Brain Struct Funct DOI 10.

1007/s00429-013-0596-5

ORIGINAL ARTICLE

Alterations of dendritic protrusions over the rst postnatal year of a mouse: an analysis in layer VI of the barrel cortex
David A. Orner Chia-Chien Chen Daniella E. Orner Joshua C. Brumberg

Received: 25 October 2012 / Accepted: 5 June 2013 Springer-Verlag Berlin Heidelberg 2013

Abstract Dendritic spines are small protrusions that serve as the principal recipients of excitatory inputs onto cortical pyramidal cells. Alterations in spine and lopodia density and morphology correlate with both developmental maturity and changes in synaptic strength. In order to better understand the developmental prole of dendritic protrusion (dendritic spines ? lopodia) morphology and density over the animals rst postnatal year, we used the Golgi staining technique to label neurons and their dendritic protrusions in mice. We focused on quantifying the density per length of dendrite and categorizing the morphology of dendritic protrusions of layer VI pyramidal neurons residing in barrel cortex using the computer assisted reconstruction program Neurolucida. We classied dendritic protrusion densities at seven developmental time points: postnatal day (PND) 15, 30, 60, 90, 180, 270, and 360. Our ndings suggest that the dendritic protrusions in layer VI barrel cortex pyramidal neurons are not static, and their density as well as relative morphological distribution change over time. We observed a signicant increase in mushroom spines and a decrease in lopodia as the animals matured. Further analyses show that as the animal mature there was a reduction in pyramidal cell dendritic lengths

overall, as well as a decrease in overall protrusion densities. The ratio of apical to basilar density decreased as well. Characterizing the prole of cortical layer VI dendritic protrusions within the rst postnatal year will enable us to better understand the relationship between the overall developmental maturation prole and dendritic spine functioning. Keywords Barrel cortex Layer VI Dendritic spines Development

Introduction There is a growing consensus that changes in cognitive abilities associated with aging are related to structural changes in cortical neurons and the networks in which they are embedded (see Power et al. 2010; Pannese 2011; Peters and Kemper 2012; Kolb and Teskey 2012; Morrison and Baxter 2012). Specically, alterations in dendritic architecture have been associated with aging and may underlie some aspects of cognitive decline (Harris and Kater 1994; Dumitriu et al. 2010; Kabaso et al. 2009; also see Pannese 2011; Dickstein et al. 2012; Morrison and Baxter 2012). Dendrites of pyramidal neurons, the most abundant cell type in the cortex, are adorned with small protrusions known as dendritic spines and lopodia that serve as their principal recipient of excitatory inputs and have been linked to neuronal learning and memory on a cellular level (Rochefort and Konnerth 2012). Alterations in dendritic protrusion (dendritic spines ? lopodia) density and morphology have been correlated with both developmental maturity and changes in synaptic strength. For example, lopodia are often considered as immature spines and become less numerous as animals age (Zuo et al. 2005b).

D. A. Orner J. C. Brumberg Neuroscience Major, Queens College, CUNY, Flushing, NY, USA D. A. Orner D. E. Orner J. C. Brumberg (&) Department of Psychology, Queens College, CUNY, 65-30 Kissena Boulevard, Flushing, NY 11367, USA e-mail: joshua.brumberg@qc.cuny.edu C.-C. Chen J. C. Brumberg Neuropsychology Ph.D. Subprogram (Psychology), The Graduate Center, CUNY, Flushing, NY, USA

