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Alterations of dendritic protrusions over the ﬁrst 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 Ó 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 ﬁlopodia density and morphology correlate with both developmental maturity and changes in synaptic strength. In order to better understand the developmental proﬁle of dendritic protrusion (dendritic spines ? ﬁlopodia) morphology and density over the animal’s ﬁrst 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 classiﬁed dendritic protrusion densities at seven developmental time points: postnatal day (PND) 15, 30, 60, 90, 180, 270, and 360. Our ﬁndings 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 signiﬁcant increase in mushroom spines and a decrease in ﬁlopodia 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 proﬁle of cortical layer VI dendritic protrusions within the ﬁrst postnatal year will enable us to better understand the relationship between the overall developmental maturation proﬁle and dendritic spine functioning. Keywords Barrel cortex Á Layer VI Á Dendritic spines Á 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). Speciﬁcally, 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 ﬁlopodia 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 ? ﬁlopodia) density and morphology have been correlated with both developmental maturity and changes in synaptic strength. For example, ﬁlopodia are often considered as immature spines and become less numerous as animals age (Zuo et al. 2005b).
D. A. Orner Á J. C. Brumberg Neuroscience Major, Queens College, CUNY, Flushing, NY, USA D. A. Orner Á D. E. Orner Á J. C. Brumberg (&) Department of Psychology, Queens College, CUNY, 65-30 Kissena Boulevard, Flushing, NY 11367, USA e-mail: email@example.com C.-C. Chen Á J. C. Brumberg Neuropsychology Ph.D. Subprogram (Psychology), The Graduate Center, CUNY, Flushing, NY, USA
Coronal slices of approximately 100–120 lm were made on a freezing cryostat (approximately -25 °C).). but of increasing importance due to the aging of the human population (see Yuste and Bonhoeffer 2004. After this drying period. PND 15. 2009). Lendvai et al. 2009).Brain Struct Funct Furthermore. stubby spines have been shown to be transitional structures that will either enlarge when strengthened. while there is little loss of mushroom or stubby spines (Dumitriu et al. the dendritic protrusion density in cortical layer V apical dendrites steadily decreased (Zuo et al. For example. It has been demonstrated that as the animals mature. To further quantify the impact of normal aging on dendritic protrusions in the mouse. sections were rinsed with distilled water and were subsequently stained in a developing solution (FD Rapid Golgi 123 . Bourne and Harris 2007). The primary somatosensory cortex in rodents contains a region called the barrel cortex. 2000. 2012) it is important to understand how their dendrites and associated protrusions develop and change over the rodent’s ﬁrst postnatal year. Yang et al. Institutional Animal Care and Use Committee and in accordance with NIH guidelines concerning the use of laboratory animals. FD Neurotechnologies) and stored at 4 °C for 1 week in darkness before slicing. 2004. pH 7. Virbac Animal Health. which processes information from their prominent facial whiskers. The morphology of each dendritic protrusion category is related to its stability and synaptic strength. 180. 270 and 360.13) or distilled H2O for 3 min (there was found to be no difference between brains processed in either solution). However.1M phosphate buffer (PB. Yasumatsu et al. or shrink due to synaptic weakening (Holtmaat et al. 2005a. 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. Thomson 2010) and has been previously used as a model to classify neurons (Chen et al. Lee et al. Slices were air dried at room temperature in darkness for at least 2–3 weeks before further processing. The slices were then placed onto triple-dipped gelatin slides and then coated with additional cryoprotectant. tissues were frozen with dry ice and quickly embedded in an optimal cutting temperature (OCT) medium. with nearly 50 % of these protrusions lost with aging being lollipop spines. while. brains were immersed in a Golgi-Cox solution comprising potassium dichromate. Richardson et al. This mixture was replaced after 12 h of initial immersion. Harris and Stevens 1989. 2003. size and shape (Peters and Kaiserman-Abramof 1970. 2010). Prior to PND 15 our staining method was ineffective. 2012a) and the later ages were chosen to effectively span the ﬁrst postnatal year. Dendritic spine loss likely occurs as a consequence of aging in most if not all mammalian species. we investigated their density and morphology over the ﬁrst postnatal year. Inc. Experimental protocols were approved by the Queens College. Given the role that dendritic protrusions have been posited to have in modifying synaptic inputs onto neurons (Kawato et al. Golgi staining At the appropriate age. 30. 