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Neuron

Article

Visual Circuit Development RequiresPatterned Activity Mediatedby Retinal Acetylcholine ReceptorsTimothy J. Burbridge,1,5 Hong-Ping Xu,1,5 James B. Ackman,1 Xinxin Ge,1 Yueyi Zhang,1 Mei-Jun Ye,2 Z. Jimmy Zhou,2

Jian Xu,4 Anis Contractor,4 and Michael C. Crair1,2,3,*1Department of Neurobiology2Ophthalmology and Visual Science3Kavli Institute for Neuroscience

Yale University School of Medicine, New Haven, CT 06510, USA4Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA5Co-first authors*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.neuron.2014.10.051

SUMMARY

The elaboration of nascent synaptic connections intohighly ordered neural circuits is an integral feature ofthe developing vertebrate nervous system. In sen-sory systems, patterned spontaneous activity beforethe onset of sensation is thought to influence thisprocess, but this conclusion remains controversial,largely due to the inherent difficulty recording neuralactivity in early development. Here, we describegenetic and pharmacological manipulations of spon-taneous retinal activity, assayed in vivo, that demon-strate a causal link between retinal waves and visualcircuit refinement. We also report a decoupling ofdownstream activity in retinorecipient regions ofthe developing brain after retinal wave disruption.Significantly, we show that the spatiotemporal char-acteristics of retinal waves affect the development ofspecific visual circuits. These results conclusivelyestablish retinal waves as necessary and instructivefor circuit refinement in the developing nervous sys-tem and reveal how neural circuits adjust to alteredpatterns of activity prior to experience.

INTRODUCTION

Neural circuit development is a complex process that integrates

cell differentiation, migration, neurite outgrowth, axon path-

finding, synapse formation, elaboration, and refinement in order

to equip the nervous system for early life behavior. A unique

feature of this process is the interaction between neuronal activ-

ity and synapse formation, which creates the opportunity for

neural network dynamics to contribute to fundamental aspects

of synapse and circuit development. In some respects, this is a

precocious example of ‘‘learning’’ in which neuronal activity

causes circuit rewiring through synaptic plasticity. Here, we

N

describe experiments that examine the link between initial in vivo

activity patterns and neural circuit refinement in the mammalian

visual system, and demonstrate that early activity patterns are

not only necessary and instructive for initial circuit development

but are optimized for the generation of distinct circuit features.

In contrast to lower vertebrates and insects, in which the

majority of initial circuit development is believed to be dependent

on activity-independent molecular factors, model circuits in a

variety of mammalian systems refine using both molecular-

and activity-dependent factors (Sperry, 1963; Sanes and Zipur-

sky, 2010; Kirkby et al., 2013). This process is best characterized

in the visual system (Huberman et al., 2008; Cang and Feldheim,

2013; Ackman and Crair, 2014), where classic experiments sug-

gest the necessity of neuronal activity for normal circuit develop-

ment (Hubel and Wiesel, 1970; Shatz and Stryker, 1988), while

more recent discoveries identify a host of molecular factors

that together play important roles in neurite targeting, elabora-

tion, and refinement (Cang and Feldheim, 2013). However, the

difficulty inherent in recording and manipulating activity in

developing animals and the continued presence of some nor-

mally targeted axons even after the removal of multiple guidance

cues (Frisen et al., 1998; Pfeiffenberger et al., 2006) leaves room

for debate over the relative contributions of activity and molecu-

lar guidance in this process.

In the visual system, classic experiments demonstrate that

visual experience fundamentally shapes circuit development

during a critical period of development (Hubel and Wiesel,

1970; Espinosa and Stryker, 2012). However, essential features

of developing mammalian neural circuits emerge before sensory

experience is possible (Rakic, 1976; Horton and Hocking, 1996;

Crair et al., 2001; Crowley and Katz, 2000), or even in the

absence of vision (Crair et al., 1998; White et al., 2001). More-

over, stereotypical features of central visual circuits endure

following the early removal of both eyes, implying that retinal

input is not necessary for the development of some circuits

(Ruthazer and Stryker, 1996; Crowley and Katz, 1999). These

apparently contradictory results cast doubt on the necessity

and relevance of neural activity in the early emergence of visual

circuits, and led to the theory that spontaneous activity in the

euron 84, 1049–1064, December 3, 2014 ª2014 Elsevier Inc. 1049

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Visual Map Refinement Requires Retinal Waves

developing retina and visual system could drive circuit refine-

ment prior to sensory experience (Galli and Maffei, 1988; Shatz

and Stryker, 1988). Pioneering experiments demonstrated that

perinatal retinas do indeed have correlated spontaneous bursts

of activity (Galli and Maffei, 1988) that are organized into large-

scale, seemingly random patterns termed ‘‘retinal waves’’ (Meis-

ter et al., 1991). Subsequent research established that this

patterned retinal activity propagates throughout the developing

visual system (Weliky and Katz, 1999; Ackman et al., 2012).

Experiments designed to determine the degree to which visual

circuit development depends on retinal waves have proven less

conclusive. Results from chronic in vivo pharmacological manip-

ulations that are known to either disrupt or agonize retinal waves

in vitro (Penn et al., 1998; Stellwagen and Shatz, 2002), or elim-

inate activity conductance through the optic nerve (O’Leary

et al., 1986b), generally support a link between retinal waves

and circuit refinement. However, the interpretation of these ex-

periments is complicated by the variable and cell-type-specific

influence of pharmacological manipulations (Sun et al., 2008a),

the potential systemic and/or neurotoxic effects of chronic injec-

tions (O’Leary et al., 1986a; Ackman et al., 2012; Shatz and

Stryker, 1988; but also see Penn et al., 1998), and only limited

knowledge about the retinal or downstream activity patterns

following these manipulations in vivo. Intriguingly, experiments

employing a toxin that kills starburst amacrine cells, the retinal

cell type known to nucleate early postnatal retinal waves

(Zhou, 1998; Zheng et al., 2006), found decorrelation of

patterned activity in vitro without any effect on visual circuit

development (Huberman et al., 2003). This work implied

that only the existence of retinal ganglion cell-bursting activity,

but not patterned retinal waves, was necessary for circuit

refinement.

Genetic models that disrupt spontaneous retinal activity

circumvent many of the experimental complications of pharma-

cological manipulations but come with their own caveats. The

most widely utilized knockout model for retinal waves are

b2�/� mice, which lack expression of the b2 subunit of the nico-

tinic acetylcholine receptor (b2-nAChR) (Bansal et al., 2000).

In vitro pharmacological manipulations demonstrate that spon-

taneous retinal waves during the first week after birth rely on

the expression of b2-nAChRs by starburst amacrine cells (Feller

et al., 1996; Zhou, 1998), and initial reports suggested that b2�/�

mice completely lack retinal waves (Bansal et al., 2000;

McLaughlin et al., 2003). These findings, along with the relative

ease and reproducibility of the b2�/� model in comparison to

chronic pharmacological injections, resulted in a host of reports

broadly linking retinal waves and b2�/� mice to altered retino-

topy, eye-specific segregation, visuotopic map alignment, and

functional deficits throughout the visual system (McLaughlin

et al., 2003; Rossi et al., 2001; Chandrasekaran et al., 2005;

Mrsic-Flogel et al., 2005; Triplett et al., 2009; Grubb and Thomp-

son, 2004; Cang et al., 2005; Stafford et al., 2009; Shah and

Crair, 2008; Wang et al., 2009).

However, subsequent reports revealed that retinal waves

actually persisted in b2�/� mice under some recording condi-

tions, perhaps even with greater frequency, though their spatio-

temporal properties were usually altered (Sun et al., 2008a; Sun

et al., 2008b; Stafford et al., 2009). This suggests that disrupted

1050 Neuron 84, 1049–1064, December 3, 2014 ª2014 Elsevier Inc.

spatiotemporal patterns of waves, and not a decrease in wave

frequency, are the likely cause of circuit refinement defects in

b2�/� mice (Godfrey et al., 2009; Stafford et al., 2009). Further

complicating matters, experiments employing b2�/� mice suffer

from the caveats typical of whole-animal knockouts, since b2-

nAChRs are normally expressed throughout the brain and

body, and glutamatergic synapse development is disrupted in

the CNS of b2�/� mice (Shah and Crair, 2008; Lozada et al.,

2012) completely apart from their likely retinal wave deficits.

