Sparsification of neuronal activity in the visual cortexat eye-openingNathalie L. Rochefort1, Olga Garaschuk1, Ruxandra-Iulia Milos1, Madoka Narushima, Nima Marandi, Bruno Pichler,Yury Kovalchuk, and Arthur Konnerth2

Center for Integrated Protein Science and Institute of Neuroscience, Technical University Munich, Biedersteinerstrasse 29, 80802 Munich, Germany

Communicated by Bert Sakmann, Max Planck Institute for Neurobiology, Martinsried, Germany, July 9, 2009 (received for review June 10, 2009)

Eye-opening represents a turning point in the function of the visualcortex. Before eye-opening, the visual cortex is largely devoid ofsensory inputs and neuronal activities are generated intrinsically.After eye-opening, the cortex starts to integrate visual informa-tion. Here we used in vivo two-photon calcium imaging to explorethe developmental changes of the mouse visual cortex by analyz-ing the ongoing spontaneous activity. We found that beforeeye-opening, the activity of layer 2/3 neurons consists predomi-nantly of slow wave oscillations. These waves were first detectedat postnatal day 8 (P8). Their initial very low frequency (0.01 Hz)gradually increased during development to �0.5 Hz in adults.Before eye-opening, a large fraction of neurons (>75%) was activeduring each wave. One day after eye-opening, this dense mode ofrecruitment changed to a sparse mode with only 36% of activeneurons per wave. This was followed by a progressive decreaseduring the following weeks, reaching 12% of active neurons perwave in adults. The possible role of visual experience for thisprocess of sparsification was investigated by analyzing dark-reared mice. We found that sparsification also occurred in thesemice, but that the switch from a dense to a sparse activity patternwas delayed by 3–4 days as compared with normally-reared mice.These results reveal a modulatory contribution of visual experienceduring the first days after eye-opening, but an overall dominatingrole of intrinsic factors. We propose that the transformation innetwork activity from dense to sparse is a prerequisite for thechanged cortical function at eye-opening.

calcium waves � cortical development � mouse � two-photonimaging � up-down states

During the first two postnatal weeks, important functionalfeatures such as retinotopic maps were shown to develop in

the visual cortex of rodents (1, 2). This development is activity-dependent and takes place before eye-opening. Intrinsic activitypatterns play a critical role in circuit refinement at this stage ofdevelopment (1–3). Activity patterns known to be present in therat visual cortex during the first postnatal week include burstswith spindle-shape field oscillations that are triggered by spon-taneous retinal bursts (4). In the ferret, also before eye-opening,long-range correlated activity is generated by intrinsic circuits inthe primary visual cortex and is modified by eye-specific con-nections from the retina via the lateral geniculate nucleus (5, 6).

Surprisingly, little is known about the spontaneous activity inthe mouse visual cortex around eye-opening. An early form ofcortical slow wave activity, the so-called early network oscilla-tions (ENOs) (7), was detected in rodents as early as embryonicday 16 (8) and persists approximately until the end of the firstpostnatal week (7, 9). The ENOs share similarities with the wavesthat were originally described in the retina (10, 11) and in thespinal cord (12). The distinctive features of ENOs are theircomplete block by anesthetics in vivo (13), their strong sensitivityto AMPA receptor antagonists and their presence during theperiod at which GABAergic transmission is depolarizing (7, 14).Such waves of activity travel over long distances within thecortex, or even from the retina to the cortex (2, 4), and play acritical role in the accurate assembly of long-range neuronal

connections (3). At the end of the first postnatal week (betweenpostnatal days P5 and P9), GABA-driven cortical giant depo-larizing potentials (GDPs) have been reported in slices of ratsomatosensory cortex (9). Importantly, to our knowledge, noth-ing is known about the activity during the second postnatal weekthat just precedes eye-opening.

In adult mammals, slow wave activity was initially found insleeping and in anesthetized animals in vivo (15). In someinstances, slow wave activity can be detected in adults also in exvivo preparations (16). The slow wave activity during early andlate developmental stages shares similarities, but has also dis-tinctive, stage-specific properties. The similarities include (i) thepresence of depolarized ‘‘Up’’ states that can be associated withmassive neuronal firing followed by relatively silent ‘‘Down’’states, (ii) Ca2� signaling during Up states and (iii) the long-range propagation of the waves involving large cortical areas (2,7). The major difference is the 15- to 20-fold higher wavefrequency in adults (0.5 Hz vs. �0.03 Hz in the cortex ofneonates) (13, 15, 17).

