Supporting InformationMoczulska et al. 10.1073/pnas.1312508110SI TextThe 2-s complex sound cue used as a conditional stimulus (CS)in the conditioning paradigms is accessible in wav-file format asAudio S1.

SI MethodsAnimal Breeding.Male CB57BL/6J mice (Charles River) in the ageof 10–14 wk were used for all immediate early gene (IEG) ex-pression and lesion experiments. Transgenic mice of line GFP-M(Tg(Thy1-EGFP)MJrs/J) (1) were bred in-house and used forspine-imaging experiments at the age of 3–6 mo. Mice werehoused in groups of 1–5 animals with a 12-h light/dark cycle, andexperiments were conducted during the light phase cycle. Allexperiments were performed in accordance with the Austrianlaboratory animal law guidelines for animal research and ap-proved by the Viennese Magistratsabteilung 58 (approval no.M58/004995/2010/6).

Auditory Cortex Lesions.Auditory cortex lesions were performed asdescribed previously (2). Briefly, mice were deeply anesthetizedand kept on a heating pad at 37 °C in a stereotaxic apparatus.The skin was disinfected, and Lidocaine/Noradrenaline was in-jected s.c. before unilateral incision. Connective tissue was re-moved from the skull and the musculus masseter dissected at itsrostral end. A small piece of bone (∼3–4 mm2) was cut out of theskull with a scalpel and put aside. The auditory cortex was le-sioned by cauterization. The procedure was repeated for thesecond hemisphere. Finally, the fragment of the skull was putback into place, fixed with bone wax, and the skin closed withtissue adhesive. Sham surgery was performed in a subset of lit-termates as a control. The procedure involved anesthesia, re-moval of the m. masseter, and reclosure of the skin; the auditorycortex was left intact. The person performing behavioral trainingand testing was blinded to the surgery condition of the individualanimals. Position and size of the lesions were documented andverified at the end of the experiment (Fig. S1).

Quantitative PCR. Quantitative PCR was performed as describedpreviously (2). Briefly, total RNA was isolated from the dissectedauditory cortex (ACx) after the memory test session. As a tem-plate, 1 μg RNA was used to generate cDNA. Quantitative PCRwas performed, and mRNA copy numbers were calculated foreach sample using the cycle threshold (Ct) value. The mRNA ofthe housekeeping gene α-tubulin was amplified in parallel andused for normalization.

Implantation of Cranial Window. Chronic cranial window implan-tation over the ACx area was performed as described previously(3). Mice were prepared for the craniotomy in the same way asfor ACx lesions. Exposed bone was smoothed using a surgicaldrill. Acrylic glue was applied on the bone area outside thewindow, and the edges of the window were gently drilled by asurgical drill. The bone covering ACx was removed. Exposedbrain was kept moisturized; a drop of low-melting agarose wasapplied on the exposed brain area and immediately covered witha round coverslip. The window was sealed with dental cement,and additional dental cement was applied on all areas of exposedbone. A custom-made head post was implanted next to thewindow and embedded with dental cement. Animals were keptin their home cages for at least 10 d before further handling.

Auditory-Cued Fear Conditioning. Auditory-cued fear conditioning(ACFC) was performed according to a previous study (2).Habituation.Mice were habituated for at least 3 d by handling andplacing them in all test environments for 5 min. No sound waspresented during habituation.Conditioning. Mice were conditioned 1 d after habituation. In theconditioning environment, lights were turned on, and a shockfloor and an alcohol scent were present. Mice were placed in thechamber directly before the start of each session. For pairedconditioning, five sound–shock pairings were presented with arandomized interstimulus interval ranging from 50 to 75 s. Forunpaired conditioning five foot shocks and five sounds werepresented in a random order, separated by at least 1 min. Thesame complex sound [2 s, spectrogram (Fig. S2A), peak level 76dB sound pressure level (SPL)] was applied for paired and un-paired conditioning for all protocols with exception of secondconditioning (Fig. 1B), where a 4-kHz pure tone was used (2 s,peak level 73 dB SPL). In some experiments (Figs. 2 and 4A),a third group of mice was placed in a conditioning chamberwithout shock or sound presentation.Memory testing. Mice were tested for freezing behavior, i.e., thesuppression of all movements except breathing, during silenceand CS presentation in the memory test chamber during amemory retention test (lights off, home cage odor, fine metal gridfloor). After a baseline period of 60–90 s, the conditioned soundwas presented in two blocks of five presentations with an in-terstimulus interval of 2 s. Blocks were given in a random orderand were separated by a randomized interval (22–37 s). CS-inducedfreezing is a behavioral readout of a fear response indicative ofsuccessful memory formation and retrieval.For IEG analysis, mice underwent paired conditioning or were

