You are on page 1of 12

Brain Struct Funct DOI 10.

1007/s00429-013-0596-5

ORIGINAL ARTICLE

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: joshua.brumberg@qc.cuny.edu C.-C. Chen Á J. C. Brumberg Neuropsychology Ph.D. Subprogram (Psychology), The Graduate Center, CUNY, Flushing, NY, USA

123

as these persistent spines are associated with life-long memories (Yang et al. mushroom spines that have relatively large spine heads are stable for weeks to months and mediate strong synapses. Slices were air dried at room temperature in darkness for at least 2–3 weeks before further processing. 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. 2009). 2009). but of increasing importance due to the aging of the human population (see Yuste and Bonhoeffer 2004. To further quantify the impact of normal aging on dendritic protrusions in the mouse. Harris and Stevens 1989. Given the role that dendritic protrusions have been posited to have in modifying synaptic inputs onto neurons (Kawato et al. PND 30 is approximately when mice reach sexual maturity. Richardson et al. It has been demonstrated that as the animals mature. Thomson 2010) and has been previously used as a model to classify neurons (Chen et al. 2009). 2004. 1984. Lee et al. with nearly 50 % of these protrusions lost with aging being lollipop spines. 2005b. while. Golgi staining At the appropriate age. 2010). Zuo et al. with a distinct topographic one-to-one relationship corresponding to the whiskers on the mystacial pad (Woolsey and Van der Loos 1970). brains were immersed in a Golgi-Cox solution comprising potassium dichromate. There were seven groups (n = 4 each) at distinct developmental time points. 90. 2003. 2005) whereas. while there is little loss of mushroom or stubby spines (Dumitriu et al. forming weaker synapses and are associated with a higher degree of plasticity (Kasai et al. Dendritic spine loss likely occurs as a consequence of aging in most if not all mammalian species. and potassium chromate. After immersion in the Golgi-Cox solution. 2010). Bourne and Harris 2007). 180. 2000. PND 15.13) or distilled H2O for 3 min (there was found to be no difference between brains processed in either solution). pH 7. Virbac Animal Health. we investigated their density and morphology over the first postnatal year. followed by storage at room temperature in darkness for 2/3 weeks. When the animals become nonresponse (by toe-pinch) brains were promptly removed and placed in either 0. are mainly short-term spines. Yang et al. 1999. Inc. Institutional Animal Care and Use Committee and in accordance with NIH guidelines concerning the use of laboratory animals. 2012a) and the later ages were chosen to effectively span the first postnatal year. which have smaller spine heads. Coronal slices of approximately 100–120 lm were made on a freezing cryostat (approximately -25 °C). dendritic protrusions are the primary site of structural plasticity in cortical pyramidal cells (Holtmaat and Svoboda 2009) and function in neural information processing. which processes information from their prominent facial whiskers. For example. Lendvai et al. PND 60 was chosen to allow for future comparisons with previous studies (Chen et al. lollipop spines.). The slices were then placed onto triple-dipped gelatin slides and then coated with additional cryoprotectant. 2012a) and will be used in the present investigation. sections were rinsed with distilled water and were subsequently stained in a developing solution (FD Rapid Golgi 123 . The barrel cortex is anatomically structured into barrel columns. Experimental protocols were approved by the Queens College. 2008. CUNY. 2012) it is important to understand how their dendrites and associated protrusions develop and change over the rodent’s first postnatal year. Takumi et al. 270 and 360. This mixture was replaced after 12 h of initial immersion. Matsuzaki et al. brains were placed in a cryoprotectant solution (FD Rapid Golgi Stain Kit. mice were anesthetized with an intraperitoneal injection of Euthasol (sodium pentobarbital. In order to prevent ice crystal damage. Dendritic protrusions have generally been classified morphologically into specific categories based on their length. 60. Feldman and Dowd (1975) reported a 20–40 % loss of protrusions as a consequence of aging. Saneyoshi et al. mercuric chloride. 2009) and study the impact of sensory experience (Chen et al.Brain Struct Funct Furthermore. 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. The morphology of each dendritic protrusion category is related to its stability and synaptic strength. or shrink due to synaptic weakening (Holtmaat et al. The primary somatosensory cortex in rodents contains a region called the barrel cortex. Upon rinsing with PB. the processes that underlie the development of dendritic protrusions and their maturation over an animal’s lifespan are not well understood. FD Neurotechnologies) and stored at 4 °C for 1 week in darkness before slicing. 30. the dendritic protrusion density in cortical layer V apical dendrites steadily decreased (Zuo et al. size and shape (Peters and Kaiserman-Abramof 1970. After this drying period. Yasumatsu et al. 2001. 2005a. However. tissues were frozen with dry ice and quickly embedded in an optimal cutting temperature (OCT) medium. 2003. The neocortex is divided into six layers and Layer VI originates cortical input to many cortical and subcortical locations (Brumberg et al.1M phosphate buffer (PB. Prior to PND 15 our staining method was ineffective. stubby spines have been shown to be transitional structures that will either enlarge when strengthened.