These and other experimental ambiguities cast some doubt on

the existence, form, and necessity of retinal waves for visual cir-

cuit development (Chalupa, 2009; Feller, 2009).

We report here on experiments examining in vivo the causal

link between retinal waves and visual circuit refinement using

a combination of imaging, anatomical circuit tracing, and phar-

macological manipulations in b2�/� and novel conditional b2-

nAChRs mutant mice. Our in vivo recordings demonstrate that

in whole-animal b2�/� mice there is a marked decrease in

patterned spontaneous retinal activity and dramatically disrup-

ted waves. We also observe surprisingly strong correlations in

spontaneous activity between the eyes and, remarkably, a de-

correlation of presynaptic (retinal) and postsynaptic (collicular)

activity in b2�/� mice. Using conditional b2-nAChRs mutant

mice, we further show that retinal, but not collicular, b2-nAChRs

are necessary for both retinal waves and visual circuit refine-

ment. Moreover, deletion of b2-nAChRs from restricted zones

in the retina eliminates retinal waves and disrupts visual circuit

refinement, but regions of the same retina with maintained

expression of b2-nAChRs continue to wave and exhibit normal

circuit refinement. Finally, we demonstrate that chronic pharma-

cological rescue of retinal wave frequency partially restores

visual circuit development in b2�/� mice. Taken together, these

results substantiate a strong causal link between b2-nAChRs in

the retina, spontaneous retinal waves, and visual circuit refine-

ment in the developing lateral geniculate nucleus (dLGN) and

superior colliculus (SC) of mice.

RESULTS

Retinal Ganglion Cell Spontaneous Activity in b2–/– MiceWeexamined spontaneous activity in retinal ganglion cells in vivo

during the first postnatal week (P3-P7) by loading retinal ganglion

cells (RGCs) with an exogenous (Calcium Green-1 Dextran) or

genetically encoded (GCaMP3/6) fluorescent calcium indicator.

Retinal waves were imaged in topographically mapped RGC

axon arbors in the SC (Figure 1). This approach reveals frequent

waves of propagating spontaneous activity that gradually en-

compasses much of the SC (Ackman et al., 2012) (Figures 1A–

1C). In mice lacking the b2 subunit of the nicotinic acetylcholine

receptor (Chrnb2) throughout the brain and body (b2�/� mice),

correlated RGC activity was typically present, but much less

frequent than in control (WT and b2+/�) mice and with noticeably

altered spatiotemporal properties (1.00 ± 0.06 waves/min in n =

27 WT SC hemispheres; 0.34 ± 0.05 waves/min in n = 56 b2�/�

hemispheres; 1.23 ± 0.08 waves/min in n = 8 b2+/� hemispheres;

mean ± SEM; p < 0.001 for the difference between WT and

b2�/�; see Movie S1 available online; Figures 1D and 2). The fre-

quency of spontaneous activity in b2�/� mice was particularly

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Figure 1. Infrequent Spontaneous Retinal Activity In Vivo in b2–/– Mice

(A) Retinal wave-imaging schematic (top) and retinotopy cartoon (bottom). Retinal waves are imaged in retinal ganglion cell axon arbors in the upper layers of the

SC, which are topographically mapped between the retina and SC (bottom). Retinal waves are imaged in RGC axon arbors in vivo through a craniotomy over the

SC. Calcium activity in RGC axons in the SC is measured either through direct injection of Calcium Green-1 Dextran into the retina or through the retina-specific

expression of GCaMP3.

(B) Example single-wave montages from P3 WT mouse (top) and P3 b2�/� mouse (bottom). Grayscale images on left show craniotomy over right and left

hemispheres of SC. All movies are acquired at 5–10 Hz. Movie frames shown in montage at 2 s intervals (top) or 1 s intervals (bottom); scale bar, 200 mm. White

arrows show onset and propagation of retinal wave front. Clear propagating retinal wave front is typical in WT mice (top) but difficult to discern in b2�/� mice

(bottom).

(C) Example raster plots of 5min recordings from P3WT (left) and b2�/� (right) mice. Each row in the raster corresponds to one 103 10 mm region of interest in the

indicated hemisphere.

(D) Spontaneous retinal activity is much less frequent in b2�/� mice than WT or b2+/� littermate controls (for this and all subsequent figures, box plots are

aggregated by SC hemisphere, box edges are first and third quartile, whiskers are 1.5 times the interquartile range, midline is median value, *p < 0.05, **p < 0.01,

***p < 0.001).

(E) Spontaneous activity by genotype and age during the first postnatal week. Coordinated activity is much less frequent in b2�/�mice than in littermate controls.

(F) Waves are much more likely to occur at the same time between the two retinas in b2�/� mice relative to littermate controls irrespective of wave frequency

(results of frequency-correcting Monte Carlo analysis described in text; Figure S1). Plot shows cumulative probability distribution of the interretina wave interval

(time interval between a wave in one retina compared to the other retina); inset box plot shows median. See also Figure S1.

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Neuron 84, 1049–1064, December 3, 2014 ª2014 Elsevier Inc. 1051

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β2-/- Retinal Wave Figure 2. Altered Spontaneous Retinal

Activity in b2–/– Mice

(A) Example single retinal wave montage from a

b2�/� mouse (scale bar, 200 mm; time interval, 1 s;

age, P6).

(B) Analysis of wave duration (in seconds) in WT,

b2�/�, and b2+/�mice reveals that waves aremuch

more brief in b2�/� relative to controls.

(C) Wave speed (mm/s) is also much faster in b2�/�

relative to controls.

(D) RGC activity is correlated over a much larger

area of the retina in b2�/� mice relative to controls.

This is quantified by comparing the size of the

wave at its peak relative to the overall spatial extent

of the wave throughout its duration (see Supple-

mental Experimental Procedures).

(E) Wave directionality plots in b2�/� andWTmice,

binned by overall wave duration. b2�/� retinal

waves show greater directional preference than

WT waves and are strongly biased in the anterior-

posterior direction.

(F) Quantification of wave direction bias index

(see Experimental Procedures) shows that waves

in b2�/� mice have a stronger direction bias than

littermate controls. See also Figure S2.

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low at the beginning of the first week after birth (Figure 1E), when

visual circuit refinement is most robust (Dhande et al., 2011), but

then gradually increased to levels that were indistinguishable

from control wave frequency 1week after birth (P7). Remarkably,

retinal spontaneous activity in b2�/� mice was notably more

correlated between the eyes than in control mice (Figures 1C

and 1F), as measured by comparing the time difference between

waves in the two hemispheres (interretina wave interval =

15.6 s ± 12.1 in n = 16 WT mice; 3.7 s ± 36.0 in n = 31 b2�/�

mice; 12.3 s ± 8.4 in n = 8 b2+/�mice; median ± median absolute

deviation; p < 0.001 for K.S. test between probability distribu-

tions in WT and b2�/� mice; Figure 1F; Supplemental Experi-

mental Procedures). A Monte-Carlo analysis of interhemispheric

wave interval confirmed that b2�/� mice were much more likely

to have significantly coincident waves in the two eyes than litter-

mate control mice (28/38 10 min epochs significantly correlated

in b2�/�; 1/16 epochs were correlated in WT; 0/6 epochs corre-

lated in b2+/� mice; see Figure S1 and Supplemental Experi-

mental Procedures). This surprising result may contribute to

the disruption of eye-specific segregation observed in b2�/�

mice, as computational (Butts et al., 2007) and experimental

(Zhang et al., 2012) results suggest that random between-eye

wave intervals encourage the refinement of these circuits.