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Furthermore, dendritic protrusions are the primary site of structural plasticity in cortical pyramidal cells (Holtmaat and Svoboda 2009) and function in neural information processing. However, the processes that underlie the development of dendritic protrusions and their maturation over an animals lifespan are not well understood, but of increasing importance due to the aging of the human population (see Yuste and Bonhoeffer 2004; Saneyoshi et al. 2010). The primary somatosensory cortex in rodents contains a region called the barrel cortex, which processes information from their prominent facial whiskers. The barrel cortex is anatomically structured into barrel columns, with a distinct topographic one-to-one relationship corresponding to the whiskers on the mystacial pad (Woolsey and Van der Loos 1970). The neocortex is divided into six layers and Layer VI originates cortical input to many cortical and subcortical locations (Brumberg et al. 2003; Thomson 2010) and has been previously used as a model to classify neurons (Chen et al. 2009) and study the impact of sensory experience (Chen et al. 2012a) and will be used in the present investigation. Given the role that dendritic protrusions have been posited to have in modifying synaptic inputs onto neurons (Kawato et al. 1984; Lee et al. 2012) it is important to understand how their dendrites and associated protrusions develop and change over the rodents rst postnatal year. Dendritic protrusions have generally been classied morphologically into specic categories based on their length, size and shape (Peters and Kaiserman-Abramof 1970; Harris and Stevens 1989; Lendvai et al. 2000; Richardson et al. 2009). The morphology of each dendritic protrusion category is related to its stability and synaptic strength. For example, stubby spines have been shown to be transitional structures that will either enlarge when strengthened, or shrink due to synaptic weakening (Holtmaat et al. 2005) whereas, mushroom spines that have relatively large spine heads are stable for weeks to months and mediate strong synapses, while, lollipop spines, which have smaller spine heads, are mainly short-term spines, forming weaker synapses and are associated with a higher degree of plasticity (Kasai et al. 2003; Takumi et al. 1999; Matsuzaki et al. 2001, 2004; Yasumatsu et al. 2008; Zuo et al. 2005a; Bourne and Harris 2007). Dendritic spine loss likely occurs as a consequence of aging in most if not all mammalian species. It has been demonstrated that as the animals mature, the dendritic protrusion density in cortical layer V apical dendrites steadily decreased (Zuo et al. 2005b; Yang et al. 2009). Feldman and Dowd (1975) reported a 2040 % loss of protrusions as a consequence of aging, with nearly 50 % of these protrusions lost with aging being lollipop spines, while there is little loss of mushroom or stubby spines (Dumitriu et al. 2010), as these persistent spines are associated with life-long memories (Yang et al. 2009). To further quantify the impact of

normal aging on dendritic protrusions in the mouse, we investigated their density and morphology over the rst postnatal year.

Materials and methods Experimental animals A total of 28 CD-1 mice of either sex between the ages of 15 and 360 postnatal days (PND) were used. There were seven groups (n = 4 each) at distinct developmental time points; PND 15, 30, 60, 90, 180, 270 and 360. Prior to PND 15 our staining method was ineffective, PND 30 is approximately when mice reach sexual maturity, PND 60 was chosen to allow for future comparisons with previous studies (Chen et al. 2012a) and the later ages were chosen to effectively span the rst postnatal year. Animals were housed in polycarbonate cages with woodchip oors on a 12 h:12 h light:dark cycle at room temperature and allowed access to rat chow and water ad libitum. Experimental protocols were approved by the Queens College, CUNY, Institutional Animal Care and Use Committee and in accordance with NIH guidelines concerning the use of laboratory animals. Golgi staining At the appropriate age, mice were anesthetized with an intraperitoneal injection of Euthasol (sodium pentobarbital, Virbac Animal Health, Inc.). When the animals become nonresponse (by toe-pinch) brains were promptly removed and placed in either 0.1M phosphate buffer (PB; pH 7.13) or distilled H2O for 3 min (there was found to be no difference between brains processed in either solution). Upon rinsing with PB, brains were immersed in a Golgi-Cox solution comprising potassium dichromate, mercuric chloride, and potassium chromate. This mixture was replaced after 12 h of initial immersion, followed by storage at room temperature in darkness for 2/3 weeks. After immersion in the Golgi-Cox solution, brains were placed in a cryoprotectant solution (FD Rapid Golgi Stain Kit, FD Neurotechnologies) and stored at 4 C for 1 week in darkness before slicing. Coronal slices of approximately 100120 lm were made on a freezing cryostat (approximately -25 C). In order to prevent ice crystal damage, tissues were frozen with dry ice and quickly embedded in an optimal cutting temperature (OCT) medium. The slices were then placed onto triple-dipped gelatin slides and then coated with additional cryoprotectant. Slices were air dried at room temperature in darkness for at least 23 weeks before further processing. After this drying period, sections were rinsed with distilled water and were subsequently stained in a developing solution (FD Rapid Golgi