ThermoFisher Scientific) and cover slipped with Permount (Sigma-Aldrich). Irwin et al. d Representative reconstruction of classification of dendritic protrusions: Stubby spines are spines with no necks and a stubbylike appearance. pyramidal cells were further examined for uncut basilar dendrites of length [100 lm that ended in a fine taper. Low magnification image a illustrating the barrel cortex and it’s six layers in between the pia and the white matter. 2009). The posterior limit of the barrel cortex was identified by the separation of the corpus callosum at the midline.Brain Struct Funct Stain Kit. 1 Golgi stained mouse brain section of the barrel cortex.4 numerical aperture (NA)) in layer VI to ensure complete staining of the cell.42 NA). At 609 oil magnification (1. 75. Mushroom spines are spines with small necks and a large often complex and irregular head. Higher magnification illustrating the pyramidal neurons of layer VI. FD Neurotechnologies) and dehydrated successively in solutions of 50. 95. Fig. Identification of the barrel field In order to accurately identify the barrel cortex as we have done previously (Chen et al.0 and 10. 1c). Inc. neurons that had truncated and/or non-tapering dendrites were not included in our analysis. The anterior limit of the barrel cortex was identified by the appearance of the anterior commissure. 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.40 NA). Finally. the neuron would be reconstructed followed by classifying the dendritic protrusions into one of five categories (see Fig. 1a). 1d. at 1009 oil magnification (1. Branched filopodia spines are bifurcated thin filopodia spines with no obvious heads 123 . 1b). 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). 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. 2000. c The scale in which spines were reconstructed from an apical dendrite of a pyramidal neuron located in layer VI. 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 sections were then defatted in a xylene substitute (SafeClearII.) and an Olympus Bx51 microscope equipped with a highresolution digital camera (Optronics Microfire). classification scheme adapted from Greenough. Neuronal selection/reconstruction/dendritic protrusion classification Imaging of neurons and their respective dendritic protrusions (spines and filopodia) was accomplished with Neurolucida 9. Only the neurons that exhibited complete Golgi impregnation with limited amount of staining artifacts were traced.0 software (MBF Bioscience. Neurons were initially imaged under 109 magnification (0. and 100 % ethanol in distilled H2O. Chen et al. the characteristic clusters of cells observed were matched with an atlas of a Golgi-stained mouse brain (Valverde 1998). The box represents the region magnified in b. Filopodia spines are single long thin protrusions with no obvious head. At this point. Lollipop spines are spines with thin necks and small heads.