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. Takumi et al. PND 60 was chosen to allow for future comparisons with previous studies (Chen et al. dendritic protrusions are the primary site of structural plasticity in cortical pyramidal cells (Holtmaat and Svoboda 2009) and function in neural information processing. 2010). 2008. 2001. Feldman and Dowd (1975) reported a 20–40 % loss of protrusions as a consequence of aging. In order to prevent ice crystal damage. Upon rinsing with PB. Matsuzaki et al. lollipop spines. forming weaker synapses and are associated with a higher degree of plasticity (Kasai et al. 2005b. When the animals become nonresponse (by toe-pinch) brains were promptly removed and placed in either 0. mushroom spines that have relatively large spine heads are stable for weeks to months and mediate strong synapses. brains were placed in a cryoprotectant solution (FD Rapid Golgi Stain Kit. and potassium chromate. Dendritic protrusions have generally been classiﬁed morphologically into speciﬁc categories based on their length. mercuric chloride. 2009). followed by storage at room temperature in darkness for 2/3 weeks. There were seven groups (n = 4 each) at distinct developmental time points. 1999. PND 30 is approximately when mice reach sexual maturity. 2012a) and will be used in the present investigation. 2009) and study the impact of sensory experience (Chen et al. The barrel cortex is anatomically structured into barrel columns. 90. Animals were housed in polycarbonate cages with woodchip ﬂoors on a 12 h:12 h light:dark cycle at room temperature and allowed access to rat chow and water ad libitum. 2003. are mainly short-term spines. CUNY. 60. 1984. mice were anesthetized with an intraperitoneal injection of Euthasol (sodium pentobarbital. which have smaller spine heads. 2005) whereas. Zuo et al. After immersion in the Golgi-Cox solution. Saneyoshi et al. the processes that underlie the development of dendritic protrusions and their maturation over an animal’s lifespan are not well understood. as these persistent spines are associated with life-long memories (Yang et al.
1a).0 and 10. The anterior limit of the barrel cortex was identiﬁed by the appearance of the anterior commissure.) and an Olympus Bx51 microscope equipped with a highresolution digital camera (Optronics Microﬁre). the characteristic clusters of cells observed were matched with an atlas of a Golgi-stained mouse brain (Valverde 1998).40 NA).Brain Struct Funct Stain Kit. At this point. ThermoFisher Scientiﬁc) and cover slipped with Permount (Sigma-Aldrich). 1c). Only the neurons that exhibited complete Golgi impregnation with limited amount of staining artifacts were traced. and 100 % ethanol in distilled H2O. Neuronal selection/reconstruction/dendritic protrusion classiﬁcation Imaging of neurons and their respective dendritic protrusions (spines and ﬁlopodia) was accomplished with Neurolucida 9. the neuron would be reconstructed followed by classifying the dendritic protrusions into one of ﬁve categories (see Fig. Each pyramidal cell for future reconstruction and dendritic spine analysis was labeled with a marker to allow for easy identiﬁcation at higher magniﬁcation (Fig. The posterior limit of the barrel cortex was identiﬁed by the separation of the corpus callosum at the midline. Neurons were initially imaged under 109 magniﬁcation (0. Irwin et al.4 numerical aperture (NA)) in layer VI to ensure complete staining of the cell. Chen et al. at 1009 oil magniﬁcation (1. d Representative reconstruction of classiﬁcation of dendritic protrusions: Stubby spines are spines with no necks and a stubbylike appearance. This was done by observing the dendrites and ensuring that they are tapered to a ﬁne point (a hallmark of a well-ﬁlled neuron and uncut dendrites). 2000.0 software (MBF Bioscience. 1b). 2009). c The scale in which spines were reconstructed from an apical dendrite of a pyramidal neuron located in layer VI. FD Neurotechnologies) and dehydrated successively in solutions of 50. 1 Golgi stained mouse brain section of the barrel cortex. Inc. Mushroom spines are spines with small necks and a large often complex and irregular head. pyramidal cells were further examined for uncut basilar dendrites of length [100 lm that ended in a ﬁne taper.42 NA). neurons that had truncated and/or non-tapering dendrites were not included in our analysis. 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. 75. 95. 1d. Lollipop spines are spines with thin necks and small heads. Layer VI neurons were identiﬁed 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. Low magniﬁcation image a illustrating the barrel cortex and it’s six layers in between the pia and the white matter. The sections were then defatted in a xylene substitute (SafeClearII. At 609 oil magniﬁcation (1. Identiﬁcation of the barrel ﬁeld In order to accurately identify the barrel cortex as we have done previously (Chen et al. The box represents the region magniﬁed in b. Higher magniﬁcation illustrating the pyramidal neurons of layer VI. classiﬁcation scheme adapted from Greenough. Branched ﬁlopodia spines are bifurcated thin ﬁlopodia spines with no obvious heads 123 . Fig. Filopodia spines are single long thin protrusions with no obvious head. Finally.