exposed to the context. On the following day or a week later, micewere kept overnight in the memory test chamber to ensure lowbasal IEG expression levels. Mice were killed 30–40 min after thememory test session, and the brains were immediately removedand auditory cortex tissue was isolated bilaterally. Mice that weredirectly taken from the home cage served as control.For behavioral experiments combined with in vivo spine im-

aging, animals underwent context exposure, paired or unpairedconditioning after habituation period. Memory was tested in micea week later. The experimenter performing imaging and spineanalysis was blinded to the behavioral treatment. Imaging sessionslasting 1 h under gas anesthesia (∼1% isoflurane) were per-formed 30 min before and 1 h after conditioning, context ex-posure or memory test. An additional imaging session wasintroduced 1 h and 30 min before the imaging session pre-ceding the memory test, and the mouse was placed in betweenin the home cage. During habituation, mice were extensivelyexposed to handling and short gas anesthesia under an analogoustime regime.Quantitative analysis of behavior. Video data were recorded duringthe memory test and analyzed by a custom-made MatLab script(MathWorks). Freezing behavior was automatically scored basedon movement rate [frame-to-frame difference, significant motionpixels (4) detected on the movies]. Baseline freezing was assessedduring silence between 30 and 60 s of each protocol run.

Intrinsic Imaging. Intrinsic signal optical imaging was performed ina sound-isolated cubicle as described previously (5). Briefly, micewere anesthetized with isoflurane and positioned below a CCDcamera. Body temperature was maintained with a heating padduring the experiment. The brain area under chronic cranial

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window was first imaged with green light (525 nm) to image thepattern of blood vessels over the brain. Subsequently, the focalplane was moved 400 μm below the blood vessels and the brainwas illuminated by a red light-emitting diode (780 nm). Soundstimuli consisted of trains of 20 white noise bursts or pure tonepips (80 ms, 2, 4, 8, and 16 kHz) separated by 20-ms smooth gapsor 2-s-long complex sounds (for spectrograms, see Fig. S2). Weused amplitude-modulated pip trains of pure tones to map thetonotopic organization of the ACx because responses to soundstimuli are strongest at the onset and offset and typically stronglyadapt during continuous presentation (6). To characterize in-trinsic sound responses elicited by stimuli resembling in theirstatistics naturally occurring sounds, we used a set of fourcomplex sounds, including the sound used as CS in the condi-tioning experiments (complex 1). Sounds were delivered witha sampling rate of 192 kHz at 80 dB SPL mean amplitude bya calibrated custom-made system consisting of a linear amplifierand a ribbon loudspeaker placed 25 cm from the mouse head(AudioComm). The baseline and response images were acquired2 s before and 2 s after sound onset for each sound presentation.For each trial, the change in light reflectance (intrinsic signal)was computed and averaged for the 30 repetitions of same soundusing custom MatLab software. Two baseline images of differentsounds were used to compute artificial blank (Fig. S2C). Intrinsicimaging of animals used for ACFC was performed during thehabituation phase.

Quantitative Analysis of Spines. We calculated the rates of spineformation and spine elimination between two imaging sessions.We considered the number of spines present uniquely in earlierthe imaging session (n disappearing), the late session (n appearing),

or in both imaging sessions (n persistent). The rates of spinesformed (Fig. 4E) and eliminated (Fig. 4F) in 2-h intervals werecalculated as follows:

rappearing =nappearing

npersistent + nappearing;   rdisappearing =

ndisappearingnpersistent + ndisappearing

:

The formation rate of persistent spines was calculated as the num-ber of spines that appeared in session 2 (s2) and were present in s3divided by the total number of spines observed in s3 (Fig. 5A):

rpersistent =nappearing in s2 and present in s3

nall in s3:

Survival rate of spines newly formed after context exposure pairedor unpaired conditioning was calculated for spines that appearedin imaging session s2 as number of spines present in session sx (s3,s4, or s5) divided by the number of spines present in the precedingsession (Fig. 5 B–D):

rsurvival sx =nappearing in s2 and present in sx

nappearing in s2 and present in sx�1:

Data Processing. Data processing, including statistical analysis(correlation coefficients, ANOVA, Wilcoxon rank-sum test), wasperformed using MATLAB, including the Statistics Toolbox(MathWorks). The graphical representation of the data wasprepared using R (7). Bar plots were generated using the R gplotpackage. Images were processed using Adobe Photoshop. Fig-ures were assembled using Adobe Illustrator.