20 2.28 4.68 6.88 745.24 ± 0.21 147.) allowed us to calculate overall dendritic protrusion densities. slender stalk.90 ± 21.g..) on a PC.53 92. 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. Dendritic protrusion types (lollipop.17 750. 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).95 ± 106.91 ± 28. which was calculated by the difference in the density at the specific postnatal dates.78 390.07 ± 21. Throughout the development of the animal. we observed a net loss of dendritic protrusions (protrusions lost/day).01 799.33 ± 13.00 136. In contrast.85 504.97 161.22 ± 0.20 ± 7.05. 2012b).63 56. Cell bodies.80 ± 69.20 ± 16. Statistical methods of analyzing dendritic protrusion density/morphology The NeuroExplorer software (MBF Bioscience.63 43. 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.34 177.04 271. For statistical analyses we conducted a mixed-factorial analyses of variance (ANOVA). To determine if certain regions of the dendritic tree were more likely to lose dendritic protrusions in older age groups.53 ± 60. 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 . followed by appropriate post hoc confirmations (Tukey HSD) between each developmental group to first determine and then confirm statistical significance (if any).78 ± 0. apical dendrites.25 3.20 ± 95.44 175.28 ± 0.01 284.03 ± 0.37 ± 24.48 ± 58.31 5. 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.47 ± 11.10 ± 31.60 465.80 ± 27. and lack a clear head.80 ± 46. stubby spines are the ones that have a short stout stalk (Fig. For each neuron the apical dendrite and the longest basilar dendrite were reconstructed to facilitate direct comparisons.87 ± 7. 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. Statistical significance was achieved if calculated p values were less than 0. Mushrooms are spines with a stalk with a conspicuous head on it. Inc. Statistical tests were run using SigmaStat (version 3.19 ± 0.20 ± 24.55 133.97 ± 0.68 ± 50.68 ± 50.95 437.84 280. branched. 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.47 ± 56.07 ± 0.13 ± 32.30 ± 0. 2a). 1d).28 2.20 705.95 ± 86. mushroom.23 2. whereas we will use the specific names (e.87 ± 0.01 320.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 251.16 2. stubby spines) when comparing morphological types. 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.11 251.46 ± 0. Filopodia are thought to be immature protrusions that have a long.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.68 163. as we have done previously (Chen et al. and the longest basilar dendrite were completely reconstructed.28 3.36 4.84 ± 32.68 181.24 2. Systat Software Inc.81 ± 0.25 ± 123.00 ± 9.37 3.87 7. Dendritic protrusions will be used to refer to all spines and filopodia on basilar or apical dendrite at specific developmental ages.06 35.72 260.45 201. mushroom vs.33 ± 20.27 ± 34.62 ± 100.25 576.5.29 ± 0.

Decreases were seen from our initial time epoch from PND 15 to PND 30. the rate of protrusion loss differs between the apical and basilar dendrites. 2c).76 ± 0.24 ± 0.19 ± 0.24).37) to PND 180 (3. As development progresses. 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. There were significant (p’s \ 0.28) from PND 90 (3. Environmental stress and gender have been shown to influence dendritic protrusion density in pyramidal cells in CA1 (Shors et al.01) changes in the density of the basilar dendrite after PND 60 (4. Fig.05) decreased to 5. 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. the density of the apical dendrite decreased significantly after PND 60 from PND 90 (3.19 ± 0.78 ± 0.06) to PND 360 (0. 2001. Plots represent population means ± one standard deviation the animal’s life (Fig. 2009.28) to PND 270 (2. Elston et al. the density ratio significantly decreased between the time points PND 15 (1.17 ± 0. Plots represent population means ± one standard deviation. c Ratio of dendritic protrusions in basilar versus apical dendrites in normal development.Brain Struct Funct Fig. after PND 30. the mean (and SD) of apical protrusion density (expressed as number of dendritic protrusions per every 10 lm dendritic length) was 6.20. after PND 270 the density on the basilar dendrite remained relatively static with no further significant decreases at PND 360 (2.18 whereas by PND 30 it had significantly (p \ 0. Thus. p [ 0. 2004. At PND 60 the spine density was 4. Leuner and Shors 2012) although in the neocortex under baseline conditions such differences have been reported to 123 . b Density of dendritic protrusions in normal development on apical and basilar dendrites of animals throughout development.10) due to decreases in basilar dendritic protrusion density and relatively smaller decreases in apical protrusion density. 2009).68. As development progressed.25) to PND 180 (2. Plots represent population means ± one standard deviation.28 ± 0. Representative micrographs were taken at 100x and then a threshold applied to remove the background for visualization purposes.23) until PND 360 (2.16). 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 first two postnatal months (Fig.29 ± 0. suggesting the rate of dendritic protrusion pruning/elimination had decreased.81 ± 0. Yang et al. We then calculated the dendritic protrusion density ratio comparing the basilar to the apical dendrite (Fig. 2 Pyramidal neuron located in layer VI.28.92 ± 0.05). 2010. as well as continuous decreases in the dendritic protrusion density on the apical dendrite from PND 60 until PND 360 (see Fig.20) to PND 270 (2. 2b). d Rate of dendritic protrusion loss in normal development on apical and basilar dendrites on animals throughout developments. which is consistent with previously published findings (Zuo et al.29 ± 0. 2005b. 2b). 2). This indicates that there was a significant amount of dendritic protrusion pruning occurring during our first time epoch. there were no significant changes in protrusion density over the next month.07 ± 0. The basilar dendrite showed similar findings.05.87 ± 0. 2d). At PND 15. However. However.46 ± 0.