An interesting feature of the slow waves recorded in the adultcortex is the sparse activation pattern with only 10% of corticalneurons firing during Up states (17). Such a sparseness of thespontaneous activity in the adult is an important prerequisite forsparse coding of sensory inputs, a general feature of informationprocessing in different sensory systems, including the visualsystem (18–21). Spontaneous synchronous activity in the adultbrain was found to strongly correlate with sensory processing aswell as with motor planning and behavioral states (22–24). In thevisual cortex of adult cats, it was shown that ongoing spontane-ous activity contributes to the variability of sensory-evokedneuronal responses (25). Spontaneous activity also plays acritical role in the coordinated memory replay in the visualcortex and in the hippocampus, during slow wave sleep (26, 27).This process is believed to be of critical importance for memoryconsolidation (26, 27).

In the present study, we investigated the specific features ofthe ongoing spontaneous activity during the postnatal develop-ment of the mouse visual cortex in vivo. We were particularlyinterested in the question of how eye-opening affects thisongoing activity. We used video-rate two-photon calcium imag-ing (28) of neural networks in vivo (29), an approach that allowsthe detection of action potential firing with a high fidelity andsensitivity (17).

Author contributions: N.L.R. and A.K. designed research; N.L.R., O.G., R.-I.M., M.N., N.M.,Y.K., and A.K. performed research; B.P. and Y.K. contributed new reagents/analytic tools;N.L.R., O.G., R.-I.M., M.N., Y.K., and A.K. analyzed data; and N.L.R., O.G., and A.K. wrote thepaper.

The authors declare no conflict of interest.

1N.L.R., O.G., and R.-I.M. contributed equally to this work.

2To whom correspondence should be addressed at: Institut fur Neurowissenschaften,Technische Universitat Munchen, Biedersteinerstrasse 29, 80802 Munchen, Germany.E-mail: arthur.konnerth@lrz.tum.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907660106/DCSupplemental.

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ResultsSlow Wave Activity in the Mouse Visual Cortex Before Eye-Opening.We stained neurons with the fluorescent Ca2� indicator OregonGreen BAPTA-1 AM (OGB-1 AM) which was deliveredthrough a patch pipette to the primary visual cortex of mice (Fig.1A). Fig. 1B shows stained cortical layer 2/3 cells located �130�m below the cortical surface of an 11-day-old (P11) C57BL/6mouse. Continuous recordings of changes in Ca2�-dependentfluorescence from a region of interest covering the entire fieldof view revealed wave-like Ca2� transients recurring at a slowrate (3 waves/min; Fig. 1 D). A more detailed analysis showedthat the wave activity was present both in neuropil and in neurons(Fig. 1 C and D). Remarkably, a large fraction of the neuronsparticipated in this spontaneous activity (Fig. 1C). A detaileddevelopmental analysis revealed that Ca2� waves were notdetected before P7 in anesthetized mice (Fig. 1E). Ca2� waveswere first detected at P8, when they occurred at a low frequency(0.5 � 0.1 waves/min). Over the next developmental days, thefrequency gradually increased, whereas the amplitude decreased(Fig. 1 E–G). After the end of the fourth postnatal week, theCa2� waves reached a frequency of �30 waves/min (or 0.5 Hz),well within the range of the electrically-recorded Up-Downstates or slow waves that were described originally by Steriadeand colleagues (15). We performed experiments in nonpig-mented BALB/c mice to relate the results to our earlier workperformed in vitro (7) and in vivo (13) as well as in pigmented

C57BL/6 mice, which exhibit a better visual detection, patterndiscrimination and visual acuity (30, 31). The frequency of Ca2�

waves was at all developmental ages not significantly different inC57BL/6 and in BALB/c mice (Student’s t test, P � 0.05, Fig. 1F and G).