In addition to a frank reduction in retinal wave frequency (Fig-

ure 1), a wide variety of spontaneous activity properties differed

between b2�/� and control mice (Figure 2; Table 1). In general,


correlated spontaneous activity in RGCs

of b2�/� mice was brief, of low amplitude,

and lacked a well-defined wave front in

comparison to littermate control mice

(Figures 2B, 2C, and S2). Retinal wave

size was also larger in b2�/� mice relative

to WT mice (Table 1; Figure S2) and pro-

duced much larger correlations across the retina relative to con-

trols (area of wave at peak/total wave area = 0.32 ± 0.09 in n = 16

WT hemispheres; 0.51 ± 0.10 in n = 31 b2�/� hemispheres; p <

0.001; Figure 2D; see Supplemental Experimental Procedures).

The brief, large, and correlated activity observed in b2�/� mice

was largely eliminated following enucleation or application of

the Gap junction antagonist meclofenamic acid (4–20 mM) to

the eye (Figure S2), consistent with in vitro experiments in peri-

natal WT and b2�/� mice (Sun et al., 2008b; Blankenship et al.,

2011; Stacy et al., 2005). It was previously reported (Stafford

et al., 2009) using in vitro recordings that retinal waves in b2�/�

mice lacked the directional bias that is observed in wild-type

mice both in vitro (Stafford et al., 2009) and in vivo (Ackman

et al., 2012). Interestingly, we observed that retinal waves in vivo

showed an even greater directional bias in b2�/�mice than inWT

mice (direction bias index 0.31 ± 0.04 in n = 16WT hemispheres;

0.61 ± 0.05 in n = 29 b2�/� hemispheres; 0.35 ± 0.05 in n = 8 b2+/�

hemispheres; mean ± SEM; p < 0.001 for difference between

b2�/� and either WT or b2+/� mice; Figures 2E and 2F). Overall,

these experiments show that correlated retinal activity is much

less frequent in b2�/� mice but partially recovers by the end of

the first postnatal week. At the same time, many properties of

the residual spontaneous retinal activity in b2�/� mice are

dramatically different fromcontrolmice,with the fast, brief, direc-

tionally biased, and more correlated waves in b2�/� mice poten-

tially all contributing to disruptions in visual circuit refinement.

Table 1. Values and Statistics for Wave Properties across Genotypes and Conditions

Measurement WT b2�/� b2+/� En1-Cre+; b2fl/� Pax6a-Cre+; b2fl/+ Pax6a-Cre+; b2fl/� b2�/� + cAMP

Retinal wave frequency

(waves/min)

1.00 ± 0.06 (n = 27) 0.34 ± 0.05 (n = 56)*** 1.23 ± 0.08 (n = 8) 1.14 ± 0.07 (n = 3) 1.01 ± 0.15 (n = 8) 1.15 ± 0.12 (n = 5)$$$

SC wave frequency

(waves/min)

1.21 ± 0.06 (n = 16) 3.28 ± 0.36 (n = 22)***,### 1.32 ± 0.06 (n = 2) 1.12 ± 0.06 (n = 5) 0.92 ± 0.11 (n = 7) 0.74 ± 0.12 (n = 7) 4.78 ± 1.18 (n = 4)***

Retinal wave speed

(mm/s)

83.2 ± 7.4 (n = 16) 180 ± 10.9 (n = 31)*** 78.7 ± 8.0 (n = 8) 86.2 ± 10.6 (n = 3) 78.6 ± 7.6 (n = 8) 207.1 ± 34.6 (n = 5)***

SC wave speed

(mm/s)

67.3 ± 7.6 (n = 16) 259 ± 13 (n = 22)***,### 66.1 ± 2.2 (n = 2) 59.8 ± 5.9 (n = 5) 67.5 ± 11.6 (n = 7) 96.7 ± 11.3 (n = 7) 254.8 ± 34.1 (n = 4)***

Retinal wave

duration (s)

20.4 ± 1.7 (n = 16) 9.6 ± 0.5 (n = 31)*** 21.3 ± 1.6 (n = 8) 23.3 ± 1.5 (n = 3) 11.4 ± 1.3 (n = 8)* 11.2 ± 0.9 (n = 5)***

SC wave duration (s) 27.7 ± 1.6 (n = 16)### 4.9 ± 0.4 (n = 22)***,## 21.7 ± 0.1 (n = 2) 26.8 ± 2.7 (n = 5) 22.6 ± 3.4 (n = 7) 12.1 ± 0.9 (n = 7)** 5.8 ± 0.4 (n = 4)***

Retinal wave size

(mm2)

0.62 ± 0.08 (n = 16) 1.08 ± 0.07 (n = 31)*** 0.99 ± 0.08 (n = 8) 0.84 ± 0.03 (n = 3) 0.38 ± 0.04 (n = 8)* 1.23 ± 0.1 (n = 5)**

SC wave size (mm2) 0.93 ± 0.05 (n = 16)# 0.45 ± 0.02 (n = 22)***,### 0.96 ± 0.07 (n = 2) 1.06 ± 0.09 (n = 5) 0.64 ± 0.12 (n = 7) 0.22 ± 0.07 (n = 7)** 0.77 ± 0.11 (n = 4)***,$$

Wave direction bias

index

0.31 ± 0.04 (n = 16) 0.61 ± 0.05 (n = 29)*** 0.35 ± 0.05 (n = 8) 0.35 ± 0.06 (n = 13) 0.26 ± 0.02 (n = 11) 0.50 ± 0.13 (n = 5)

Retinal ROI wave

frequency (waves/min)

0.69 ± 0.09 (n = 16) 0.39 ± 0.05 (n = 32)** 0.94 ± 0.1 (n = 8)

Retinal ROI interwave

interval (s)

105 ± 13.4 (n = 16) 169 ± 17.1 (n = 32)* 68.3 ± 6.9 (n = 8)

Retinal ROI wave

duration (s)

0.99 ± 0.04 (n = 16) 1.02 ± 0.02 (n = 32) 1.14 ± 0.07 (n = 8)*

Retinal ROI wave

amplitude (DF/F)

0.032 ± 0.002 (n = 16) 0.021 ± 0.001 (n = 32)*** 0.024 ± 0.001(n = 8)*


(waves/min) P3

1.00 ± 0.14 (n = 6) 0.27 ± 0.02 (n = 4)** 1.48 ± 0.13 (n = 2)


(waves/min) P4

0.84 ± 0.14 (n = 6) 0.19 ± 0.05 (n = 11)** 1.12 ± 0.02 (n = 2)


(waves/min) P5

0.99 ± 0.12 (n = 6) 0.18 ± 0.04 (n = 15)***


(waves/min) P6

1.09 ± 0.09 (n = 5) 0.33 ± 0.07 (n = 17)*** 1.30 ± 0.10 (n = 2)


(waves/min) P7

1.14 ± 0.07 (n = 4) 0.81 ± 0.17 (n = 9) 1.00 ± 0.10 (n = 2)

Measurement descriptions for each row are located in the first column, and the genotypes and conditions follow to the right. Measurements are aggregated by hemisphere and reported asmeans

(± SEM). Statistical significance was determined by using one- or two-way analysis of variance (ANOVA) along with Tukey’s honest significant difference (HSD) post hoc test for reported p values.

Asterisks denote significant difference from the most comparable wild-type measurement. Pound signs denote significant difference between most comparable retinal and SC wave recordings

(pre- versus postsynaptic wave differences). Dollar signs denote significant difference in b2�/� wave properties after retinal injection of CPT-cAMP.

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VisualM

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Figure 3. Spontaneous Activity in the SC Is Largely Independent of Retinal Activity in b2–/– Mice

(A) Neuronal activity is measured in SC neurons by crossing En1-Cre to floxed GCaMP3 mice or loading OGB1AM-dye into the SC.