lollipop and filopodia protrusions were most prevalent across all age groups. filopodia.2 % (PND 15) suggesting that within our data set gender had a relatively minimal impact on our results.96). 1d). Stubby. mushroom (14. Dendritic protrusion morphology We also assessed potential changes in protrusion morphology over development. 3 Distribution of dendritic protrusions during normal development. We grouped protrusions with similar morphologies (see Fig.61).62). stubby (42. which resulted in five morphologically distinct subgroups: branched. Figure 3b shows the distribution of the different morphological groups of dendritic protrusions in basilar dendrites across age groups. Fig.92 % ± 2. stubby (43. and branched protrusions when comparing their densities at PND 15 with those observed at PND 30.Brain Struct Funct be minimal (Teskey et al. lollipop (21.31).2 % (PND 360) to 7.54 % ± 1. filopodia.74 % ± 0. mushroom (14.67 % ±1.06). 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. stubby. There was relatively no change in spine morphology distribution after PND 90 on the basilar dendrites. and branched filopodia. The mean percent morphological distribution for basilar PND 15 mice were: branched (5. Distribution of Layer VI dendritic protrusions on apical (a) and basilar (b) dendrites during normal development 123 . filopodia (15. Figure 3a shows the distribution of different categories of dendritic protrusions in apical dendrites across development. Similar to apical dendrites. Similarly there were further decreases when comparing PND 30 to PND 90. stubby. filopodia.18 % ± 2. lollipop (20. filopodia (16.04 % ± 3.86). lollipop and filopodia mushrooms were most prevalent at PND 15 in layer VI dendrites.45 % ± 1. and branched filopodia of protrusions and relatively no change in spine morphology distribution after PND 90 on the dendrites. Thus.42 % ± 1.56 % ± 2.49 % ± 1. over development in basilar dendrites there was an increase in lollipop and mushroom spines and a decrease in stubby. 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. filopodia.91). Over development there was an increase in the relative percentage of lollipop and mushroom spines and a decrease in stubby. It was observed in both apical and basilar dendrites that there is a significant decrease in stubby.61). Mean percent distribution for apical PND 15 animals were: branched (5. The variance from the mean across our entire data set ranged from 2. 1999).62).07). lollipop and mushroom.

