm and extend the fact that retinal projections to the dLGN do not segregate normally in NEUROSCIENCE these mutants. In the ␤2Ϫ/Ϫ mutants, binocular overlap within Fig. 5. Effect of curare and 18 ␤-GA on the retinal waves. (A) WT and mutant the geniculate persists for several days longer than in WT mice retinas exhibited retinal waves when placed in a MEME bath solution. Appli- and the segregation pattern that is ultimately attained in these cation of nicotinic acetylcholine blocker curare reduced the retinal waves in animals is clearly aberrant, with the ipsilateral input becoming WT P5 mice, but had no effect on the firing patterns in the mutants. Appli- patchy rather than confined to a well localized region of the cation of gap junction blocker 18 ␤-GA had no effect on retinal waves of WT P5 mice, but application on the ␤2Ϫ/Ϫ P5 mouse retina caused an alteration in dLGN as is the case in the WT animal (Fig. S2). the firing pattern of mutants. These findings suggest that retinal waves These findings have important implications for the field of visual recorded during the first week in the mutant retinas are driven by gap development. First, the results demonstrate that the abnormalities Ϫ Ϫ junctions. (B) The retinal wave frequency (waves/min) in P4–5 WT and ␤2Ϫ/Ϫ noted in the ␤2 / mutants in the segregation of left and right eye mutant. Numbers indicate the number of retinas recorded. Data are given as projections and retinotopic order are not caused by a lack of retinal mean Ϯ SEM. Brackets mean the connected two columns were significantly waves. Clearly, these mutants should not be used for assessing the different based on the Kruskal-Wallis test with *, P Ͻ 0.05. role of retinal waves in studies of visual system development. The ␤2Ϫ/Ϫ mutants are also not suitable for assessing the role of correlated activity in the development of retinal pathways ؊/؊ ␤ Retinal Waves in 2 Mutants and WT Animals Are Apparently because the correlated discharge patterns of neighboring Generated by Different Mechanisms. To investigate the underlying RGCs in these mice are essentially the same as in WT animals, mechanisms that regulate the high-frequency retinal waves whereas those of cells spatially dispersed are more highly ␤ Ϫ/Ϫ detected in 2 mutants, we explored the effects of pharma- correlated than in WT mice. cological agents on the MEA recordings. We first tested curare, Taken together with the results of a previous study showing the nicotinic acetylcholine receptor blocker, to assess whether that eye-specific inputs can occur when retinal discharges were ␤ Ϫ/Ϫ the retinal waves in 2 mutants were related to any residual decorrelated by treating the developing retina with a cholinergic acetylcholine activities. In P4 and P5 WT animals, application of cell immunotoxin (32), the present findings indicate that retinal 50 ␮M curare substantially reduced or completely blocked waves are neither necessary nor sufficient for the formation of retinal waves (Fig. 5A). This result is consistent with the well segregated retinogeniculate projections. established role of nicotinic acetylcholine receptors in mediating .such activity during this developmental period (28). By contrast, Characteristics of Individual RGC Firing in WT and ␤2؊/؊ Mutant Mice Ϫ Ϫ application of curare to ␤2 / mutant retinas had no detectable At the same time it is important to acknowledge that our results do effect on wave activities. These results demonstrate that cholin- not necessarily rule out a role for activity in the formation of ergic activities cannot account for the retinal waves in the ␤2Ϫ/Ϫ segregated eye-specific retinogeniculate projections because we mutants, and that different mechanisms are used to generate have also documented a number of significant differences between waves in the mutant and WT retinas. A difference in underlying the ␤2Ϫ/Ϫ mutants and the WT animals in the properties of retinal mechanisms was further supported by comparing the effects of waves and the discharges of individual cells. The most obvious

Sun et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13641 Downloaded by guest on October 2, 2021 difference is that in both the Xu and the Picciotto mutants retinal the figures of those papers. In the present study we recorded waves are at a higher frequency than in the retinas of the WT between 45 and 110 cells per retinal segment. One or more of the animals. A previous study on developing ferrets showed that foregoing methodological factors could account for the fact that binocular injections of agents that elevate cAMP, thereby increas- retinal waves in the ␤2Ϫ/Ϫ mutants were evident in the present ing the frequency of retinal waves, resulted in normal segregation study but not in earlier studies. Given the large number of of retinogeniculate projections (9). By contrast, the segregation of methodological differences it is problematic to specify what retinal projections of the ␤2Ϫ/Ϫ mutants is delayed