At later developmental stages, Ca2� transients recorded invivo in the somata of neurons in the visual cortex resultexclusively from neuronal firing (32), similarly to neurons inother cortical areas (17, 29). To test whether this is the case alsoat early stages of development we combined cell-attached andwhole-cell recordings with Ca2� imaging in vivo (Fig. 2A). Usinga video rate two-photon imaging microscope (see ExperimentalProcedures), single action potential-evoked Ca2� transients werereliably detected and the amplitude of Ca2� transients correlatedwith the number of action potentials (R2 � 0.98 see Fig. 2 B andC) (17). The median number of action potentials per Ca2�

transient at P11-P12, before eye-opening, was found to besignificantly higher (Kolmogorov-Smirnov test, Z � 5.331, P �0.001) than that at P14–P16, after eye-opening (Fig. 2D). Theseresults were in agreement with the larger amplitudes of Ca2�

waves recorded before P12 (see Fig. 1G).The temporal relation between the electrical activity of single

neurons and the Ca2� waves was determined in experiments inwhich current clamp whole-cell recordings were combined withtwo-photon Ca2� imaging. Fig. 2E illustrates an experimentperformed in a P17-old mouse. Whole-cell recordings from a

Fig. 1. Spontaneous Ca2� waves in the developing mouse visual cortex in vivo. (A) Combined fluorescence/transmitted light micrograph of a coronal brain slicefrom an 11-day-old C57BL/6 mouse taken after an in vivo experiment. The area stained in vivo with OGB-1 is shown in green. (B) An in vivo micrograph showingan area of layer 2/3 in the primary visual cortex where recordings illustrated in D were made. (C) Active cells (in red) during a representative Ca2� wave at P11.(D) Upper: spontaneous Ca2� waves, recorded from a region of interest covering the entire frame shown in B and C. Lower: cellular responses during the Ca2�

wave marked with an asterisk in the upper trace, in seven neurons (x) and in the neuropil (N) (regions of interest are indicated in C). The Average trace (red) isthe mean of the seven neuronal responses. (E) Developmental profile of the spontaneous Ca2� waves. Each trace is a recording from a large region of interest(e.g. D, upper trace), at different ages. (F) Frequencies of spontaneous Ca2� waves in C57BL/6 and BALB/c mice before eye-opening (P10–11) and 6 weeks aftereye-opening (P48–79), (C57BL/6 mice: 10 animals/P10–11, 5/P48–P79; BALB/c mice: 9/P10–11, 5/P48–P79). (G) Frequencies (filled circles) and amplitudes (opensquares) of spontaneous Ca2� waves at different ages. Data from C57BL/6 and BALB/c mice were pooled, because for each age-group there was no significantdifference (Student’s t test) between both strains (BALB/c mice: 5 animals/P8, 5/P9, 4/P10, 5/P11, 5/P12–P13, 5/P14–P15, 4/P16–P19, 5/P20–29, 4/P30–P39,5/P48–P79; C57BL/6 mice: 5/P8–P9, 5/P10, 5/P11, 13/P12–P13, 13/P14–P15, 5/P16–P19, 10/P20–29, 5/P30–P39, 5/P48–P79). Here and below error bars representstandard error of the mean.

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layer 2/3 neuron showed the highly characteristic Up-Downstate-related membrane potential deflections (Fig. 2 E and F)known to occur during slow wave activity in the visual cortex aswell as in other brain regions (17, 18, 24). At this developmentalstage (P16–P18), �50% (47 � 11%, n � 9 cells in 7 mice) of theUp-states in a given neuron were associated with action potentialfiring. During Down-states, single spikes were observed onlyoccasionally, and spike bursts were virtually never encountered.The onset of the Up-states correlated closely with the onset ofthe Ca2� waves recorded from the surrounding neuropil (Fig.2E). These results, in agreement with previous findings in themotor and the somatosensory cortex (17), identify the neuropilCa2� waves as a reliable reporter for the Up-Down state activity.

Sparsification of Spontaneous Neuronal Activity After Eye-Opening.Before eye-opening, a striking feature of the spontaneous ac-tivity was the fact that most neurons participated in each wavewith a Ca2� transient (Fig. 3 A, C, and E). Thus, at P8–P11, theaverage fraction of active neurons per wave was 74.5 � 4.6%.This dense activation pattern is in striking contrast to the sparseneuronal activity that is found in adults (Fig. 3B, D, and E).Closer inspection of the developmental profile (Fig. 3F) showedthat on the day of eye-opening, a process of sparsification wasinitiated leading to a strong decrease in the fraction of activecells per wave from 59.8 � 3.6% at the day of eye-opening to36.0 � 1.7% only 1 day later. This sparsification was nearlycompleted within 1 week after eye-opening and reached a levelof �12% of active cells per wave in adults (Fig. 3E, fraction ofactive cells per wave was 17.5 � 1.9% at P20–29 and 12.3 � 1.7%at P48–79). As previously reported for the adult somatosensoryand frontal cortices (17, 33), we found that at all developmentalages �10% of the visual cortex neurons were ‘silent’ and did notdisplay any calcium transient during our periods of recording (�

3 min). The other neurons were active at characteristic age-dependent frequencies (from 1.1 � 0.19 waves/min at P10 to4.05 � 0.49 waves/min at P48–60).