there is a decrease in the dendritic spine density. and dendritic length as an animal gets older (Feldman and Dowd 1975). Similar to Benavides-Piccione et al. In general it was observed that protrusions were more likely to be initially lost at the most distal locations of the dendrites. Over development (PND 15 to 360) there was a significant increase in lollipop spines in both the apical and basilar dendrites (p’s \ 0. 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. and overall. 1992). 2007. (2007) and Dumitriu et al. Simultaneously. 5). it is possible that a subset of the stubby spines.001).05). However.833). filopodia. mushroom spines were lost more proximally than distally across age groups (mixed ANOVA. our results found that lollipop spines remained relatively stable with insignificant changes in morphology as the animal aged. Koch and Zador 1993. 1997. 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. we found that in normal development. Koch et al. particularly the thin subtypes. which have narrow necks. filopodia. across all age groups. (2012). Tukey’s post hoc tests. Specifically.15 % and the basilar dendrite decreased 35. 2009). and filopodia protrusions were mainly lost while larger mushroom spines and lollipop spines remained the dominant spines. The apex of the dendritic length in both apical and basilar dendrites was seen at PND 90. 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. the rate of loss is greater for basilar dendrites. Specifically. and human studies (Jacobs et al.119). it was observed in both the apical and basilar dendrites (Figs. 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. stubby. after PND 90 the distribution of stubby. 6b). in basilar dendrites. whereas we examined pyramidal neurons from layer VI of the rodent barrel cortex. 4. which is consistent with previous observations found in aging rat. Specifically. Dumitriu et al. 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. (2010) focused on pyramidal cells that were located in primate prefrontal cortex layer III. 6a) were combined with the apical and longest basilar dendrite reconstructions (see ‘‘Materials and methods’’) and we quantified their lengths.833). This is a time point after the start of dendritic protrusion loss was first observed the PND 15 to PND 30 epoch. 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. 2006. stubby. Consistent with previous experiments. this may suggest an increase in synaptic efficiency as a result of cortical maturation. Duan et al. we found a lower protrusion density and shorter length of dendrites in basilar compared with apical dendrites. PND 60 to PND 90 (p = 0. 123 .434). In contrast. as well as a decline in small and short dendritic protrusions in both basal and apical dendrites and an increase in lollipop spines. 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. macaque monkeys. In contrast. and branched protrusions remained stable throughout the remainder of the first postnatal year (p’s [ 0. that there was a larger loss of dendritic protrusions distally than proximally in lollipop. branched.405) and PND 90 to PND 180 (p = 0. This discrepancy might be due to the different locations and species of the experiments since both Hao et al. the average total length of the apical dendrite decreased 34. mushroom spines significantly increased in apical dendrites with the exception of PND 15 to PND 30 (p = 0. The ratio of basilar spine density to apical spine density decreased as well. 2013). However. followed by a decrease until PND 360 for both the apical and basilar dendrites (see Fig. Furthermore. we sought to determine if the changes were uniform across the extent of the dendrite or regionalized (e. mushrooms significantly increased in all groups except for PND 15 to PND 30 (p = 0. p’s \ 0. we observed a significant increase in mushroom spines and a decrease in filopodia as the animals matured. distal vs. Dumitriu et al.g. Given that we observed a significant increase in mushroom spines and a decrease in filopodia as the animals matured.09 %. are what other studies have categorized as filopodia (Zuo et al.05). Given the noted increase in mushroom spines. Representative complete reconstructions (Fig. proximal locations). 2003. Methodologically. and PND 90 to PND 180 (p = 0. Further analysis shows that across age groups there was a reduction in overall spine densities. 2010).Brain Struct Funct However. our results extend on these findings by showing how different morphological types of dendrites change as a function of age. Dendritic length We examined if dendritic length also changed as a function of developmental age. We found that the average total dendritic length increased until PND 90.

e branched filopodia. and thus it is possible that spines were incorrectly assigned to a particular group (e. 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. two spines 123 . a lollipop spines. 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.. 2009). Note that spine loss is most profound at the more distal locations as the animal matures 2005a. b mushroom spines. especially given that the morphological distribution of our stubby spines ? filopodia yielded similar proportions of total dendritic protrusions to these previously published studies. The plot represents the mean number of dendritic protrusions between 10 lm concentric spheres on the apical dendrites during normal development. 4 Dendritic protrusions vary as a function of distance from the soma on the apical dendrite.Brain Struct Funct Fig. c stubby spines.g. b). d filopodia. f overall protrusion density.

c stubby spines. Previous studies in the rodent neocortex have suggested that apical and basilar dendrites should be treated as separate components. 2002.Brain Struct Funct Fig. Note that protrusion loss is most profound at the more distal locations as the animal matures close together may appear as a stubby spine). f overall protrusion density. 2005). d filopodia. such errors. b mushroom spines. if any. 2010). 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. e branched filopodia. 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. persistent spines in vivo are mushroom-type spines (Trachtenberg 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. Holtmaat et al. as they often respond to the same 123 . However. should be consistent across developmental ages.