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Stain Kit, FD Neurotechnologies) and dehydrated successively in solutions of 50, 75, 95, and 100 % ethanol in distilled H2O. The sections were then defatted in a xylene substitute (SafeClearII, ThermoFisher Scientic) and cover slipped with Permount (Sigma-Aldrich). Identication of the barrel eld In order to accurately identify the barrel cortex as we have done previously (Chen et al. 2009), the characteristic clusters of cells observed were matched with an atlas of a Golgi-stained mouse brain (Valverde 1998). The anterior limit of the barrel cortex was identied by the appearance of the anterior commissure. Layer VI neurons were identied by their relative location inferior to the large pyramidal cells found in layer V and the white matter at the limit of the cortical plate (Fig. 1a). The posterior limit of the barrel cortex was identied by the separation of the corpus callosum at the midline. Neuronal selection/reconstruction/dendritic protrusion classication Imaging of neurons and their respective dendritic protrusions (spines and lopodia) was accomplished with

Neurolucida 9.0 and 10.0 software (MBF Bioscience, Inc.) and an Olympus Bx51 microscope equipped with a highresolution digital camera (Optronics Microre). Neurons were initially imaged under 109 magnication (0.4 numerical aperture (NA)) in layer VI to ensure complete staining of the cell. This was done by observing the dendrites and ensuring that they are tapered to a ne point (a hallmark of a well-lled neuron and uncut dendrites); neurons that had truncated and/or non-tapering dendrites were not included in our analysis. Only the neurons that exhibited complete Golgi impregnation with limited amount of staining artifacts were traced. Each pyramidal cell for future reconstruction and dendritic spine analysis was labeled with a marker to allow for easy identication at higher magnication (Fig. 1b). At 609 oil magnication (1.42 NA), pyramidal cells were further examined for uncut basilar dendrites of length [100 lm that ended in a ne taper. Finally, at 1009 oil magnication (1.40 NA), the dendrite would be examined to ensure that its dendritic protrusions had been thoroughly labeled with the GolgiCox solution to reveal their distinct morphologies (Fig. 1c). At this point, the neuron would be reconstructed followed by classifying the dendritic protrusions into one of ve categories (see Fig. 1d, classication scheme adapted from Greenough; Irwin et al. 2000, Chen et al.

Fig. 1 Golgi stained mouse brain section of the barrel cortex. Low magnication image a illustrating the barrel cortex and its six layers in between the pia and the white matter. The box represents the region magnied in b. Higher magnication illustrating the pyramidal neurons of layer VI. c The scale in which spines were reconstructed from an apical dendrite of a pyramidal neuron located in layer VI. d Representative reconstruction of classication of dendritic

protrusions: Stubby spines are spines with no necks and a stubbylike appearance. Lollipop spines are spines with thin necks and small heads. Mushroom spines are spines with small necks and a large often complex and irregular head. Filopodia spines are single long thin protrusions with no obvious head. Branched lopodia spines are bifurcated thin lopodia spines with no obvious heads

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Brain Struct Funct Table 1 Dendritic parameters

PND 15 Animals (n) Neurons Average total # of dendritic protrusions Apical Basilar Average total length (lm) Apical Basilar Average density (protrusions/ 10 lm) Apical Basilar 251.68 50.87 7.22 0.31 5.24 0.68 6.03 0.36 4.28 0.28 4.81 0.28 3.29 0.37 3.30 0.25 3.07 0.28 2.78 0.20 2.97 0.23 2.46 0.16 2.87 0.24 2.19 0.20 705.95 86.11 251.68 50.88 745.90 21.04 271.25 123.17 750.62 100.01 284.10 31.01 799.95 106.84 280.07 21.25 576.48 58.45 201.84 32.85 504.13 32.44 175.91 28.60 465.53 60.68 163.37 24.95 437.47 56.68 181.27 34.78 390.20 95.97 161.80 69.01 320.80 46.00 136.33 13.72 260.80 27.53 92.47 11.34 177.20 24.63 56.00 9.21 147.20 16.63 43.20 7.55 133.33 20.06 35.87 7.08 4 15 PND 30 4 15 PND 60 4 15 PND 90 4 15 PND 180 4 15 PND 270 4 15 PND 360 4 15