(B and C) (B) Example SC wave activity montages in WT (top, P7) and b2�/� (bottom, P3) mice. Activity in WTmice (top) has a clear propagating wave front (white

arrows). SC activity in b2�/� mice displays two characteristic patterns, infrequent ‘‘large’’ waves (bottom panel, one wave in each hemisphere) that are similar to

retinal activity in b2�/�mice (Figure 1) and (C)more-frequent, highly stereotyped and bilaterally correlated ‘‘small’’ waves that propagate rapidly in the anterior and

medial SC (P3-4). Scale, 200 mm.

(D) Example raster plots of SC activity in WT (top, P3) and b2�/� mice (bottom, P3).

(E) Summary quantification of retinal (Ret) and SC wave frequency in WT and b2�/� mice. Waves measured in SC neurons in b2�/� mice are much more frequent

than retinal waves in b2�/� mice or waves in WT mice.

(F) There is a strong and similar correspondence betweenwave size and duration in both RGCs and SC neurons inWTmice (WT retinal wave size versus duration,

r = 0.71, n = 390 waves; WT SC wave size versus duration, r = 0.63, n = 298 waves, p < 0.001).

(G) Retinal wave size and duration are unrelated in b2�/�mice (r = 0.23, n = 338 waves), but SCwave size and duration remain correlated (r = 0.62, n = 878 waves,

p < 0.001), leaving spontaneous activity totally mismatched between RGCs and SC neurons in b2�/� mice. See also Figure S3.

Neuron


Activity in SCNeuronsDoesNotMatchRetinal Activity inb2–/– MiceSpontaneous retinal waves are thought to guide the refinement

of developing neural circuits through a Hebb-based mechanism

that relies on the correlation between presynaptic retinal gan-

glion cell activity and postsynaptic neuronal activity. In WT

mice, retinal waves produce corresponding waves of activity in

postsynaptic neurons (Ackman et al., 2012). To determine

whether these correlations persist in b2�/� mice in light of the


disrupted retinal wave activity, we examined spontaneous activ-

ity in SC neurons using exogenous application of a calcium

fluorophore (OGB1AM) and/or genetically encoded calcium indi-

cators expressed in SC neurons but not the retina (En1-Cre X

floxed-GCaMP3; Figure 3). In WT mice, the spatial and temporal

properties of spontaneous activity was very similar in recordings

of presynaptic retinal afferents and postsynaptic neurons in the

SC (Figures 3B and 3G; Movie S2), consistent with a dominant

role for retinal waves in driving postsynaptic activity in the SC

Neuron


before eye opening (Ackman et al., 2012). Remarkably, in b2�/�

mice there was a dramatic difference in presynaptic activity re-

corded in retinal afferents and postsynaptic activity observed

in SC neurons. In the SC of b2�/� mice, in addition to larger

and faster but infrequent waves, as found in retinal afferents of

b2�/� mice (Figures 2 and 3B), we observed a new, distinct

form of spontaneous activity that was frequent, very rapid, and

largely confined to the midline between the two hemispheres

(Movie S2; Figures 3C and S3). This unexpected ‘‘extra’’ pattern

of spontaneous activity accounted for the vast majority of the

postsynaptic activity in the SC of b2�/� mice and dramatically

increased the overall frequency of spontaneous activity to levels

well above that observed even in WT SC (Figures 3D and 3E). As

with activity recorded from retinal ganglion cell afferents, most

spontaneous activity parameters recorded from SC neurons

were significantly different in b2�/� mice relative to WT mice.

This included wave size, duration, and speed (Figures 3F and

S3; Table 1), with all of the parameters consistent with the

appearance in b2�/� SC neurons of very small, transient, and

fast midline waves, which were not typically observed in WT

mice (Figure S3). For example, inWTmice, there was a clear cor-

respondence between wave size and wave duration, with

smaller waves being of shorter duration and larger waves having

longer duration in both presynaptic RGC afferents and postsyn-

aptic SC neurons (Figure 3F). In sharp contrast, in b2�/� mice a

significant correlation between wave size and duration was lack-

ing for RGC activity (Figure 3G), while in b2�/� SC neurons the

appearance of an extra class of very small and brief waves

restored the correspondence between wave size and duration

and completely disrupted the match between presynaptic RGC

activity and postsynaptic SC waves found in WT recordings.

In summary, these data indicate that coordinated sponta-

neous retinal activity is dramatically reduced in frequency in

b2�/� mice, and that the properties of the remaining sponta-

neous activity are fundamentally altered. In the SC, an apparently

new form of spontaneous activity appears in b2�/� mice that is

markedly different in form from both WT waves and the altered

presynaptic activity observed in b2�/� RGCs, potentially due to

homeostatic pressures in the absence of coordinated sponta-

neous drive from the retina.

b2-nAChRs in the SC Are Not Required for Normal WaveActivityb2-nAChRs are expressed throughout the developing nervous

system and are known to contribute to glutamatergic synapse

development in the CNS (Lozada et al., 2012). The very unusual

properties of spontaneous retinal waves and the mismatched

activity in developing SC neurons in whole-animal b2�/� mice

in vivo create some ambiguity about the role of retinal waves

and the specific contribution of b2-nAChRs in the retina or SC

to visual map development. To resolve this ambiguity, we gener-

ated mice with loxP sites flanking exon 5 of Chrnb2 (Figures 4A

and 4B), the largest exon of the b2-nAChR gene encoding half of

the N-terminal ligand binding domain and three of four trans-

membrane domains. We confirmed that germline deletion of

exon 5 produced functional null receptors in the retina (Figure 4C)

and recapitulated visual system anatomical phenotypes previ-

ously reported in whole-animal b2�/� mice (Figure S4). We

N

then crossed floxed b2-nAChR mice to Cre recombinase lines

that are specific to either midbrain postsynaptic (En1-Cre+;

b2fl/� or SCb2-cKO mice, Figure 4D) or retinal presynaptic

(Pax6a-Cre+;b2fl/� or Retb2-cKOmice, Figures 5 and 6) cell pop-

ulations. Crossing midbrain-specific En1-Cre mice to floxed b2-

nAChR mice to generate SCb2-cKO mice completely eliminated

b2-nAChR mRNA expression in the SC, but retinal mRNA levels

were unchanged (Figure 4E). Wave properties in SC neurons

in vivo in the SCb2-cKO mice were indistinguishable from WT

mice (Figure 4F), with normal frequency, duration, and speed,

but were significantly different from b2 whole-animal knockout

(b2�/�) waves (Figure S5). Anatomical analysis of retinocollicular

projections using focal retinal injections of DiI in the retina (to

assess retinotopic map formation in the SC) or whole-eye injec-

tions of CTB-Alexa 488 and 555 (to study eye-specific segrega-

tion in the SC) at P6 revealed that retinotopic mapping and

eye-specific segregation in SCb2-cKO mice were indistinguish-

able from control mice at postnatal day 8 (Figures 4G, 4H, and

S5). These experiments demonstrate that b2-nAChRs in SC

neurons have no effect on spontaneous activity patterns or early

visual circuit development.

b2-nAChRs in the Retina Are Required for Normal WaveActivityTo examine the role of retinal b2-nAChRs on retinal wave propa-

gation and visual circuit development, we generated retina-spe-

cific b2-nAChRs mutants by crossing Pax6a-Cre mice with

floxed-b2 mice (Pax6a-Cre+;b2fl/� or Retb2-cKO mice). Cre re-

combinase expression inPax6a-Cremice is high in theperipheral

retina, but weak in the central retina along a dorsal-ventral strip

(Marquardt et al., 2001; Figure 5A). This delimits the retina into

three broad subregions, with RGCs in the temporal and nasal

retina, which project to the rostral and caudal SC, expressing

Cre recombinase, while RGCs in the central retina, which project

to a medial-lateral strip in the middle of the SC, were largely

spared of Cre recombinase activity and serve as an internal con-

trol (Figure 5A). The clear borders between Cre-expressing sub-

regions of the retina permit the identification of regions of the SC

that lackb2-nAChRexpressingRGCs (rostral andcaudal SC) and

control (mid-SC) RGC axon-projecting regions that maintain

b2-nAChR expression (Figure 5A, bottom). Remarkably, wave

activity in Retb2-cKO mice observed in both RGC axons and

SC neurons was limited to the medial-lateral strip in the SC con-

taining RGCs with maintained b2-nAChR expression (Figures 5D

and 5E), and waves neither nucleated nor propagated into the

knockout regions of the retina (Movie S3). A quantitative analysis

of wave frequency in the rostral and caudal SC shows almost no

wave activity in these regions in Retb2-cKOmice, while wave ac-

tivity in the medial SC was unchanged relative to control mice in

Retb2-cKOmice (Figure 5F; Table 1). Interestingly, thewaves that

persisted in Retb2-cKO mice were spatiotemporally similar to

waves observed in control littermate (Pax6a-Cre+;b2fl/+) and

WTmice (Figures 5G–5I andS6; Table 1),with aclear propagating

wave front and wave speeds that were comparable to WT and

control littermate waves (Figure 5G; Table 1). Wave size was

significantly smaller in Retb2-cKO than littermate control, b2�/�

and WT mice (Figure 5H; Table 1), and wave duration was

much shorter than inWT and littermate control mice (Figure S6A;