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3. Loewenstein Y, Kuras A, Rumpel S (2011) Multiplicative dynamics underlie theemergence of the log-normal distribution of spine sizes in the neocortex in vivo.J Neurosci 31(26):9481–9488.

4. Kopec CD, et al. (2007) A robust automated method to analyze rodent motion duringfear conditioning. Neuropharmacology 52(1):228–233.

5. Bathellier B, Ushakova L, Rumpel S (2012) Discrete neocortical dynamics predictbehavioral categorization of sounds. Neuron 76(2):435–449.

6. Bartho P, Curto C, Luczak A, Marguet SL, Harris KD (2009) Population coding of tonestimuli in auditory cortex: Dynamic rate vector analysis. Eur J Neurosci 30(9):1767–1778.

7. R Development Core Team (2012) R: A Language and Environment for StatisticalComputing (R Foundation for Statistical Computing, Vienna).

8. Paxinos G, Franklin KBJ (2001) The Mouse Brain In Stereotaxic Coordinates (AcademicPress, San Diego), 2nd Ed.

9. Govindarajan A, Israely I, Huang SY, Tonegawa S (2011) The dendritic branch is thepreferred integrative unit for protein synthesis-dependent LTP. Neuron 69(1):132–146.

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11. Branco T, Häusser M (2010) The single dendritic branch as a funda-mental functional unit in the nervous system. Curr Opin Neurobiol 20(4):494–502.

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Fig. S1. Histological verification of size and location of ACx lesions. (A) Coronal brain slices stained with nuclear marker DAPI with representative lesions ofthe ACx indicated by green arrowheads. (B) We analyzed the position and size of the lesioned area in the majority of lesioned mice (n = 11) (2). (Upper Left)Side view of a brain with lesioned area indicated by green arrowheads. (Lower Left) Same view of the brain with a template indicating the position of the ACxthat was constructed from a lateral projection of a mouse brain atlas and superimposed (8). The outline of the lesioned area is indicated by a black line. (UpperRight) Same view of the brain with the aligned outlines of the lesions in the left hemisphere from all analyzed mice. (Lower Right) Same analysis but showingsuperimposed lesion outlines in the right hemisphere. (C) Quantification of the average size of the lesioned area that was located within and outside of theACx according to the template. The size of the ACx template corresponds to 3.4 mm2. Bars represent mean ± SEM.

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Fig. S2. Quantification of sound-evoked intrinsic signals in the ACx. (A) Spectrogram representations of auditory stimuli used for intrinsic imaging. Pure tonesconsisted of 2-s-long sine waves of 2, 4, 8, or 16 kHz that were amplitude modulated at 10 Hz. Complex sounds consisted of 2-s-long snippets of arbitrary animalcalls or music pieces played fourfold faster than original sampling rate to optimally cover the hearing range of mice. All stimuli were calibrated to decibel SPLaverage intensity. (B) Average signal amplitude observed in response to pure tones and complex sounds normalized to largest observed mean response.Complex sounds with a broad frequency content strongly activate ACx, consistent with a tonotopic organization (n = 8 mice). Bars represent mean ± SEM. (C)Correlation analysis of the spatial structure in response patterns. Average cross-correlation matrix for all response images evoked by several complex sounds,pure tones, and artificial blank (Methods). Warm colors indicate high degree of similarity of spatial response patterns. The cluster of high correlations in thebottom right corner indicates that response patterns elicited by complex sounds are more similar to each other. Highest similarity in patterns evoked by puretone stimuli is observed with pure tone response patterns of similar frequency. Complex sound 1 marked with red color was used as CS in all conditioningexperiments. Other complex sounds 2–4 were only used for intrinsic imaging experiments.