Data represents population means one standard deviation

2012b) by labeling each type of dendritic protrusion with a unique marker. Fifteen neurons were identied from four animals at each age (34 neurons/animal) and they were reconstructed and their dendrites and protrusions were quantied (see Table 1). For each neuron the apical dendrite and the longest basilar dendrite were reconstructed to facilitate direct comparisons. Statistical methods of analyzing dendritic protrusion density/morphology The NeuroExplorer software (MBF Bioscience, Inc.) allowed us to calculate overall dendritic protrusion densities. Protrusion density was calculated as the total number of protrusions on a dendrite divided by the length of that dendrite (lm) and then expressed as the number of protrusions per 10 lm of dendrite. Cell bodies, apical dendrites, and the longest basilar dendrite were completely reconstructed. Dendritic protrusion types (lollipop, mushroom, branched, lopodia and stubby) were marked on the apical and the basilar dendrites from neurons at different developmental time points throughout the rst postnatal year of the animal (Fig. 2a). Mushrooms are spines with a stalk with a conspicuous head on it. Filopodia are thought to be immature protrusions that have a long, slender stalk, and lack a clear head. In contrast, stubby spines are the ones that have a short stout stalk (Fig. 1d). Dendritic protrusions will be used to refer to all spines and lopodia on basilar or apical dendrite at specic developmental ages, whereas we will use the specic names (e,g., mushroom vs. stubby spines) when comparing morphological types, as we have done previously (Chen et al. 2012b). The mean and standard deviation (mean SD) were computed for the spine density

(dendritic protrusions per 10 lm) for each dendritic protrusion type for both the apical and basilar layer VI dendrites. To determine if certain regions of the dendritic tree were more likely to lose dendritic protrusions in older age groups, we performed a Sholl analysis on all reconstructed neurons (1953) in which the cell body is centered in the middle of concentric spheres that are separated by 10 lm. We quantied the number and length of dendrites in each sphere as well as the density and type of dendritic protrusion as a function of developmental age and distance from the soma. Throughout the development of the animal, we observed a net loss of dendritic protrusions (protrusions lost/day), which was calculated by the difference in the density at the specic postnatal dates. The metric was calculated by determining the spine density difference between any two consecutive time points and dividing by the number of days between the two ages. For statistical analyses we conducted a mixed-factorial analyses of variance (ANOVA), followed by appropriate post hoc conrmations (Tukey HSD) between each developmental group to rst determine and then conrm statistical signicance (if any). Statistical signicance was achieved if calculated p values were less than 0.05. Statistical tests were run using SigmaStat (version 3.5, Systat Software Inc.) on a PC.

Results Density of dendritic protrusions decreases across the rst postnatal year Within layer VI pyramidal cells it was observed that dendritic protrusion density decreased across the rst year of

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Fig. 2 Pyramidal neuron located in layer VI. Representative dendritic micrographs a demonstrating the presence of dendritic protrusions of different morphological types on apical dendrites from postnatal day 15 through postnatal day 360. Representative micrographs were taken at 100x and then a threshold applied to remove the background for visualization purposes. b Density of dendritic protrusions in normal development on apical and basilar dendrites

of animals throughout development. Plots represent population means one standard deviation. c Ratio of dendritic protrusions in basilar versus apical dendrites in normal development. Plots represent population means one standard deviation. d Rate of dendritic protrusion loss in normal development on apical and basilar dendrites on animals throughout developments. Plots represent population means one standard deviation

the animals life (Fig. 2). Decreases were seen from our initial time epoch from PND 15 to PND 30, as well as continuous decreases in the dendritic protrusion density on the apical dendrite from PND 60 until PND 360 (see Fig. 2b). At PND 15, the mean (and SD) of apical protrusion density (expressed as number of dendritic protrusions per every 10 lm dendritic length) was 6.19 0.18 whereas by PND 30 it had signicantly (p \ 0.05) decreased to 5.24 0.68. This indicates that there was a signicant amount of dendritic protrusion pruning occurring during our rst time epoch, which is consistent with previously published ndings (Zuo et al. 2005b; Elston et al. 2009, 2010; Yang et al. 2009). However, after PND 30, there were no signicant changes in protrusion density over the next month. At PND 60 the spine density was 4.28 0.28, suggesting the rate of dendritic protrusion pruning/elimination had decreased. As development progressed, the density of the apical dendrite decreased signicantly after PND 60 from PND 90 (3.29 0.37) to PND 180 (3.07 0.28) to PND 270 (2.92 0.23) until PND 360 (2.87 0.24). The basilar dendrite showed similar ndings. There were signicant (ps \ 0.01) changes in the density of the basilar dendrite after PND 60 (4.81 0.28) from PND 90 (3.29 0.25) to PND 180