mushroom vs.28 4.10 ± 31.30 ± 0.95 437. followed by appropriate post hoc conﬁrmations (Tukey HSD) between each developmental group to ﬁrst determine and then conﬁrm statistical signiﬁcance (if any). Statistical tests were run using SigmaStat (version 3.33 ± 13.87 7.07 ± 0.29 ± 0. whereas we will use the speciﬁc names (e.80 ± 27. as we have done previously (Chen et al. 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.17 750.81 ± 0.00 136.19 ± 0.55 133.85 504.62 ± 100.16 2.78 390.84 280.97 ± 0.72 260. We quantiﬁed 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.21 147. 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.06 35.33 ± 20. For statistical analyses we conducted a mixed-factorial analyses of variance (ANOVA).87 ± 7.25 ± 123.11 251.24 ± 0.04 271.46 ± 0.80 ± 69.78 ± 0. 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.28 ± 0. In contrast. stubby spines are the ones that have a short stout stalk (Fig.20 705.53 ± 60.63 56.68 6. For each neuron the apical dendrite and the longest basilar dendrite were reconstructed to facilitate direct comparisons.07 ± 21.25 3.95 ± 106. slender stalk.00 ± 9.08 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. apical dendrites.47 ± 56. 2a).37 3.03 ± 0.20 ± 7.91 ± 28.20 ± 16. Results Density of dendritic protrusions decreases across the ﬁrst postnatal year Within layer VI pyramidal cells it was observed that dendritic protrusion density decreased across the ﬁrst year of 123 .22 ± 0. ﬁlopodia and stubby) were marked on the apical and the basilar dendrites from neurons at different developmental time points throughout the ﬁrst postnatal year of the animal (Fig. Filopodia are thought to be immature protrusions that have a long.90 ± 21. 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. Inc.63 43. Statistical methods of analyzing dendritic protrusion density/morphology The NeuroExplorer software (MBF Bioscience.88 745.25 576. we observed a net loss of dendritic protrusions (protrusions lost/day).27 ± 34.44 175. stubby spines) when comparing morphological types.05.34 177.24 2. and the longest basilar dendrite were completely reconstructed. 1d).g.31 5. Systat Software Inc. which was calculated by the difference in the density at the speciﬁc postnatal dates. and lack a clear head. branched.20 ± 95. To determine if certain regions of the dendritic tree were more likely to lose dendritic protrusions in older age groups.28 3.) on a PC.68 181.5.01 799.01 320.36 4. Fifteen neurons were identiﬁed from four animals at each age (3–4 neurons/animal) and they were reconstructed and their dendrites and protrusions were quantiﬁed (see Table 1).13 ± 32.95 ± 86. Statistical signiﬁcance was achieved if calculated p values were less than 0.53 92. mushroom.. Dendritic protrusion types (lollipop.97 161. Mushrooms are spines with a stalk with a conspicuous head on it.01 284. 2012b).23 2.45 201.68 163.37 ± 24.84 ± 32. Dendritic protrusions will be used to refer to all spines and ﬁlopodia on basilar or apical dendrite at speciﬁc developmental ages.) allowed us to calculate overall dendritic protrusion densities.68 ± 50.87 ± 0. Throughout the development of the animal.47 ± 11.20 2.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 251.60 465.20 ± 24.48 ± 58.68 ± 50.80 ± 46.28 2. Cell bodies.