A

B C

D

E

F

G

H

Figure 4. Conditional Deletion of b2-

nAChRs from the SC Has No Effect on

Wave Activity, Retinotopy, or Eye-Specific

Segregation

(A) Design of floxed b2-nAChR mice (also see

Experimental Procedures and Figure S4).

(B) Southern blot showing WT, b2�/�, and floxed-

b2 (b2fl/fl) bands (WT = 8.0 kB; b2�/� = 5.8 kB;

b2fl/fl = 4.7 kB).

(C) Knockout of b2-nAChR subunit eliminates

response to a nicotinic acetylcholine receptor

agonist (DMPP) in starburst amacrine cells in the

retina of a P5 mouse, 1 mM, 300 ms puff.

(D) Expression of En1-Cre, visualized through lacZ

or GFP reporter mice, at embryonic and early

postnatal ages. En1-Cre was used to delete b2-

nAChR subunits from the midbrain, but not the

retina, of b2fl/� mice.

(E) Reverse transcription PCR reveals that mRNA

for b2-nAChR is absent from the SC of En1-

Cre+;b2fl/� (SCb2-cKO) mice but is spared in the

retina at P6-7.

(F) Example montage of SC wave activity in SCb2-

cKOmice (P5 example). Waves in SCb2-cKOmice

are indistinguishable from waves in WT mice in

both frequency and spatiotemporal properties

(see Figure S4 and text).

(G) Retinotopy is unaltered in SCb2-cKO mice.

Shown are example target zones (yellow spots) in

the SC (white dotted outlines) following focal in-

jections of DiI into the ventral-temporal (left-top) or

dorsal (left-bottom) retina. Summary quantification

of target zone size shown on the right for ventral-

temporal retinal injections (right, top) and dorsal

injections (right, bottom). Scale, 500 mm.

(H) Eye-specific segregation is unaltered in SCb2-

cKO mice. Example ipsi (top, grayscale) and

contra (green) with ipsi (red) projections together

(bottom) in sagittal sections through the SC.

Summary quantification of eye segregation (right),

measured as the fraction of SGS layer (dotted

outline) with ipsilateral label. Scale, 500 mm. See

also Figures S4 and S5.

Neuron


Table 1), presumably because wave propagation was truncated

upon entering the null region of the retina in Retb2-cKO mice.

Wave direction bias in Retb2-cKO was also indistinguishable


from WT and littermate control mice, and

significantly less than the bias in b2�/�

mice (Figure 5I; Table 1). These data

demonstrate that regions of the retina

that lack expression of b2-nAChRs also

lack wave activity, while normal propa-

gating retinal waves persist, in the

same retinas, where b2-nAChR expres-

sion persists.

We next used anatomical techniques to

examine visual circuit development in the

Retb2-cKO mice. To examine retinotopic

map development, we made targeted

focal injections ofDiI around the periphery

of the retina (Figure 6A). InRetb2-cKOmice, projections to theSC

and dLGN from dorsal RGCs, which lack Cre recombination and

maintain normal retinal wave activity, were indistinguishable from

A B C

D E

F G H I

Figure 5. Conditional Deletion of b2-nAChRs from the Retina Eliminates Waves in the Retina and SC

(A) Region-specific expression of Pax6a-Cre in the retina (GFP reporter, top panel; cartoon, middle panel). Cre is expressed in the peripheral two-thirds of

the retina in Pax6a-Cre+ mice, and GFP fluorescence in the SC (bottom panel) shows corresponding Pax6a-Cre+ (anterior and posterior) and Pax6a-Cre�

(medial-lateral strip) RGC axon projection regions in the SC.

(B) Example retinal wave montage from Pax6a-Cre+;b2-nAChRfl/+ control (heterozygous) mouse (P7).

(C) Map of average activity in this control example reveals that waves extend throughout the SC.

(D) Example retinal wave montage from a Retb2-cKO (Pax6a-Cre+;b2-nAChRfl/�) mouse (P5).

(E) Map of average activity shows that retinal waves in Retb2-cKOmice are confined to regions of the retina lacking Cre expression (Pax6a-Cre�). Scale, 200 mm.

Retinal wave activity in Retb2-cKO mice shows a sharp border at the posterior edge of the Cre� region, which is absent in control (Pax6a-Cre+;b2-nAChRfl/+)

littermate mice. Note that the cortex progressively covers the anterior-lateral aspect of the SC during the first week after birth.

(F) Regional analysis of wave frequency in Retb2-cKO and littermate control mice demonstrates that wave activity is restricted to the Cre� region in Retb2-cKO

mice, but is spread evenly in littermate control mice.

(G) Wave speed in Retb2-cKO mice is similar to WT and littermate control mice and much slower than b2�/� mice.

(H) Wave size is smaller in Retb2-cKO (Pax6a-Cre+;b2-nAChRfl/�) relative to WT, b2�/�, and littermate control mice.

(I) Wave direction bias index in Retb2-cKO (Pax6a-Cre+;b2-nAChRfl/�) mice is indistinguishable fromWT and littermate control mice (Pax6a-Cre+;b2-nAChRfl/+),

but b2�/� waves are more directionally biased than any other group. See also Table 1 and Figure S6.

Neuron


littermate control mice at P8 (Figures 6B and S6C). In stark

contrast, RGC projections from ventral-temporal and ventral-

nasal retina in Retb2-cKO mice, with robust Cre recombination

and no retinal wave activity, were dramatically disrupted. Thus,

regions of the retina in Retb2-cKO with normal retinal waves

N

maintained normal retinotopic projections, while retinotopic

refinement in regions of the retina without retinal waves was pro-

foundly disrupted (Figures 6A and 6B). These experiments pro-

vide a strong causal link between retinal wave activity and the

development of normal retinotopic projections.


A C

B

D

Figure 6. Conditional Deletion of b2-nAChRs from the Retina Disrupts Circuit Development in a Region-Specific Manner

(A) Cartoon illustrating region-specific retinotopic mapping defects in WT, b2�/�, and Retb2-cKO mice.

(B) Retb2-cKO retinotopy is specifically disrupted in regions of the SC (ventral-nasal, left panels; ventral-temporal, right panels) that receive projections from

RGCs that lack b2-nAChR subunit expression and waves at P8. Retinotopy of RGCs from the dorsal retina in Retb2-cKOmice, which maintain expression of b2-

nAChRs and retinal waves, is unperturbed relative to littermate controls (middle panels). Bottom row of histograms shows summary quantification of retinotopic

defects in Retb2-cKO relative to littermate control mice for ventral-temporal (left), dorsal (middle), and ventral-nasal (right) retinocollicular projections.

(C) Eye-specific segregation is disrupted in the SC of Retb2-cKO mice relative to littermate control mice at P8. Grayscale panels show ipsilateral projections at

twomagnifications, and color panels show ipsi (red) and contra (green) projections (scale, 500 mm). Summary quantification of eye segregation in the SC shown in

histograms on right.