Arellano et al.Brain Struct Funct Fig. 1995. 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. 2003. 2009.C.B. Both apical and basilar dendrites decrease in length as a function of development. We thank Dr. Jiang J. Furthermore. 2012a). Benavides-Piccione 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. Metz et al. Carolyn Pytte and Dr. References Araya R. Across age groups we saw the first signs of spine loss at the most distal regions. Brumberg for helpful comments on the manuscript. 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. 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). our results show that specific classes of protrusions (e. Chen et al. Eisenthal KB. 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. This suggests that developmental changes may be localized to specific neuronal arborizations. but they did not look at the consequence of aging. Similarly. 6 Dendritic length decreases with age. 2012b). Acknowledgments The work was supported by a DSC award to C-C Chen and PSC-CUNY 62750-00 40 and NS058758 to J. Our results show that the developmental morphological changes are localized to particular regions across age groups. BenavidesPiccione et al. 2012.. Arellano et al. and similar phenomena has been observed in mouse genetic knockout models as well (Chen et al. 2007. Garrett and Wellman 2009. 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. Stephan F. 2007. our findings in the current study have shown that the rate of protrusion loss is faster in basilar compared with apical dendrites. Our study clearly showed that as development progresses spines proximal to the soma are more likely to be retained. 2012).g. Yuste R (2006) The spine neck filters membrane potentials. Proc Natl Acad Sci USA 103(47): 17961–17966 123 .