Role of Visual Experience. To determine whether sparsificationrequires visual experience we examined mice that were rearedfrom birth in the dark. In these mice, sparsification also occurredin the absence of visual experience. The activity patterns re-corded before eye-opening (P11) as well as those recorded 2(P20–29) or more than 7 weeks (P48–79) after eye-opening,were similar in normally and in dark-reared mice (Fig. 4F andFig. S1). Specifically, the number of active cells per wave wasvirtually identical at these later developmental stages (Fig. 4F).However, the inspection of the activity patterns recorded 1, 2,and 3 days after eye-opening revealed a strong differencebetween normally and dark reared mice (Fig. 4 A–E). Fig. 4 A–Dshows an example of activity patterns recorded in mice of thesame age (P15) with the same number of days after eye-opening(�3 days) but one reared in normal conditions and the otherreared in the dark. The fraction of active cells per wave wasmarkedly higher (1.8 times higher) in dark-reared than innormally-reared mice (Fig. 4 E and F). Thus, in dark-rearedanimals, the switch from a dense activity pattern to a sparse oneoccurred 4–5 days after eye-opening instead of occurring 1 dayafter eye-opening as in normally-reared mice. These resultsindicate that visual experience is not required for the process ofsparsification, but has a strong modulatory effect at the onset ofvision.

DiscussionBy using two-photon calcium imaging of neural networks in vivo,we identified in this study two distinct modes of network activityof layer 2/3 neurons in the mouse primary visual cortex. A dense

Fig. 2. Cellular properties of neuronal Ca2� signals during slow wave activity. (A and B) Simultaneous recordings of spontaneous Ca2� transients and underlyingaction potential firing in loose-seal cell-attached configuration (B) in a layer 2/3 neuron (A) in the primary visual cortex of a C57BL/6 15-day-old mouse.Spontaneously occurring action potentials (APs) are indicated in red. (C) Relation between the number of action potentials and the amplitude (�F/F) of thecorresponding Ca2� transient (P14-P16 C57BL/6 mice, seven cells as indicated). Dotted line indicates the linear least squares fit to the data. (D) Box-and-whiskerplot (Left) and cumulative functions (Right) illustrating the distribution of number of action potentials per calcium transient at P11–P12 (5 cells) and P14–P16(12 cells). The median number of action potentials per Ca2� transient at P11–P12, before eye-opening, was significantly higher (Kolmogorov-Smirnov test, Z �5.331, P � 0.001) than that at P14–P16, after eye-opening. (E) Simultaneous whole-cell patch-clamp recordings of electrical activity (Top) from a layer 2/3 cellwithin the field of view and Ca2� recordings from the surrounding neuropil (Lower). Note that the Up-Down states are strictly associated with the Ca2� waveactivity in the neuropil. Mouse age was P17. (F) Histogram of the membrane potential values of the same neuron as in E.

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mode is present before eye-opening, when the cortex generatesand receives largely intrinsic signals, and a sparse mode developsafter eye-opening, when the cortex processes both intrinsic andvisually driven signals. This sparsification process also occurs indark-reared animals, indicating a critical role of intrinsic factors

for this process. However, we find that the process of sparsifi-cation is modulated by visual experience during the first 4 daysafter eye-opening.

This study reveals the presence of a wave-like network activityat P8–P9, at a time point which is �4–7 days before eye-opening.These waves recur at very slow rates in the subdelta range andare produced by simultaneously active neurons. We show thatthis slow wave activity is present in the visual cortex during aperiod (from P8–9 to eye-opening) that corresponds to stage IIIof the retinal waves (2). As previously shown during the firstpostnatal week for stage II retinal waves (4), it is possible thatstage III retinal waves also trigger the slow waves at the cortical