(D) Eye-specific segregation is also disrupted in in the dLGN of Retb2-cKO mice relative to littermate control mice at P8. Ipsilateral projection shown in red and

contralateral in green. Overlap of the two eye projections in the dLGN is shown in white (scale, 500 mm). Histograms on right show summary quantification of eye

segregation in the dLGN. In all histograms, Retb2-cKO are shown in red; littermate controls are shown in black and green.

Neuron


We next examined the emergence of eye-specific segregation

in the SC and dLGN of Retb2-cKO and littermate control mice.

As with the development of retinotopy, eye-specific segregation

in the SC was disrupted in Retb2-cKO mice in comparison to all

littermate control mice at P8 (Figure 6C). Moreover, eye-specific

segregation in the dLGN, like in the SC, was significantly worse in

Retb2-cKO mice than in littermate controls (Figure 6D). These

experiments provide definitive support of a necessary role for


retinal waves in the emergence of both retinotopy and eye-spe-

cific segregation in the developing SC and dLGN.

Rescuing Retinal Waves Restores Eye-SpecificSegregation in b2–/– MiceOur data suggest that the loss of spontaneous retinal activity

leads to axon refinement and neural circuit defects in whole-

animal (b2�/�) and retina-specific (Retb2-cKO) b2-nAChR

A

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WT saline β2-/- saline β2-/- cAMP

dLGN

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β2-/-

ipsi (cAMP)

ipsi (saline)SO

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P4 β2-/-

0 100 200 300

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3000 100 200Time (s)

RH

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*****

6 55

Figure 7. Eye-Specific Segregation Is

Partially Rescued after Increasing Fre-

quency of Retinal Spontaneous Activity in

b2–/– Mice

(A) Experimental schematic. Activity recordings are

made in the SC of whole-animal b2�/� mice before

and after double-eye injection of CPT-cAMP

(2 mM, �500 nL).

(B) Example wave activity raster plot from 5 min

recording before (top) and after (bottom) double

eye CPT-cAMP injection. Wave frequency is

increased in the SC contralateral to the eye that

receives CPT-cAMP.

(C) Summary of effects of CPT-cAMP on wave

frequency in b2�/� mice. Application of CPT-cAMP

to b2�/� mice restores wave frequency to levels

comparable toWTmice, which is much higher than

in b2�/� mice.

(D) Wave speed in b2�/� CPT-cAMP-treated ret-

inas is no different from wave speed in b2�/� mice

without CPT-cAMP.

(E) Wave duration in b2�/� CPT-cAMP-treated

retinas is no different from wave duration in b2�/�

mice without CPT-cAMP.

(F and G) (F) Eye-specific segregation is partially

rescued at P8 in the SC and dLGN (G) of b2�/�mice

after 5 days of binocular CPT-cAMP treatment

(from P2-P6).

(H) In contrast, retinotopic mapping in the SC is

unaffected in bothWT and b2�/�mice after chronic

CPT-cAMP treatment at P8 (scale, 500 mm). See

also Figure S7.

Neuron


conditional knockout mice. To directly test this hypothesis, we

attempted to ‘‘rescue’’ spontaneous retinal waves in b2�/�

mice and examine the effects on in vivo activity patterns and

RGC axon refinement. CPT-cAMP increases the frequency of

retinal waves in neonatal ferret in vitro (Stellwagen et al., 1999)

and improves eye-specific segregation in mice in vivo (Xu

et al., 2011a). Application of CPT-cAMP to the retina of b2�/�

mice dramatically increased the frequency of in vivo sponta-

neous retinal activity (Movie S4; Figures 7A–7C). Interestingly,

Neuron 84, 1049–1064, D

while the frequency of spontaneous retinal

activity in b2�/� mice following treatment

with CPT-cAMP increased to levels that

were indistinguishable from control mice

(p = 0.951; Figure 7C; Table 1), the waves

remained highly correlated between the

eyes (Figure S7), and the spatiotemporal

properties of the induced retinal activity

remained disrupted. In particular, retinal

wave speed, duration, and size (Figures

7D, 7E, and S7; Table 1) were similar

with and without CPT-cAMP treatment in

b2�/�mice, but were significantly different

from control mice. Chronic daily treatment

of b2�/� mice with CPT-cAMP in both

eyes (vitreal application of 2mM CPT-

cAMP from P2-P6) significantly improved

eye-specific segregation in both the SC

(Figure 7F) and dLGN (Figure 7G) in comparison to saline-treated

b2�/� mice at P8. However, retinotopic refinement was not

improved by CPT-cAMP treatment in either b2�/� mice or WT

mice (Figure 7H), suggesting that CPT-cAMP’s effect is not a

generalized consequence of actions on neurite outgrowth and

branching (Nicol et al., 2007) but is limited to eye-specific

segregation. These data provide strong evidence that a

threshold level of coordinated spontaneous retinal activity is

necessary for the emergence of eye-specific segregation, but

ecember 3, 2014 ª2014 Elsevier Inc. 1059

Neuron


appropriate spatiotemporal patterns of retinal waves are

required for retinotopic refinement.

DISCUSSION

Molecular and activity-dependent factors act together during

development to guide the formation of refined neural circuits.

Here, we describe experiments that examined the role of retinal

b2-nAChRs and spontaneous retinal waves in visual circuit

development. We focused our experiments on characterizing

the properties of spontaneous activity in the intact, unanesthe-

tized visual system in vivo, because previous in vitro experiments

produced conflicting results about the role of b2-nAChRs in

retinal wave generation (Bansal et al., 2000; McLaughlin et al.,

2003; Sun et al., 2008b; Stafford et al., 2009), and our own exper-

iments show that the spatiotemporal properties of retinal waves

differ to some extent in vivo and in vitro (Ackman et al., 2012). We

observed that the frequency of activity and the dynamics of

retinal waves were dramatically altered in b2�/� (whole animal

knockout) mice. Moreover, unlikeWTmice, spontaneous activity

in neurons in the developing SC no longer resembled retinal ac-

tivity in b2�/�mice, suggesting that neurons in the SC develop an

intrinsic pattern of activity in the absence of adequate retinal

drive.We also used conditional genetic deletion of the b2 subunit

to demonstrate that midbrain b2-nAChRs are unimportant for

retinal waves and anatomical circuit refinement, but that retinal

knockout of b2-nAChRs eliminated waves in both RGCs and

SC neurons and greatly disrupted visual circuits in a region-spe-

cific manner. We finally showed that boosting retinal wave fre-

quency in b2�/� mice using a pharmacological approach was

sufficient to partially rescue eye-specific segregation in both

the dLGN and SC, but had no effect on retinotopy. Overall, our

experiments underscore the strong causal link between retinal

waves and normal visual circuit development, specifically impli-

cate retinal b2-nAChRs in this process, and distinguish between

a role for overall retinal wave frequency and dynamics in distinct

aspects of visual circuit refinement in the developing dLGN and

SC of mice.

Spontaneous Activity In Vivo versus In VitroRetinal waves are a network property governed by cellular phys-

iology and circuit dynamics under broad neuromodulatory influ-

ence. In this context, it is perhaps not surprising that some

aspects of wave dynamics differ in vitro and in vivo. These differ-

ences may be exacerbated in mice (like the b2�/� mice) in which

the main neurotransmitter system responsible for the predomi-

nant form of network activity is mutated. Under in vitro condi-

tions, reports variously suggest that retinal waves in b2�/�

mice are either absent (Bansal et al., 2000; McLaughlin et al.,

2003), present at a much higher frequency (Sun et al., 2008b),

or present with a modestly reduced frequency (Stafford et al.,

2009). Our data clearly demonstrate that waves are much less

frequent in vivo in b2�/� mice than WT mice, which is consistent

with Stafford et al. (2009). However, when observed in vitro,

waves in b2�/�mice lack directional bias, but are of similar dura-

tion as waves in WT mice (Stafford et al., 2009). Neither of these

network properties was observed in vivo, as retinal waves in

b2�/� mice are much shorter in duration (Figure 2), and show


an even more pronounced directional bias than WT mice (Fig-

ure 2). Other b2�/� wave properties observed in vitro are similar

to what we observed in vivo, including wave dependence onGap

junctions (Sun et al., 2008b) and stronger correlations over a

larger area of the retina (Stafford et al., 2009; Sun et al.,

2008b). We also observed a remarkable increase in wave corre-

lations between the eyes in b2�/� mice, which obviously could

not be predicted from in vitro experiments. Finally, we note

that the somewhat-contradictory reports on the role of retinal

waves in visual circuit development (e.g., Penn et al., 1998; Hu-

berman et al., 2003; Sun et al., 2008b; Chalupa, 2009; Feller,

2009) rely in part on extrapolating in vitro analysis of retinal

network dynamics after pharmacologic or genetic manipulations

to the in vivo condition. Our data suggest that this inference is

potentially problematic, and highlight the importance of exam-

ining network properties of spontaneous activity in an intact,

in vivo condition.