76 ± 0. Decreases were seen from our initial time epoch from PND 15 to PND 30.19 ± 0. which is consistent with previously published ﬁndings (Zuo et al.28) from PND 90 (3.10) due to decreases in basilar dendritic protrusion density and relatively smaller decreases in apical protrusion density. 2d).17 ± 0.20. We then calculated the dendritic protrusion density ratio comparing the basilar to the apical dendrite (Fig. the density of the apical dendrite decreased signiﬁcantly after PND 60 from PND 90 (3. suggesting the rate of dendritic protrusion pruning/elimination had decreased. Plots represent population means ± one standard deviation the animal’s life (Fig.37) to PND 180 (3. Plots represent population means ± one standard deviation.81 ± 0. Representative micrographs were taken at 100x and then a threshold applied to remove the background for visualization purposes. 2009).29 ± 0. 2 Pyramidal neuron located in layer VI. This indicates that there was a signiﬁcant amount of dendritic protrusion pruning occurring during our ﬁrst time epoch.46 ± 0.23) until PND 360 (2. 2001. However.01) changes in the density of the basilar dendrite after PND 60 (4.05) decreased to 5. 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. As development progressed.Brain Struct Funct Fig.18 whereas by PND 30 it had signiﬁcantly (p \ 0.28.24). The basilar dendrite showed similar ﬁndings.25) to PND 180 (2. 2005b.16).28) to PND 270 (2. Finally.29 ± 0. c Ratio of dendritic protrusions in basilar versus apical dendrites in normal development.05. Plots represent population means ± one standard deviation. However. b Density of dendritic protrusions in normal development on apical and basilar dendrites of animals throughout development.28 ± 0. after PND 270 the density on the basilar dendrite remained relatively static with no further signiﬁcant decreases at PND 360 (2. 2004. At PND 15.92 ± 0. 2b). 2010. the density ratio signiﬁcantly decreased between the time points PND 15 (1. Fig. 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 (p’s \ 0.19 ± 0.20) to PND 270 (2. At PND 60 the spine density was 4. p [ 0.06) to PND 360 (0. Leuner and Shors 2012) although in the neocortex under baseline conditions such differences have been reported to 123 . Yang et al. d Rate of dendritic protrusion loss in normal development on apical and basilar dendrites on animals throughout developments. 2c).87 ± 0.07 ± 0. As development progresses. Environmental stress and gender have been shown to inﬂuence dendritic protrusion density in pyramidal cells in CA1 (Shors et al. 2b). there were no signiﬁcant changes in protrusion density over the next month.24 ± 0. after PND 30. 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 ﬁrst two postnatal months (Fig. 2009. the rate of protrusion loss differs between the apical and basilar dendrites.68. There were signiﬁcant (p’s \ 0. as well as continuous decreases in the dendritic protrusion density on the apical dendrite from PND 60 until PND 360 (see Fig. Thus. 2). Elston et al.78 ± 0.05). the mean (and SD) of apical protrusion density (expressed as number of dendritic protrusions per every 10 lm dendritic length) was 6.
The variance from the mean across our entire data set ranged from 2. Our ﬁndings 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. Mean percent distribution for apical PND 15 animals were: branched (5. 1999). It was observed in both apical and basilar dendrites that there is a signiﬁcant decrease in stubby. ﬁlopodia.67 % ±1. and branched ﬁlopodia.06). lollipop and ﬁlopodia mushrooms were most prevalent at PND 15 in layer VI dendrites.07).45 % ± 1. 1d).31). over development in basilar dendrites there was an increase in lollipop and mushroom spines and a decrease in stubby. lollipop (20.61). lollipop and mushroom.92 % ± 2.18 % ± 2. Dendritic protrusion morphology We also assessed potential changes in protrusion morphology over development. lollipop (21. 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.04 % ± 3.61).74 % ± 0. and branched protrusions when comparing their densities at PND 15 with those observed at PND 30. ﬁlopodia. Fig. 3 Distribution of dendritic protrusions during normal development.62).56 % ± 2.Brain Struct Funct be minimal (Teskey et al. mushroom (14. mushroom (14. ﬁlopodia (15. Distribution of Layer VI dendritic protrusions on apical (a) and basilar (b) dendrites during normal development 123 .54 % ± 1.91). Similar to apical dendrites. stubby. Figure 3a shows the distribution of different categories of dendritic protrusions in apical dendrites across development. ﬁlopodia.62). lollipop and ﬁlopodia protrusions were most prevalent across all age groups. stubby (42. Similarly there were further decreases when comparing PND 30 to PND 90.42 % ± 1.86). The mean percent morphological distribution for basilar PND 15 mice were: branched (5. which resulted in ﬁve morphologically distinct subgroups: branched. There was relatively no change in spine morphology distribution after PND 90 on the basilar dendrites. stubby. Stubby.49 % ± 1. Thus. We grouped protrusions with similar morphologies (see Fig. ﬁlopodia (16. stubby (43. Figure 3b shows the distribution of the different morphological groups of dendritic protrusions in basilar dendrites across age groups. and branched ﬁlopodia of protrusions and relatively no change in spine morphology distribution after PND 90 on the dendrites.2 % (PND 15) suggesting that within our data set gender had a relatively minimal impact on our results. Over development there was an increase in the relative percentage of lollipop and mushroom spines and a decrease in stubby.2 % (PND 360) to 7. ﬁlopodia.96).