(2.78 0.20) to PND 270 (2.46 0.16). However, after PND 270 the density on the basilar dendrite remained relatively static with no further signicant decreases at PND 360 (2.19 0.20, p [ 0.05). We then calculated the dendritic protrusion density ratio comparing the basilar to the apical dendrite (Fig. 2c). As development progresses, the density ratio signicantly decreased between the time points PND 15 (1.17 0.06) to PND 360 (0.76 0.10) due to decreases in basilar dendritic protrusion density and relatively smaller decreases in apical protrusion density. We compared the dendritic protrusion density between apical and basilar dendrites and found that until PND 90 the basilar dendrites had a higher density and then afterwards the apical dendrite had a higher density (ps \ 0.05, Fig. 2b). Thus, the rate of protrusion loss differs between the apical and basilar dendrites. Finally, we computed the rate of dendritic protrusion loss (see Materials and methods) for both the apical and basilar dendrites and saw that the greatest rates of elimination were during the rst two postnatal months (Fig. 2d). Environmental stress and gender have been shown to inuence dendritic protrusion density in pyramidal cells in CA1 (Shors et al. 2001, 2004; Leuner and Shors 2012) although in the neocortex under baseline conditions such differences have been reported to

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be minimal (Teskey et al. 1999). To examine this post hoc we compared the protrusion density of each dendrite to the population mean for that dendrite type (apical or basilar) at each developmental age. The variance from the mean across our entire data set ranged from 2.2 % (PND 360) to 7.2 % (PND 15) suggesting that within our data set gender had a relatively minimal impact on our results. Dendritic protrusion morphology We also assessed potential changes in protrusion morphology over development. We grouped protrusions with similar morphologies (see Fig. 1d), which resulted in ve morphologically distinct subgroups: branched, lopodia, stubby, lollipop and mushroom. Figure 3a shows the distribution of different categories of dendritic protrusions in apical dendrites across development. Mean percent distribution for apical PND 15 animals were: branched (5.74 % 0.91), lopodia (15.67 % 1.62), stubby (42.92 % 2.86), lollipop (21.49 % 1.96), mushroom (14.18 % 2.06). Stubby, lollipop and lopodia protrusions were most prevalent across all age groups. Figure 3b shows the distribution of the different morphological groups of dendritic protrusions in basilar dendrites across

age groups. The mean percent morphological distribution for basilar PND 15 mice were: branched (5.54 % 1.61), lopodia (16.56 % 2.31), stubby (43.04 % 3.07), lollipop (20.42 % 1.62), mushroom (14.45 % 1.61). Similar to apical dendrites, stubby, lollipop and lopodia mushrooms were most prevalent at PND 15 in layer VI dendrites. Over development there was an increase in the relative percentage of lollipop and mushroom spines and a decrease in stubby, lopodia, and branched lopodia. There was relatively no change in spine morphology distribution after PND 90 on the basilar dendrites. Thus, over development in basilar dendrites there was an increase in lollipop and mushroom spines and a decrease in stubby, lopodia, and branched lopodia of protrusions and relatively no change in spine morphology distribution after PND 90 on the dendrites. Our ndings suggest that the dendritic protrusions in layer VI of barrel cortex pyramidal neurons are not static and their density and relative morphological distribution change over time. It was observed in both apical and basilar dendrites that there is a signicant decrease in stubby, lopodia, and branched protrusions when comparing their densities at PND 15 with those observed at PND 30. Similarly there were further decreases when comparing PND 30 to PND 90.