Fig. 3. Sparsification of spontaneous neuronal activity after eye-opening. (Aand B) Recordings from layer 2/3 in the visual cortex of a P11 (A) and a P26C57BL/6 mouse (B). The two images in the top row indicate the regions ofinterest. The two images in the middle row display active cells (in red) duringa representative Ca2� wave (dotted lines) at P11 and P26, respectively. Bottomrow: wave-associated Ca2� transients in individual neurons. Note the pro-nounced increase in the frequency of neuropil wave activity at P26. (C and D)Dot plot representation of cellular responses during eight consecutive Ca2�

waves at P11 and P26, respectively (same experiments as those shown in A andB). (E) Fraction of cells active during a given Ca2� wave as a function ofpostnatal age. Data obtained from both C57BL/6 and BALB/c mice werepooled, because for each age-group there was no significant difference(Student’s t test) between both strains (same experiments as those shown inFig. 1G). (F) Fraction of active cells per Ca2� wave in relation to the day ofeye-opening. The day of eye-opening (P12–14) is indicated in the graph as day0. All results were obtained in C57BL/6 mice [eyes closed (-1 to �3); 6 animals;day of eye-opening: 6 animals; eyes opened (�1 through � 4 days): 6, 5, 4, and4 animals, respectively). The corresponding postnatal ages of the animals areindicated for each group in parenthesis. The asterisks indicate significance(Kolmogorov-Smirnov test, P � 0.005).

Fig. 4. Sparsification is modulated by visual experience. (A and B) Wave-associated Ca2� transients in individual layer 2/3 neurons in a control (A) andin a dark-reared (B) 15-day-old mouse, 3 days after eye-opening. The twoimages in the top row indicate the regions of interest. Bottom row: wave-associated Ca2� transients in individual neurons. Active cells during a repre-sentative Ca2� wave (indicated by dotted lines in the bottom row) are markedin red. (C and D) Dot plot representation of cellular responses during consec-utive waves in a control (C) and in a dark-reared (D) 15-day-old mouse (sameexperiments as in A and B). (E and F) Fraction of active cells per Ca2� wave inrelation to the day of eye-opening. The day of eye-opening is indicated as day0, the days before and after eye-opening are indicated by �1 to �3 and by �1, �2, and so on, respectively. (E) Each circle indicates the percentage obtainedin one animal. (F) Mean values of the fraction of active cells per Ca2� wave incontrol and dark reared mice. All results were obtained in C57BL/6 mice.Normally reared mice (NR), same number of animals as in Fig. 3E—(F). Darkreared (DR) mice, eyes closed (-1 to �3): 5 animals; day of eye-opening: 5animals; eyes opened (�1 through � 3 days): 5, 4, and 7 animals, respectively;eyes opened (�4–5 through � 43–75 days): 5, 5, 7, and 10 animals, respec-tively). The asterisks indicate significance in F (Kolmogorov-Smirnov test, P �0.01). The corresponding postnatal ages of the animals are indicated for eachgroup in parenthesis.

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level during the second postnatal week. It has been suggestedthat spontaneous activity is involved in the wiring of the distinctpattern of connectivity in the cortex (1, 2). In the mammalianvisual system, as in the developing Xenopus retino-tectal system(34), spontaneous activity, through activation of NMDA recep-tors, may regulate axonal growth and, thereby, the pattern ofinnervation territories in the cortex. Such early forms of pat-terned spontaneous activities promote the establishment of thecoarse connectivity and of sensory maps in the visual system (1,35, 36).

A remarkable aspect of the cortical slow wave activity beforeeye-opening is the fact that the majority of layer 2/3 neurons isengaged in each wave of activity. Such a dense activation patternhad not been observed before in vivo and is in remarkablecontrast to the sparse activity found at later stages of develop-ment. Our analysis of mice at defined days before and aftereye-opening revealed that the process of sparsification is stronglyaccelerated at the day of eye-opening. Irrespective of the bio-logical age of the mouse—P12, P13, or P14—there is a suddendrop in the fraction of active cells per wave, from 60% at the dayof eye-opening to 36% one day later (Fig. 3F). This suggests thateye-opening directly affects the process of sparsification. A roleof visual experience is further supported by the observation thatduring the first 3 days after eye-opening the fraction of activecells per wave is significantly higher in dark-reared mice com-pared with normally-reared animals (Fig. 4 E and F). However,the finding that sparsification eventually develops in animals thatwere reared in the dark indicates that intrinsic factors play adecisive role in the process of sparsification. In these animals, thedelayed decrease in the percentage of active cells per wave at 4–5days after eye-opening (from 55.6 � 3.4% to 32.2 � 3.4%, Fig.4F) is as sudden as that observed in control animals at 1 day aftereye-opening (from 59.8 � 3.6% to 36.0 � 1.7%, Fig. 4F). Thissimilarity suggests that the sudden reduction is part of anintrinsic developmental program that, in normal conditions, isjust triggered by visual stimuli. The period of eye-opening ischaracterized not only by a switch in the cortical firing mode, butalso by a change in retinal firing.