Koch and Zador 1993. Simultaneously. it was observed in both the apical and basilar dendrites (Figs. stubby. 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. Given the noted increase in mushroom spines. (2012). macaque monkeys. in basilar dendrites. we sought to determine if the changes were uniform across the extent of the dendrite or regionalized (e. (2007) and Dumitriu et al. However. However. 5). PND 60 to PND 90 (p = 0. 2009). particularly the thin subtypes. 2007. 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.001). In contrast. Koch et al. our results extend on these ﬁndings by showing how different morphological types of dendrites change as a function of age.833). 2006. Representative complete reconstructions (Fig. the rate of loss is greater for basilar dendrites. 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. Tukey’s post hoc tests. Spine neck width and length has long been associated with the efﬁciency of synaptic transmission with wider and shorter spine necks being associated with more efﬁcient transmission of synaptic signals (Araya et al. In contrast.09 %. 2010). ﬁlopodia. This is a time point after the start of dendritic protrusion loss was ﬁrst observed the PND 15 to PND 30 epoch. The apex of the dendritic length in both apical and basilar dendrites was seen at PND 90. we found a lower protrusion density and shorter length of dendrites in basilar compared with apical dendrites.Brain Struct Funct However. Dumitriu et al. and branched protrusions remained stable throughout the remainder of the ﬁrst postnatal year (p’s [ 0. are what other studies have categorized as ﬁlopodia (Zuo et al. Similar to Benavides-Piccione et al. Consistent with previous experiments. after PND 90 the distribution of stubby. we observed a signiﬁcant increase in mushroom spines and a decrease in ﬁlopodia as the animals matured. which is consistent with previous observations found in aging rat.g. Discussion Our ﬁndings 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. and human studies (Jacobs et al. we found that in normal development. that there was a larger loss of dendritic protrusions distally than proximally in lollipop.05). mushroom spines were lost more proximally than distally across age groups (mixed ANOVA. 2013). In general it was observed that protrusions were more likely to be initially lost at the most distal locations of the dendrites. 6b). it is possible that a subset of the stubby spines. Further analysis shows that across age groups there was a reduction in overall spine densities. mushrooms signiﬁcantly increased in all groups except for PND 15 to PND 30 (p = 0. our results found that lollipop spines remained relatively stable with insigniﬁcant changes in morphology as the animal aged. These results are in agreement with previous studies performed in monkeys which found that spine densities and length were signiﬁcantly reduced in apical dendrites with aging (Kabaso et al.434). 123 . Furthermore. proximal locations). across all age groups. p’s \ 0. 2003. distal vs. as well as a decline in small and short dendritic protrusions in both basal and apical dendrites and an increase in lollipop spines. whereas we examined pyramidal neurons from layer VI of the rodent barrel cortex. 1992). Given that we observed a signiﬁcant increase in mushroom spines and a decrease in ﬁlopodia as the animals matured. and PND 90 to PND 180 (p = 0. 2010) that show spine loss is a consequence of aging although recent reports suggest at least some cellular phenotypes do not show signiﬁcant losses (Mostany et al. and ﬁlopodia protrusions were mainly lost while larger mushroom spines and lollipop spines remained the dominant spines. (2010) focused on pyramidal cells that were located in primate prefrontal cortex layer III. and dendritic length as an animal gets older (Feldman and Dowd 1975). and overall. Over development (PND 15 to 360) there was a signiﬁcant increase in lollipop spines in both the apical and basilar dendrites (p’s \ 0. 6a) were combined with the apical and longest basilar dendrite reconstructions (see ‘‘Materials and methods’’) and we quantiﬁed their lengths. which have narrow necks. Speciﬁcally. 1997. this may suggest an increase in synaptic efﬁciency as a result of cortical maturation. mushroom spines signiﬁcantly increased in apical dendrites with the exception of PND 15 to PND 30 (p = 0.05). Methodologically. Dendritic length We examined if dendritic length also changed as a function of developmental age. Dumitriu et al. stubby. followed by a decrease until PND 360 for both the apical and basilar dendrites (see Fig. We found that the average total dendritic length increased until PND 90. This discrepancy might be due to the different locations and species of the experiments since both Hao et al.405) and PND 90 to PND 180 (p = 0.119). Duan et al. The ratio of basilar spine density to apical spine density decreased as well. branched. 4.15 % and the basilar dendrite decreased 35. Speciﬁcally. the average total length of the apical dendrite decreased 34.833). ﬁlopodia. Speciﬁcally. there is a decrease in the dendritic spine density.