J Comp Neurol 386:661–680 Kabaso D. Nature 429:761–766 McAllister AK. PMID: 22819970 Dickstein DL. Nature 404(6780):876–881 Leuner B. Hara Y. Fujita I (2010) Spinogenesis and pruning from early visual onset to adulthood: an intracellular injection study of layer III pyramidal cells in the ventral visual cortical pathway of the macaque monkey. J Neurosci 29:3271–3275 Elston GN. Nakahara H (2003) Structure-stability-function relationships of dendritic spines. Hof PR (2003) Age-related dendritic and spine changes in corticocortically projecting neurons in macaque monkeys. Brain Struct Funct 217(2):435–446. DeFelipe J (2012) Age-based comparison of human dendritic spine structure using complete three-dimensional reconstructions. Front Neurosci 1:131–143 Benavides-Piccione R. Schall M (1997) Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study. Fenstermaker V. experience. Yasumatsu N. Teskey GC (2012) Age. Hamzei-Sichani F.1016/j. Svoboda K (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Crump KL.021 Matsuzaki M. Cereb Cortex 19:2248–2268 Kasai H. Nat Rev Neurosci 13(4):240–250 Mostany R. Martina M (2009) Morphological and functional reorganization of rat medial prefrontal cortex in neuropathic pain. Brown TH (1992) Dendritic spines: convergence of theory and experiment. Kasai H (2001) Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Trachtenberg JT. Science 256:973–974 Kolb B. Janssen WG. Trends Neurosci 26:360–368 Kawato M. Lu HC. Cereb Cortex 10:1038–1044 Jacobs B. Lou W. Am J Anat 127:321–355 Peters A. anxiety. Morrison JH. Rapp PR. J Neurobiol 56:95–112 ´¨ Lee KF. Cereb Cortex. Oxford University Press. Oxford. Stevens JK (1989) Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. Kaiserman-Abramof IR (1970) The small pyramidal neuron of the rat cerebral cortex: the perikaryon. Fujita I (2009) Spinogenesis and pruning scales across functional hierarchies.1093/cercor/bhs154 Bourne J. Noguchi J. Honkura N. Knott G. and the changing brain. Neuron 45:279–291 Irwin SA. Hof PR.09. doi:10. PNAS 106(7):2423–2428 Morrison JH. Yuste R. Matsuzaki M. Hamaguchi T. Zador A (1993) The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization. Portera-Cailliau C (2013) Altered synaptic dynamics during normal brain aging. Nemoto T. Yau HJ. Kaufmann J. Janssen WG. Dev Psychobiol 54(3):311–325 Konur S.neuroscience.1155/2012/ 704103 Lendvai B. Weaver CM. J Comp Neurol 512:726–746 Chen CC. Katz LC (1995) Neurotrophins regulate dendritic growth in developing visual cortex. Lasley BL. Harris KM (2007) Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol 17:381–386 Brumberg JC. Yuste R (2003) Systematic regulation of spine head diameters and densities in pyramidal neurons from juvenile mice. Tam D. Soares C. Ha pp 1–35 Garrett JE. Shepherd GM. Brumberg JC (2012b) mGluR5 knockout mice display increased dendritic spine densities. Stern EA. Lou W. Knott GW. J Neurosci 13:413–422 Koch C. and dendritic spines: what are the connections? Neuroscience. Kasthuri N. Dowd C (1975) Loss of dendritic spines in aged cerebral cortex. Rocher AB. Yuste R (2003) Morphological and physiological characterization of layer VI corticofugal neurons of mouse primary visual cortex. Chen B. Morrison JH (2007) Interactive effects of age and estrogen on cognition and pyramidal neurons in monkey prefrontal cortex. J Neurophysiol 89:2854–2867 Chen CC. Yuste R (2007) Ultrastructure of dendritic spines: correlation between synaptic and spine morphologies. Shors TJ (2012) Stress. Spruston ¨ usser M (eds) Dendrites. Wearne SL (2009) The electrotonic structure of pyramidal neurons contributing to prefrontal cortical circuits in macaque monkeys is significantly altered in aging. Maco B. Brumberg JC (2009) Morphological heterogeneity of layer VI neurons in mouse barrel cortex. J Neurosci 30:7507–7515 Elston GN. Centeno MV. Be ıque JC (2012) Examining form and function of dendritic spines. Gan WB (2002) Long-term dendritic spine stability in the adult cortex. dendrites and spines. Abrams S. Annu Rev Psychol 17:341–371 Harris KM. Neuroscience. Neural Plast. Fernaud-Espinosa I. Coskren PJ.Brain Struct Funct Arellano JI. Macedo A. Wellman CL (2009) Chronic stress effects on dendritic morphology in medial prefrontal cortex: sex differences and estrogen dependence. Kasai H (2004) Structural basis of long-term potentiation in single dendritic spines.2012. Zador A. N. Hof PR (2012) Dendritic spine changes associated with normal aging. Galvez R. injury. Harris KM (1999) Dendrite structure. Cereb Cortex 20: 1398–1408 Feldman ML. Miyashita Y. Ellis-Davies GC.1016/j. Kater SB (1994) Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Lo DC. Cereb Cortex 13:950–961 Dumitriu D. Iino M. Driscoll L. Apkarian AV. Neurosci Lett. Okamoto T. Anat Embryol 148:279–301 Fiala JC. Rapp PR. J Neurosci 9:2982–2997 Holtmaat A. Wearne SL. Nat Neurosci 4:1086–1092 Matsuzaki M. Brain Struct Funct 216:85–89 Peters A. Greenough WT (2000) Dendritic spine structural anomalies in fragile-X mental retardation syndrome. Rabinowitz D. Morrison JH (2010) Selective changes in thin spine density and morphology in monkey prefrontal cortex correlate with aging-related cognitive impairment. Brumberg JC (2012a) Sensory deprivation differentially impacts the dendritic development of pyramidal versus non-pyramidal neurons in layer 6 of mouse barrel cortex. Nat Rev Neurosci 10:647–658 Holtmaat AJ. Oga T. Defelipe J. doi: 10. 2012:704103.neuroscience.1007/s00429-0110342-9 Chen CC. Nature 420:812–816 Hao J. Baxter MG (2012) The ageing cortical synapse: hallmarks and implications for cognitive decline. Tsukahara N (1984) Quantitative analysis of electrical properties of dendritic spines. In: Stuart G. Hao J. Neuron 15: 791–803 Metz AE. Neurobiol Aging 33(10):2357–2372 123 . doi:10. Henry BI. Anstey JE. Luebke JI. Benavides-Piccione R. Murakami F. J Neurosci 33(9):4094–4104 Pannese E (2011) Morphological changes in nerve cells during normal aging. Wilbrecht L. doi:10. Biol Cybern 50(6):447–454 Koch C. Neuroscience 162(1):195–207 Grutzendler J. Ellis-Davies GC. doi:10.04. Pinhas A. E-pub. Zhang X. Kemper T (2012) A review of the structural alterations in the cerebral hemispheres of the aging rhesus monkey.077 Duan H. Oga T. Hof PR. Svoboda K (2005) Transient and persistent dendritic spines in the neocortex in vivo. Proc Natl Acad Sci USA 104:11465–11470 Harris KM. 2012. Robles V. Svoboda K (2000) Experiencedependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.