Fig. 3 Distribution of dendritic protrusions during normal development. Distribution of Layer VI dendritic protrusions on apical (a) and basilar (b) dendrites during normal development

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However, after PND 90 the distribution of stubby, lopodia, and branched protrusions remained stable throughout the remainder of the rst postnatal year (ps [ 0.05). In contrast, mushroom spines signicantly increased in apical dendrites with the exception of PND 15 to PND 30 (p = 0.119), PND 60 to PND 90 (p = 0.434), and PND 90 to PND 180 (p = 0.833). However, in basilar dendrites, mushrooms signicantly increased in all groups except for PND 15 to PND 30 (p = 0.405) and PND 90 to PND 180 (p = 0.833). Over development (PND 15 to 360) there was a signicant increase in lollipop spines in both the apical and basilar dendrites (ps \ 0.05). Given that we observed a signicant increase in mushroom spines and a decrease in lopodia as the animals matured, we sought to determine if the changes were uniform across the extent of the dendrite or regionalized (e.g. distal vs. proximal locations). Sholl analysis (1953) data were gathered to assess the relative location of each protrusion type and what the impact of aging was on their relative distribution along a dendrite. In general it was observed that protrusions were more likely to be initially lost at the most distal locations of the dendrites. Specically, it was observed in both the apical and basilar dendrites (Figs. 4, 5), that there was a larger loss of dendritic protrusions distally than proximally in lollipop, stubby, lopodia, branched, and overall. In contrast, mushroom spines were lost more proximally than distally across age groups (mixed ANOVA, Tukeys post hoc tests, ps \ 0.001). Dendritic length We examined if dendritic length also changed as a function of developmental age. Representative complete reconstructions (Fig. 6a) were combined with the apical and longest basilar dendrite reconstructions (see Materials and methods) and we quantied their lengths. We found that the average total dendritic length increased until PND 90, followed by a decrease until PND 360 for both the apical and basilar dendrites (see Fig. 6b). The apex of the dendritic length in both apical and basilar dendrites was seen at PND 90. This is a time point after the start of dendritic protrusion loss was rst observed the PND 15 to PND 30 epoch. Furthermore, across all age groups, the average total length of the apical dendrite decreased 34.15 % and the basilar dendrite decreased 35.09 %.

Discussion Our ndings demonstrate that the dendritic protrusions in layer VI barrel cortex pyramidal neurons are not static and their density and relative morphological distribution change over time. Consistent with previous experiments,

we found that in normal development, there is a decrease in the dendritic spine density, and dendritic length as an animal gets older (Feldman and Dowd 1975), our results extend on these ndings by showing how different morphological types of dendrites change as a function of age. Specically, we observed a signicant increase in mushroom spines and a decrease in lopodia as the animals matured. Similar to Benavides-Piccione et al. (2012), we found a lower protrusion density and shorter length of dendrites in basilar compared with apical dendrites, as well as a decline in small and short dendritic protrusions in both basal and apical dendrites and an increase in lollipop spines. These results are in agreement with previous studies performed in monkeys which found that spine densities and length were signicantly reduced in apical dendrites with aging (Kabaso et al. 2009). Spine neck width and length has long been associated with the efciency of synaptic transmission with wider and shorter spine necks being associated with more efcient transmission of synaptic signals (Araya et al. 2006; Koch and Zador 1993; Koch et al. 1992). Given the noted increase in mushroom spines, which have narrow necks, this may suggest an increase in synaptic efciency as a result of cortical maturation. Further analysis shows that across age groups there was a reduction in overall spine densities. The ratio of basilar spine density to apical spine density decreased as well, which is consistent with previous observations found in aging rat, macaque monkeys, and human studies (Jacobs et al. 1997; Duan et al. 2003; Dumitriu et al. 2010) that show spine loss is a consequence of aging although recent reports suggest at least some cellular phenotypes do not show signicant losses (Mostany et al. 2013). Our results show that the rate of loss is different for the apical and basilar dendrites which might suggest that certain inputs are more susceptible to being lost during normal aging. Specically, the rate of loss is greater for basilar dendrites. However, in contrast to patterns seen in aging macaque monkey prefrontal cortex (PFC) where thin spines (what we term lollipop spines) are mainly lost while the larger mushroom spines remain (Hao et al. 2007; Dumitriu et al. 2010), our results found that lollipop spines remained relatively stable with insignicant changes in morphology as the animal aged. Simultaneously, stubby, and lopodia protrusions were mainly lost while larger mushroom spines and lollipop spines remained the dominant spines. This discrepancy might be due to the different locations and species of the experiments since both Hao et al. (2007) and Dumitriu et al. (2010) focused on pyramidal cells that were located in primate prefrontal cortex layer III, whereas we examined pyramidal neurons from layer VI of the rodent barrel cortex. Methodologically, it is possible that a subset of the stubby spines, particularly the thin subtypes, are what other studies have categorized as lopodia (Zuo et al.