f overall protrusion density. b).. b mushroom spines.Brain Struct Funct Fig. two spines 123 . 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. especially given that the morphological distribution of our stubby spines ? ﬁlopodia yielded similar proportions of total dendritic protrusions to these previously published studies. and thus it is possible that spines were incorrectly assigned to a particular group (e. a lollipop spines. The plot represents the mean number of dendritic protrusions between 10 lm concentric spheres on the apical dendrites during normal development.g. 4 Dendritic protrusions vary as a function of distance from the soma on the apical dendrite. d ﬁlopodia. e branched ﬁlopodia. Note that spine loss is most profound at the more distal locations as the animal matures 2005a. 2009). 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. c stubby spines.
Brain Struct Funct Fig. as they often respond to the same 123 . 2005). should be consistent across developmental ages. 5 Dendritic protrusions vary as a function of distance from the soma on the basilar dendrite. However. f overall protrusion density. 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. Interestingly. such errors. b mushroom spines. c stubby spines. Holtmaat et al. Our ﬁndings are consistent with ﬁndings that mushroom spines are characterized as being stable and are consistent with two-photon studies showing that the large majority of stable. Note that protrusion loss is most profound at the more distal locations as the animal matures close together may appear as a stubby spine). if any. persistent spines in vivo are mushroom-type spines (Trachtenberg et al. The plots represent the protrusion density on basilar dendrites during normal developments a lollipop spines. d ﬁlopodia. 2002. Previous studies in the rodent neocortex have suggested that apical and basilar dendrites should be treated as separate components. 2010). e branched ﬁlopodia.
Stephan F.C. Garrett and Wellman 2009.Brain Struct Funct Fig. but they did not look at the consequence of aging.B. Consistent with this is the ﬁnding that separate locations (proximal and distal) of the dendritic arbor have distinct changes in response to an extrinsic cue (Yacoubian and Lo 2000). Both apical and basilar dendrites decrease in length as a function of development. 2012. Characterizing the proﬁle of dendritic protrusions within layer VI over the ﬁrst postnatal year provides a better understanding of the developmental maturation of dendritic protrusions. our results show that speciﬁc classes of protrusions (e. and similar phenomena has been observed in mouse genetic knockout models as well (Chen et al. 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. Yuste R (2006) The spine neck ﬁlters membrane potentials. 2012b). Metz et al. 2007. Arellano et al. 2003. 2012a). We thank Dr. Benavides-Piccione et al. 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.g. Arellano et al. 2009. our ﬁndings in the current study have shown that the rate of protrusion loss is faster in basilar compared with apical dendrites. Brumberg for helpful comments on the manuscript. 6 Dendritic length decreases with age. Chen et al. This suggests that developmental changes may be localized to speciﬁc neuronal arborizations. Acknowledgments The work was supported by a DSC award to C-C Chen and PSC-CUNY 62750-00 40 and NS058758 to J.. Eisenthal KB. Representative complete reconstructions show less elaborate dendrites as a function of age (a) which is quantiﬁed in b Data represent population means ± one standard deviation experimental manipulation in different manners (McAllister et al. mushrooms) are more likely to be retained throughout the ﬁrst postnatal year suggesting that these spines may be involved with synaptic pathways that are not easily altered such as long term memories. 1995. Furthermore. 2007. BenavidesPiccione et al. Similarly. Jiang J. Proc Natl Acad Sci USA 103(47): 17961–17966 123 . Our results show that the developmental morphological changes are localized to particular regions across age groups. Carolyn Pytte and Dr. References Araya R. 2012). Our study clearly showed that as development progresses spines proximal to the soma are more likely to be retained. Across age groups we saw the ﬁrst signs of spine loss at the most distal regions.
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