In normally-reared mice, retinal waves begin to break down atP15, just after eye-opening. By P21, waves are not present anymore, and the activity pattern is comparable with that observedin adults (37). Although the precise, day-by-day changes of thepattern of spontaneous retinal activity between P15 and P21 areunknown, it is remarkable that the retina reaches, as the cortex,at �1 week after eye-opening a level of maturation that is similarto that found in adults (37). In addition, in dark-reared mice asimilar time course for the disappearance of retinal waves wasobserved. Remarkably, at P15, dark-reared retinas occasionallyshowed abnormally long periods of relative inactivity, not seenin controls (37). In view of the evidence that during the firstpostnatal week retinal waves trigger the activity in the visualcortex (4), it remains to be determined whether the changes ofspontaneous retinal activity around eye-opening contribute tothe sparsification of cortical activity.

Further support for the decisive role of intrinsic factors comesfrom the finding that the same level of sparseness is eventuallyreached in mice with and without visual experience (for exampleat P57–89, 9.6 � 0.9% of active cells per wave in control miceand 12.0 � 1.2% in dark reared mice, no significant differencebetween both groups). It seems that these unknown intrinsicfactors exert their action not only in the visual cortex, but alsoin other cortical areas, at least for layer 2/3 neurons. We haveobtained evidence for such a switch from dense to sparse activityalso in the auditory cortex (Fig. S2), whereas others have shownsparse neuronal activity at late developmental stages in thesomatosensory (17) and the motor cortex (17, 38).

In conclusion, we demonstrate that the activity of the visualcortex undergoes profound functional changes at the onset of

vision. In the present work, we investigated the properties of thecircuitry by analyzing the spontaneous slow wave activity. Theobserved sparse signaling may be of direct significance forinformation processing during slow-wave sleep in the visualcortex (26, 27). It is likely, however, that the developmentalchanges of the circuitry are also essential for the function of thevisual cortex during signal processing in behaving animals. Wepropose that the rapid development of visual performanceduring the first 8–10 days after eye-opening [see for example (39,40)] is based, at least in part, on the sparsification of layer 2/3neurons spontaneous activity in the primary visual cortex.

Experimental ProceduresAnimals. All experimental procedures were performed in accordance withinstitutional animal welfare guidelines and were approved by the state gov-ernment of Bavaria, Germany. Litters of C57BL/6 (7- to 89-day-old) and BALB/cmice (7- to 65-day-old) were kept under 12 h/12 h light/dark cycles except fordark-rearing experiments. The day of birth (P0) was accurately ascertained aswell as the day of eye-opening. For this, the eyes were checked four times perday (at 8 AM, 1 PM, 6 PM, 8–9 PM) from the age of P10 on, and the eyes wereconsidered opened as soon as we observed the initial break in the membranesealing the eyelids. For dark-reared mice, the same procedure was performedwith the help of infrared viewers.

Surgical Procedures. Surgery was performed in accordance with institutionalanimal welfare guidelines as described previously (29). Briefly, the mice wereplaced onto a warming plate (38°C) and anesthetized by inhalation of 1.5%isoflurane (Curamed) in pure O2. The depth of anesthesia was assessed bymonitoring the tail-pinch reflex and the respiration rate. After removing theskin, the skull was gently thinned under a dissecting microscope using dentaldrills. The custom-made recording chamber (41) was then glued to the skullwith cyanoacrylic glue (UHU). The mouse was transferred into the set-up,placed onto a warming plate (38°C) and continuously supplied with 0.9–1.1%isoflurane in pure O2. The recording chamber was perfused with warm (37°C)extracellular perfusion saline containing (in mM): 125 NaCl, 4.5 KCl, 26NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 20 glucose, pH 7.4, whenbubbled with 95% O2 and 5% CO2. For two-photon imaging of corticalneurons a small craniotomy (�1 mm) was performed above an area devoid ofbig blood vessels using a thin (30 G) injection needle.