and occurs in an factor or combination of factors could account for the earlier aberrant patchy pattern despite the occurrence of faster and more failure to record retinal waves in the mutants. frequent retinal waves in these animals. The discharge properties of individual cells were also found to Materials and Methods differ between the mutants and the WT mice, with some All experiments were performed in accordance with National Institutes of parameters significantly higher and others lower than normal. Of Health and institutional guidelines regarding animal use and were approved particular relevance is the incidence of cells that fired at fre- by the campus animal use and care committee of the University of California, Ϫ Ϫ quencies Ͼ10 Hz. In a previous study (33), this parameter was Davis. The Picciotto ␤2 / mutant line was a kind gift from M. Picciotto (Yale shown to differentiate mouse mutants with retinogeniculate University School of Medicine, New Haven, CT) (27), and the Xu mutant line projections that failed to segregate normally from those of was derived from embryos (ES Cell Line ID 00211-UNC) supplied by the Mutant Mouse Regional Resource Centers, University of California, Davis. Neonatal animals in which segregation was normal. Whereas the distri- mice were administered a lethal i.p. dose (0.05–0.1 ml) of Fatal Plus (Vortech bution of the percentage of cells firing at a rate Ͼ10 Hz in the Ϫ Ϫ Pharmaceuticals) at the time of tissue collection. All mutant mice used in this ␤ / Ϫ Ϫ two 2 mutants showed substantial overlap with the WT study were confirmed to be ␤2 / by genotyping. animals (Fig. S3), we observed a total lower average percent of mutant cells that met this criterion. Therefore, it seems unlikely Immunohistochemistry. Eyes from WT and mutant animals were enucleated that this factor alone could account for the abnormal retino- then fixed in 4% paraformaldehyde (PFA) in PBS for 45 min, cryoprotected in Ϫ Ϫ geniculate segregation pattern observed in the ␤2 / mutants. If a 25% sucrose solution, then embedded in OCT (Ted Pella). Transverse sections neuronal activity plays a role in the formation of eye-specific were cut at 10 ␮m on a Leica) cryostat. Unfixed brains of WT and mutants were inputs, our results suggest that a combination of parameters, embedded in OCT, frozen on dry ice, and sectioned at 15 ␮m. Before process- rather than retinal waves or high-frequency firings alone, is ing for immunohistochemistry, brain sections were fixed in 4% PFA for 2–3 min instrumental to this process. then washed in PBS. Retinal and brain sections were blocked in 10% normal donkey serum, 2% BSA, and 0.3% Triton X-100 in PBS, then incubated in blocking solution overnight at 4°C with a mAb Anti-Chrnb2 (M270:1:250 ؊/؊ ␤ Retinal Waves Are Mediated by Different Circuits in the 2 n8408, Sigma), followed by PBS washes then incubation with Alexa Fluor 594 Mutants. During the developmental period when eye-specific pro- fluorescent secondary (1:500; Invitrogen) for 1 h. Nuclei were visualized with jections are formed, retinal waves in the WT mouse, as in other DAPI (1:500; KPL). For control slides, primary antibodies were omitted. Images species, reflect primarily cholinergic activity of starburst amacrine were acquired on an Olympus FV500 confocal microscope. Brightness and cells (28). Pharmacological treatments that impact cholinergic contrast were adjusted by using Photoshop (Adobe Systems). activity have been shown to interfere with wave activity (25), and depletion of starburst amacrine cells by immunotoxin treat- Tracing and Analysis of Retinogeniculate Projections. WT and Picciotto and Xu ment decorrelates the discharges of neighboring ganglion cells in mutants were sampled from P1 to adulthood (P1, P3, P5, P8, P12, P20, and adult the developing ferret retina (32). In line with these observations, with n ϭ 4–5 for each age). Injections and analysis were performed as described application of curare to the WT retina was found to abolish or (17, 32). substantially decrease retinal waves. In sharp contrast, applica- tion of curare had no appreciable effect on retinal waves in the Tissue Preparation and MEA Recording. The tissue preparation and MEA ␤ Ϫ/Ϫ recording procedures were similar to that described (36). Briefly, after eutha- 2 mutants. In these mutants, retinal waves were effectively nasia the eyes were enucleated, and the retinas were isolated and stored in abolished by application of a drug that blocks gap junctions. This buffered and oxygenated Minimum Essential Medium Eagle (MEME) (M7278; drug had relatively modest effect on activity in the WT retina, thus Sigma–Aldrich) at room temperature. The retinas were cut into 5- to 8-mm2 Ϫ/Ϫ a different circuit mediates waves in the ␤2 mutants than in the rectangles. A piece of retina was placed ganglion cell layer down onto a WT retina, and an up-regulation of gap junctions seems to occur in 60-channel MEA (MultiChannel Systems), held in place