Welker E. Van der Loos H (1970) The structural organization of layer IV in the somatosensory region (S1) of mouse cerebral cortex: the description of a cortical field composed of discrete cytoarchitectonic units. Petersen SE (2010) The development of human functional brain networks. EMBO Rep 13(8):699–708. Soderling TR (2010) Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways. Feng G. Chen BE. Nat Neurosci 3:342–349 Yang G. Hutchinson JE. Blundon JA. Falduto J (2001) Sex differences and opposite effects of stress on dendritic spine density in the male versus female hippocampus. Xu ZC (2009) Diversity and fluctuation of spine morphology in CA1 pyramidal neurons after transient global ischemia. Lin A. Bayazitov IT. Konnerth A (2012) Dendritic spines: from structure to in vivo function. Fair DA. Leuner B (2004) The opposite effects of stress on dendritic spines in male vs. J Neurosci 29(20):6406–6417 Rochefort NL. Gan WB (2009) Stably maintained dendritic spines are associated with lifelong memories. Bonhoeffer T (2004) Genesis of dendritic spines: insights from ultrastructural and imaging studies. Ottersen OP (1999) Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Schlaggar BL. Nat Rev Neurosci 5:24–34 Zuo Y. Neuron 46(2):181–189 123 . Nat Neurosci 2:618–624 Teskey GC.Brain Struct Funct Power JD. Noguchi J. doi:10. Front Neuroanat 4:1 Trachtenberg JT. Eur J Neurosci 19(1):145–150 Takumi Y. Kolb B (1999) Sex differences in cortical plasticity and behavior following anterior cortical kindling in rats. J Neurosci Res 87:61–68 Saneyoshi T.1038/ embor. J Anat 87(4):387–406 Shors TJ.2012. Curr Opin Neurobiol 20(1):108–115 Sholl DA (1953) Dendritic organization in the neurons of the visual and motor cortices of the cat. Yang G. Fan Y. Sanes JR. Chang P. Zakharenko SS (2009) Connectivity patterns revealed by mapping of active inputs on dendrites of thalamorecipient neurons in the auditory cortex. Svoboda K (2002) Long-term in vivo imaging of experiencedependent synaptic plasticity in adult cortex. Kasai H (2008) Principles of long-term dynamics of dendritic spines. Pan F. Nature 462(7275): 920–924 Yasumatsu N. a review. Falduto J. Austria Woolsey TA.102 Ruan YI. Knott GW. Zhigang L. J Neurosci 21(16):6292–6297 Shors TJ. Neuron 67: 735–748 Richardson RJ. Cereb Cortex 9(7):675–682 Thomson AM (2010) Neocortical layer 6. Miyazaki T. Laake P. J Neurosci 28:13592–13608 Yuste R. Nature 436(7048):261–265 Zuo Y. Matsuzaki M. Ramirez-Leon V. Gan WB (2005b) Development of long-term dendritic spine stability in diverse regions of cerebral cortex. female rats are NMDA receptordependent. Kwon E. Nature 420: 788–794 Valverde F (1998) Golgi atlas of the postnatal mouse. Fortin DA. Rinvik E. Gan WB (2005a) Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex. Chua C. Lo DC (2000) Truncated and full-length TrkB receptors regulate distinct modes of dendritic growth. Zou B. Brain Res 17(2):205–242 Yacoubian TA. Springer.