In Vivo Ca2� Imaging of Network Activity. The position of the primary visualcortex was located according to brain atlas coordinates (Bregma �3 to �4.5mm, 2–3 mm lateral to the midline for P18–P65 (42)). In younger animals, theposition of the primary visual cortex (0–1 mm anterior to the lambda suture,1.5–3 mm lateral to the midline) was identified according to the Golgi atlas ofthe postnatal mouse brain (43). In all experiments the correct location of theimaged neurons was confirmed in vivo by their responses to light flashesand/or posthoc by imaging of the stained brain area (e.g., Fig. 1A). Similarprocedures were used to identify the location of the primary auditory cortex(Bregma �2.2 to �3.6 mm, 4–4.5 mm lateral to the midline) (42).

The brain area of interest was stained in vivo with the fluorescent Ca2�

indicator dye Oregon Green BAPTA-1 (OGB-1) (29). Two-photon imaging wasperformed with a custom-build video-rate two-photon microscope based ona resonance scanner (44) and a mode-locked femtosecond pulse laser, oper-ating at 710–920 nm wavelength (MaiTai, Spectra Physics). The scanner wasmounted on an upright microscope (BX61WI, Olympus) equipped with water-immersion objectives (60, 1.0 NA Nikon, or 40, 0.8 NA Olympus). Becauseisoflurane is known to act differently in rodents of different ages, we adjustedits concentration in each experiment such that the animals were continuouslybreathing at a rate of 90–110 per minute. In general, we started recordings at1.1% isoflurane in pure O2 at P17–P65 (1.1–1.2% at P7–P16) and graduallydecreased its concentration to levels at which the breathing rate was constant,just above the threshold for awakening.

Cell-Attached and Whole-Cell Patch Clamp Recordings in Vivo. Loose-sealcell-attached and whole-cell patch clamp recordings were performed using anEPC-9 patch clamp amplifier (HEKA) as described in (17, 18). All data werefiltered at 3–10 kHz and digitized at 5–20 kHz. The pipette solution forloose-seal cell-attached recordings contained (in mM): 150 NaCl, 2.5 KCl, 10HEPES, 2 Ca2Cl, 1 MgCl2, and 20 glucose, pH 7.4. The pipette solutionfor whole-cell patch clamp recordings contained (in mM): 148 K-Gluconate,10 Hepes, 10 NaCl, 0.5 MgCl2, 4 Mg-ATP, 0.4 Na-GTP, and 0.05 Alexa Fluor 594,pH 7.3.

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Image Analysis. The image analysis was performed off-line in two steps. First,the ImageJ software (http://rsb.info.nih.gov/ij/) was used for drawing regionsof interest (ROIs) around cell bodies and around a large area of cell-freeneuropil. Astrocytes were excluded from the analysis based on their selectivestaining by sulforhodamine 101 (45, 46), brighter appearance after stainingwith OGB1-AM (17) and their specific morphology with clearly visible pro-cesses (see for example on the left edge of Fig. 1B). The presence of glialprocesses was assessed by inspecting 3D-stacks (30 �m below and upper theimaged plane) obtained routinely at the end of each experiment. Fluores-cence values from all ROIs were extracted. In the next step, custom maderoutines of the Igor Pro software (Wavemetrics) were used for the detectionof wave-associated Ca2� transients in individual neurons and in the neuropil.Background fluorescence, measured in blood vessel lumen, was subtractedfrom all signals. All signals were expressed as relative fluorescence changes

(dF/F) after background subtraction. All dF/F traces were smoothed with abinomial filter (time window � 0.3 s). Each smoothed trace was subtractedfrom the original dF/F trace, resulting in the ‘baseline noise’ trace. Calciumtransients were automatically detected with a template-matching algorithm,taking into account their sharp rise. They were accepted as signals if theiramplitude was three times larger than the standard deviation of the corre-sponding baseline noise values. After the automatic analysis, all traces werecarefully inspected again to correct for false positive or negative signals.

ACKNOWLEDGMENTS. We thank Jia Lou for her help with the figures. Thiswork was supported by grants of the Deutsche Forschungsgemeinschaft (SFB596, GA 654/1–1, HO 2156/2–1) and the Bundesministerium fur Bildung undForschung (NGFN-2). M.N. was supported by a fellowship from the JapanSociety for the Promotion of Science.

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