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Fig. 4 Dendritic protrusions vary as a function of distance from the soma on the apical dendrite. The plot represents the mean number of dendritic protrusions between 10 lm concentric spheres on the apical dendrites during normal development. a lollipop spines, b mushroom

spines, c stubby spines, d lopodia, e branched lopodia, f overall protrusion density. Note that spine loss is most profound at the more distal locations as the animal matures

2005a, b), especially given that the morphological distribution of our stubby spines ? lopodia yielded similar proportions of total dendritic protrusions to these previously published studies. In contrast to the above mentioned studies we used the Golgi method which has been shown in comparison to neurons stained intracellularly to slightly underestimate overall spine densities (Ruan et al. 2009). It

is possible due to the resolution of the light microscope that we are incorrectly estimating the distribution of different dendritic protrusions since protrusions projecting vertically up or down relative to the objective could be obscured by the parent dendrite or mischaracterized (if only their distal end is observed, and thus it is possible that spines were incorrectly assigned to a particular group (e.g., two spines

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Fig. 5 Dendritic protrusions vary as a function of distance from the soma on the basilar dendrite. The plots represent the protrusion density on basilar dendrites during normal developments a lollipop

spines, b mushroom spines, c stubby spines, d lopodia, e branched lopodia, f overall protrusion density. Note that protrusion loss is most profound at the more distal locations as the animal matures

close together may appear as a stubby spine). However, such errors, if any, should be consistent across developmental ages. Interestingly, despite this limitation the overall distribution of spine morphologies that we observed is comparable to what has been observed using electron microscopy (reviewed in Fiala and Harris 1999) and also seen with other more modern microscopy techniques (Dumitriu et al. 2010). Our ndings are consistent with

ndings that mushroom spines are characterized as being stable and are consistent with two-photon studies showing that the large majority of stable, persistent spines in vivo are mushroom-type spines (Trachtenberg et al. 2002; Holtmaat et al. 2005). Previous studies in the rodent neocortex have suggested that apical and basilar dendrites should be treated as separate components, as they often respond to the same

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Brain Struct Funct Fig. 6 Dendritic length decreases with age. Both apical and basilar dendrites decrease in length as a function of development. Representative complete reconstructions show less elaborate dendrites as a function of age (a) which is quantied in b Data represent population means one standard deviation

experimental manipulation in different manners (McAllister et al. 1995; Arellano et al. 2007; Benavides-Piccione et al. 2012; Garrett and Wellman 2009; Metz et al. 2009; Chen et al. 2012a). Similarly, our ndings in the current study have shown that the rate of protrusion loss is faster in basilar compared with apical dendrites. Across age groups we saw the rst signs of spine loss at the most distal regions. Consistent with this is the nding that separate locations (proximal and distal) of the dendritic arbor have distinct changes in response to an extrinsic cue (Yacoubian and Lo 2000), and similar phenomena has been observed in mouse genetic knockout models as well (Chen et al. 2012b). This suggests that developmental changes may be localized to specic neuronal arborizations. Our results show that the developmental morphological changes are localized to particular regions across age groups. Previous studies in the neocortex of mice have shown a lack of correlation between the spine size and the distance from the soma (Konur et al. 2003; Arellano et al. 2007; BenavidesPiccione et al. 2012), but they did not look at the consequence of aging. Our study clearly showed that as development progresses spines proximal to the soma are more likely to be retained.

Characterizing the prole of dendritic protrusions within layer VI over the rst postnatal year provides a better understanding of the developmental maturation of dendritic protrusions. Furthermore, our results show that specic classes of protrusions (e.g., mushrooms) are more likely to be retained throughout the rst postnatal year suggesting that these spines may be involved with synaptic pathways that are not easily altered such as long term memories. The knowledge gained by understanding spine morphology and development should help pave the way for therapeutics aimed at synaptic preservation or regeneration in both normal aging and in disease states.
Acknowledgments The work was supported by a DSC award to C-C Chen and PSC-CUNY 62750-00 40 and NS058758 to J.C.B. We thank Dr. Carolyn Pytte and Dr. Stephan F. Brumberg for helpful comments on the manuscript.

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