Examining Retinal Waves in RGC Axon Arbors in the SCWe examined the spatiotemporal dynamics of retinal waves by

measuring activity in retinal axon arbors in the SC. Differences

in RGC axon arbors in b2�/� mice relative to WT mice (Dhande

et al., 2011) are unlikely to contribute significantly to altered

retinal wave dynamics observed in b2�/� mice for a number of

reasons. First, axon arbors in b2�/� mice are expanded relative

to WT mice at P8, but are indistinguishable from WT mice at P4

(Dhande et al., 2011), whereas we observed that retinal waves

are altered in b2�/� mice throughout the first week after birth.

Similarly, RGC axon arbors in WT mice refine significantly

throughout the first postnatal week (Dhande et al., 2011), but

wave dynamics are no different in WT mice at P3 than at P7.

Second, enlarged RGC axon arbors in b2�/� mice should have

no effect on retinal wave duration, whereas we observed spon-

taneous activity that was dramatically shorter in duration in

b2�/�mice relative to control mice. Third, the dynamics of retinal

waves (speed, size, directionality, etc.) in WT mice measured in

RGC axon terminals in the SC are very similar to the wave prop-

erties measured in SC neurons, suggesting that RGC axon

arbors do not significantly distort retinal waves. Finally, and

perhaps most tellingly, total wave size (area) is about 30 times

larger than the area of a given RGC axon arbor in both b2�/�

and WT mice (Dhande et al., 2011). Thus, wave size is governed

much more by the area of wave propagation in the retina than it

is by the size of a given RGC axon arbor in the SC. Similarly, the

speed of a propagating wave in the retina, as measured in

the SC, is not significantly altered by changes in the size of the

RGC axon arbor in the SC, since the axon arbor is so much

smaller (and the Ca2+ transients associated with the wave pass-

ing through much briefer) than the total area consumed by a

wave. Perhaps the only wave parameter that we expect may

be altered by having a larger axon arbor is the size of the

wave front as it propagates across the SC, and in fact it is often

difficult to even distinguish a clear wave front in b2�/� mice in

comparison to WT mice (Figure 1B). Overall, these various lines

of evidence suggest that the altered wave dynamics we

observed in RGC axon terminals in the SC of b2�/� mice reflect

genuine differences in spatiotemporal patterns of activity in the

retina.

Neuron


Why Are Visual Maps Disrupted in b2–/– Mice?Whole-animal b2�/� mice show a remarkably strong anatomical

and functional phenotype, and have become the most

commonly used ‘‘retinal wave mutants’’ for the study of activ-

ity-dependent visual circuit refinement (Kirkby et al., 2013;

Cang and Feldheim, 2013; Ackman and Crair, 2014). Despite

the severity of their visual circuit disruption and their popularity

as a model system, the specific cause of the various b2�/�

mutant phenotypes was unclear. Our experiments specifically

localize the cause of visual circuit and functional deficits in

b2�/�mice to disruption in the retina and retinal activity, but a pri-

ori we still imagine four distinct factors that may individually or

in combination explain the visual circuit phenotypes observed

in b2�/� mice. First, the dramatically reduced wave frequency,

especially early in the first week when RGC axon elaboration is

at its peak (Dhande et al., 2011), may disrupt circuit refinement.

Second, the dissociation between presynaptic (retinal) waves

and postsynaptic (SC neuron) activity may interfere with Hebbian

(‘‘fire together, wire together’’) mechanisms for circuit refine-

ment. Third, the infrequent coordinated spontaneous ‘‘wave’’

activity that persists in the retina of b2�/� mice has severely dis-

rupted spatiotemporal properties, which may negatively influ-

ence normal circuit refinement. Finally, the few retinal waves

that do persist in b2�/� mice are highly correlated between the

two retinas, which may also disrupt circuit refinement, particu-

larly with respect to eye-specific projections. Our experiments

using chronic CPT-cAMP application to boost retinal wave

frequency in b2�/� mice specifically implicate retinal wave

frequency for developmental disruptions in eye-specific

segregation (Figure 7). However, CPT-cAMP application did

not completely correct eye-specific segregation in b2�/� mice,

and retinotopic circuits remained totally disrupted, which sug-

gests that both a minimum level of spontaneous coordinated

wave activity as well as specific patterns of spontaneous activity

are together necessary for normal visual circuit refinement

(Zhang et al., 2012; Xu et al., 2011a; Godfrey et al., 2009; Butts

et al., 2007).

Changes in Spontaneous Activity in the SC of b2–/– MiceSpontaneous retinal waves in b2�/�mice are GAP junctionmedi-

ated, significantly reduced in frequency, and gradually recover to

near WT levels by the end of the first postnatal week after birth.

This suggests that compensatory mechanisms encourage the

persistence of coordinated activity in the retina, despite their

dramatically altered form relative to WT mice. There is normally

a strong correspondence between spontaneous (presynaptic)

retinal wave activity and (postsynaptic) SC neuron activity, while

in b2�/�mice there is amarked disconnect between retinal activ-

ity and collicular neuron activity. The patterned spontaneous

activity in the SC of b2�/� mice dissociated from retinal activity

is strikingly similar to the pronounced rhythmic spiking activity

observed in the dLGN of young enucleated ferrets (Weliky and

Katz, 1999), which may explain the preservation of structured

geniculocortical and corticocortical connections in enucleated

ferrets (Ruthazer and Stryker, 1996; Crowley and Katz, 1999).

The persistent correlated spontaneous activity in the dLGN of

enucleated ferrets relies on feedback projections from the visual

cortex (Weliky and Katz, 1999). Similarly, descending cortical

N

input to the SCmay be the source of some of the collicular activ-

ity in b2�/� mice. However, these projections normally don’t

emerge until P6-P8 (Triplett et al., 2009), while the unusual SC

activity is already present at P3 in b2�/� mice. Dynamic reorga-

nization on a network level may be a common phenomenon in

the early neonatal brain (Stacy et al., 2005; Kirkby and Feller,

2013), and could facilitate large-scale compensatory rewiring

in response to disruptions in neural activity resulting from a vari-

ety of neurodevelopmental insults and disorders (Ebert and

Greenberg, 2013).

Region-Specific EffectsRegions of the retina with persistent b2-nAChR expression (in

Retb2-cKO mice) maintain relatively normal wave frequency

and spatiotemporal properties both in the retina and target re-

gions of the SC (Figure 5), whereas b2-nAChR ‘‘null’’ regions of

the retina are nearly devoid of any spontaneous activity (but

see Stacy et al., 2005). Anatomical projections in Retb2-cKO

mice are indistinguishable from WT mice only in the regions of

the retina that maintain normal waves, strengthening the causal

link between retinal waves and normal visual circuit develop-

ment. It is notable that waves in the null part of the retina are

nearly absent, whereas in b2�/� mice waves are significantly

reduced in frequency but persist, albeit with quite abnormal

spatiotemporal dynamics. This suggests that compensatory

mechanisms keep track of activity across the entire (or large

swaths of the) retina, and maintain Gap junction-mediated

waves in b2�/� mice (Stacy et al., 2005; Kirkby and Feller,

2013) but are silent in b2-nAChR null regions of Retb2-cKOmice.