with a piece of dialysis response to the deletion of Chrnb2. These results are reminiscent membrane (Spectrapore 132130; Spectrum), and superfused with buffered of the findings of Stacy et al. (34), who showed that in a choline MEME at 1–2 ml/min at 37°C. We used MEME because this solution has been acetyltransferase conditional mutant gap junctions are required for commonly used in previous studies of retinal development (36–38). The array retinal waves. The present results and the findings of Stacy et al. (34) electrodes were 30 ␮m in diameter, arranged on an 8 ϫ 8 rectilinear grid with ␮ suggest that a homeostatic mechanism regulates wave activity in the 200- m interelectrode spacing. Analog data were acquired at 20 kHz per channel simultaneously from each of the 60 electrodes for 15–20 min. Overall developing retina. See also ref. 35 for an example of nonsynaptic firing rates of the ensemble appeared stable over this time period. Curare and mechanisms that correlate spontaneous activity in the developing 18 ␤-GA were from Sigma-Aldrich. mammalian retina. It remains to be established which retinal cell ␤ Ϫ/Ϫ types manifest up-regulated gap junctions in the 2 mutants. Spike Identification. Before sorting spike events, the data were digitally filtered with a 125-Hz high-pass filter. A threshold of six times the standard Comparison with Earlier Studies That Reported a Lack of Retinal deviation of the channel was set for each channel, and 1 ms of data before Ϫ Ϫ Waves. The present results showing that the retinas of the ␤2 / a threshold-crossing event and 4 ms after the threshold event were stored mutants manifest robust waves contrast with the results of for each negative-slope event. These candidate spike waveforms were then previous studies that the Xu ␤2Ϫ/Ϫ mutants lack retinal waves sorted with the OfflineSorter (Plexon), using the first three principal (15, 17, 19). A comparison of the methods we used with those of components of the spike waveforms. Coincident events within 0.5 ms of the earlier studies reveals a host of methodological differences, one another that occurred on all electrodes were attributed to perfusion including such factors as the drugs used to anesthetize the noise and removed. Clusters were first identified by using an EM cluster algorithm by Shoham et al. (40) and then manually edited for clustering animals, the bath solutions used to maintain the retinas (see Fig. errors. S4), the bath temperature, the temperature at which the isolated retinas were maintained, the time period the isolated retinas MEA Statistical Analyses. Burst analysis. The burst duration was measured by were stored before commencing recordings, and the MEA size using the burst analysis algorithm provided by Neuroexplorer (Nex Technol- and configuration. Moreover, previous studies recorded 10–65 ogies), which scans the spike train until an interspike interval is found that is cells in each retinal segment, but only 4–8 cells were shown in less than or equal to an interval (I1) ϭ 0.1 s. Interspike intervals of Ͻ I2 ϭ 1 were

13642 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807178105 Sun et al. Downloaded by guest on October 2, 2021 included in the burst, whereas an interspike interval of Ͼ I2 denoted the end duration of the recording in s, Na(0, T) and Nb (0, T) are the total number of of the burst. The algorithm then merged all bursts with an interval Ͻ I3 ϭ 5s spikes from cell a and b during the recording, and 2 ϫ w is the width of the and removed bursts with a duration Ͻ I4 ϭ 0.5 s or with fewer than I5 ϭ 4 correlation window. Nab was computed by using w ϭ 0.1 s, and the cross- spikes. For normal distributions, Student’s t tests were applied to the data for correlation function was binned at 0.05 s. group comparison, otherwise a Kruskal–Wallis test was used. Wave frequency analysis. Naı¨veexperimenters visually inspected the raw mul- Correlation analysis. To quantify the degree of correlated firing between tielectrode analog data and counted wave events directly. A wave was de- recorded pairs of cells, all cross-correlation functions were calculated and fined as coincident bursting on four or more electrode sites. assigned a CI. The CI measures the likelihood relative to chance that a pair of cells fired together within a particular time window. The CI was computed as ACKNOWLEDGMENTS. We thank Xianghong Shan and Kimberly Zhou for Ϫ ϩ ϫ technical assistance and Dr. Marina Picciotto for generously donating Chrnb2 described by Wong et al. (7) by using the following formula: Nab( w, w) mutant mice. This research was supported by grants from Research to Prevent ϫ ϫ ϫ Ϫ ϩ T/(Na(0, T) Nb(0, T) 2 w), where Nab( w, w) is the number of spike pairs Blindness and National Institutes of Health Grants EY016182 (to L.M.C. and from cells a and b for which cell b fires within w seconds of cell a, T is the D.K.W.) and EY003991 and P30 EY12576 (to L.M.C.).

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