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Fig. S3. Additional analysis of spine turnover rates. (A) Overview of the structure of the dataset acquired in mice from the paired conditioning group. (B)Same as A for dataset from unpaired conditioning group. (C) Same as A for dataset from context exposure group. (D) Analysis of mean spine formation rates inindividual dendrites observed in the three groups of mice for the three 2-h intervals (same as Fig. 4D). (E) Same data as D but combined across individualdendrites within same group and time point (s1→s2: paired: 16.1%, unpaired: 11.2%, context: 8.0%; χ2 test: P < 0.001; significant pairwise post hoc χ2 tests:paired vs. context, P < 0.001; paired vs. unpaired, P < 0.05; s3→s4: paired: 7.1%, unpaired: 7.9%, context: 6.5%; χ2 test: P = 0.72, n.s.; s4→s5: paired: 8.2%,unpaired: 7.6%, context: 5.8%; χ2 test: P = 0.38, n.s.). (F) Analysis of mean spine elimination rates in individual dendrites observed in the three groups of micefor the three 2-h intervals (same as Fig. 4E). (G) Same data as F, but again combined across individual dendrites within same group and time point (s1→s2:paired: 10.7%, unpaired: 16.0%, context: 7.5%; χ2 test: P < 0.001; significant pairwise post hoc χ2 tests: unpaired vs. context, P < 0.001; paired vs. unpaired, P <0.001; s3→s4: paired: 10.7%, unpaired: 9.5%, context: 8.7%; χ2 test: P = 0.54, n.s.; s4→s5: paired: 7.3%, unpaired: 5.6%, context: 6.9%; χ2 test: P = 0.96, n.s.).Asterisks indicate significant pairwise differences. Fraction of appearing (gray bars) and disappearing (white bars) spines shown for individual dendritesgrouped by neurons in paired (H), unpaired (I), and context exposure (J) experimental groups. The fraction of spines appearing on a given dendrite wascalculated by dividing the number of new spines by the total spine number observed in s2. Level of spines disappearing from the population was calculated bydividing the number of disappearing spines by the total number of spines observed on dendritic segment in s1. Total numbers of spines observed on dendritein imaging session s1 are indicated on white bars, and total numbers of spines observed in s2 are indicated on gray bars. Numbers of appearing and dis-appearing spines on a dendrite are positively correlated in paired (Pearson correlation coefficient: r = 0.38), unpaired (r = 0.44), and context exposure (r = 0.66)groups, consistent with a synaptic reorganization that is clustered on individual sections of the dendritic tree (9–12).

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Fig. S4. Survival rates of preexisting spines. (A) Mean survival probability of spines present at the beginning of the experiment in s1 in the three groups ofmice. Survival probability was calculated for the 2-h interval until s2 (s1→s2; paired: 88.3 ± 1.4%, unpaired: 85.6 ± 2.5%, context: 91.7 ± 1.2%; one-way ANOVA,P = 0.06, n.s.). Number of analyzed dendrites: 18 (paired), 19 (unpaired), 17 (context). (B) Similar analysis of survival probability of spines present in s1 as shownin A; however, survival was calculated for the duration of a week (s1→s3; paired: 59.9 ± 3.3%, unpaired: 56.1 ± 2.0%, context: 68.3 ± 2.7%; one-way ANOVA,P < 0.01; significant post hoc Wilcoxon rank-sum test: unpaired vs. context, P < 0.01). Asterisk indicates significant difference. Number of analyzed dendrites:14 (paired), 12 (unpaired), 15 (context).

Fig. S5. Model of structural synaptic changes accompanying paired and unpaired conditioning. Differential effects on synaptic populations were observedafter paired and unpaired conditioning. Paired conditioning correlated with a temporary increase in spine formation. These spines could be a part of a memorytrace and serve to encode novel associative memory. An excess of dendritic spines is removed after a longer period leading to a constant number of spines inthe population. Memory recall induces IEG expression but is not associated with structural rearrangements as observed during initial memory formation.Unpaired conditioning correlates with a temporary increased rate of spine elimination. Again, after a week, a similar number of spines is observed as beforeconditioning, indicating homeostatic mechanisms working at longer time scales than the immediate changes in rates following early after conditioning. Notethat this schematic illustrates only the conditioning-induced component of spine turnover. For clarity, the schematic does not reflect basal spine turnover,which significantly impacts the structure ACx connectivity independent of explicit learning experience (3).

Audio S1. The 2-s complex sound cue used as CS in the conditioning paradigms is accessible in wav file format.

Audio S1

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