Bilateral Correlations in Retinal Activity in b2–/– MiceWe previously reported that WT mice exhibited unanticipated,

but weak, bilateral correlations in retinal wave activity (Ackman

et al., 2012). These bilateral correlations were dramatically

enhanced in b2�/� mice relative to control mice (Figure 2). Simi-

larly, retinal waves in WT mice exhibit a marked directional bias,

which is also enhanced in b2�/� mice. The directional bias may

be related to the tendency for waves to nucleate in the ventral-

temporal retina, which could be enhanced in b2�/� mice. The

origin of the bilaterally correlated activity also remains uncertain,

but we note that bilateral activity sometimes (though not always)

appeared to begin in one retina and then propagate with a small

temporal lag to the contralateral retina (e.g., Figure 3B). This sug-

gests that direct synaptic communication between the retinas in

neonatal mice may be the origin of some correlated bilateral ac-

tivity (Bunt and Lund, 1981; Ackman et al., 2012). b2�/� mice

may have more correlated binocular activity through enhanced

retino-retinal connections or by virtue of the larger waves

observed in b2�/� mice more frequently engaging retino-retinal

communication. Bilateral correlations in spontaneous activity

are unlikely to be the (sole) origin of disrupted visual circuit devel-

opment in b2�/�mice because CPT-cAMP application improved

eye-specific segregation by enhancing retinal wave frequency,

but spontaneous activity remained correlated between the two

eyes even after this pharmacological manipulation (Figure S7).

Finally, we note that the partial recovery of eye-specific segrega-

tion, but not retinotopy, in the SC and dLGN of b2�/� mice

treated with CPT-cAMPmice further suggests that the presence,


Neuron


frequency, and spatiotemporal pattern of retinal waves are all

important for normal visual circuit refinement, and that different

aspects of circuit development are dependent on different

features of spontaneous activity (Zhang et al., 2012; Xu et al.,

2011b).

ConclusionsIn summary, these experiments establish a firm causal link be-

tween nicotinic acetylcholine receptors, retinal waves, and visual

circuit development and demonstrate that spontaneous retinal

waves are necessary and instructive for circuit refinement in

the developing nervous system. They also validate the use of

b2�/� mice as a model for disrupted spontaneous activity during

development, thoughmultiple altered features of retinal waves in

these mice may contribute to circuit refinement defects. The

persistence of patterned spontaneous activity in retinorecipient

regions of the brain following the withdrawal of retinal input,

as was observed in b2�/� mice and enucleated ferrets (Weliky

and Katz, 1999), suggests that homeostatic pressures work

to preserve network activity and adapt to the loss of sensory

drive, thus generating structured neural circuits that reflect the

altered neonatal activity patterns. These results also suggest

that normal variations in spontaneous activity can lead to the

emergence of a range of functional circuits, and that disrupted

patterns of spontaneous activity early in development may play

an important role in the etiology of circuit-based neurodevelop-

mental disorders.

EXPERIMENTAL PROCEDURES

Animals

We created a line of floxed b2-nAChR mice in order to conditionally and spe-

cifically delete b2-nAChR2. b2-floxed mice were made following published

strategies (Xu et al., 2009; Xu et al., 2011b) using DNA from a BAC clone con-

taining large fragments of the Chrnb2 genomic DNA from the 129SvEv mouse

genome. loxP sites were inserted surrounding exon 5, the largest coding exon

(970 bp) of the gene, and conditional deletion after crossing to Cre recombi-

nase mice was confirmed using standard reverse transcriptase PCR (see

Supplemental Experimental Procedures; Figures 4–6 and S4). Littermate con-

trols blind to the genotype were used everywhere, except in Figure S5, where,

due to the difficulty of the experiments and breeding scheme, pregenotyping

was used to focus on En-Cre+;b2f/� mice.

In Vivo Calcium Imaging

Spontaneous activity was recorded from neonatal mice as has been described

in detail previously (Ackman et al., 2012; also see Supplemental Experimental

Procedures). Calcium dyes were introduced either through injection of exoge-

nous calcium dye (Calcium Green-1 Dextran, Invitrogen #C-6765, or Oregon

Green 488 Bapta-1 AM, Invitrogen #O-6807) into the retina/SC, or induction

of genetically encoded GCaMP through either viral injection in the retina

(GCaMP6, Penn Vector Core, AV-1-PV2833) or crossing of a floxed GCaMP3

mouse (Ai38, Jackson Laboratory #014538) to either retina (Rx-Cre, Dhande

et al., 2012)- or midbrain (En1-Cre, Dhande et al., 2012)- specific Cre lines. Af-

ter retinal injections, calcium dye was allowed to fill retinal ganglion cell axons

for at least 2 (Calcium Green-1) or 4 (viral GCaMP6) days. Imaging of activity

was performed on unanesthetized, head-fixed mice after a 1–2 hr recovery

from isoflurane anesthesia and the surgical procedure to expose the SC

(recovery time being directly dependent on the duration of the procedure).

Pharmacological manipulation of in vivo spontaneous activity was achieved

by reanesthetizing animals using 3% isoflurane for 5–10 min, exposing the

eye, and injecting �500 nL per eye with a pulled glass micropipette. After in-

jection the eyelid was closed and covered in ophthalmic ointment and a brief


local anesthetic, and the animal returned to 0% isoflurane before being

allowed to recover at least 20–30 min before resumption of imaging. Movies

were analyzed using overlaid 10 mm square regions of interest (ROIs), as has

been described previously (Ackman et al., 2012; also see Supplemental Exper-

imental Procedures).

Wave Property Analyses

Interretina wave interval was measured by determining the difference between

the time of the wave peak in opposing hemispheres in the same animal. Wave

direction bias index was computed by grouping individual wave directions into

30� bins and calculating a vector sum of the angle and length of directions

within each hemisphere. Recordings fromGCaMP-loaded cells were preferred

wherever possible for wave property analyses, as cell loading was found to be

much more uniform and consistent between hemispheres and animals.

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures, Supplemental Experimental

Procedures, and four movies and can be found with this article at http://dx.doi.

org/10.1016/j.neuron.2014.10.051.

AUTHOR CONTRIBUTIONS

M.C.C., T.J.B., and H.-P.X. designed the experiments. J.X., A.C., Y.Z., and

M.C.C. created the b2-nAChRfl/fl mice. T.J.B. performed in vivo imaging

experiments. H.-P.X. performed anatomical tracing and in vitro retinal wave

recordings. M.-J.Y. and Z.J.Z. performed electrophysiological confirmation

of b2-nAChR subunit functional knockout. J.B.A., M.C.C., X.G., and T.J.B.

designed analysis routines. T.J.B. and H.-P.X. performed primary analysis of

SC recordings and anatomical tracing, respectively, and T.J.B. and X.G. per-

formed secondary data analysis on SC recordings. T.J.B. and M.C.C. wrote

the manuscript with comments from all other authors.

ACKNOWLEDGMENTS

We would like to thank all members of the Crair lab for their helpful comments

on the work and manuscript. We would also like to thank Profs. In-Jung Kim,

Jonathan Demb, Ed Ruthazer, and Thomas Biederer for their helpful com-

ments on the manuscript, and Profs. Carlos Portera-Cailliau and Chinfei

Chen for methodological advice. This work was supported by NIH Grants

RR19895, RR029676–01 for the Yale University Biomedical High Performance

Computing Center and NIH grants P30 EY000785, T32 NS007224, R01

EY015788 and R01 EY023105 to M.C.C, R01 EY017353, and T32 EY022313

to Z.J.Z., and in part by an unrestricted Department of Ophthalmology grant

from Research to Prevent Blindness (RPB), Inc. M.C.C. also thanks the family

of William Ziegler III for their support.

Accepted: October 22, 2014

Published: November 20, 2014

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