Neuroscience Letters 589 (2015) 88–91

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Research article

Fractal dimension of apical dendritic arborization differs in the superficial and the deep pyramidal neurons of the rat cerebral neocortex Nela Puˇskaˇs a,∗ , Ivan Zaletel a , Bratislav D. Stefanovic´ a , Duˇsan Ristanovic´ b a b

Institute of Histology and Embryology “Aleksandar Ð. Kosti´c”, School of Medicine, University of Belgrade, Viˇsegradska 26, 11000 Belgrade, Serbia Department of Biophysics, School of Medicine, University of Belgrade, Viˇsegradska 26, Belgrade, Serbia

h i g h l i g h t s • • • •

Comparison and distinguishing of superficial and deep neocortical pyramidal cells. Morphology and complexity analysis of arborization of the apical dendrite. Topological parameters of homogeneity of apical dendrites arborization. Fractal dimension of apical dendrite of superficial cells is higher than in deep ones.

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Article history: Received 8 December 2014 Received in revised form 10 January 2015 Accepted 16 January 2015 Available online 17 January 2015 Keywords: Golgi technique Rat neocortex Cortical pyramidal neurons Apical dendrite Dendritic morphology Dendrite complexity Fractal analysis

a b s t r a c t Pyramidal neurons of the mammalian cerebral cortex have specific structure and pattern of organization that involves the presence of apical dendrite. Morphology of the apical dendrite is well-known, but quantification of its complexity still remains open. Fractal analysis has proved to be a valuable method for analyzing the complexity of dendrite morphology. The aim of this study was to establish the fractal dimension of apical dendrite arborization of pyramidal neurons in distinct neocortical laminae by using the modified box-counting method. A total of thirty, Golgi impregnated neurons from the rat brain were analyzed: 15 superficial (cell bodies located within lamina II–III), and 15 deep pyramidal neurons (cell bodies situated within lamina V–VI). Analysis of topological parameters of apical dendrite arborization showed no statistical differences except in total dendritic length (p = 0.02), indicating considerable homogeneity between the two groups of neurons. On the other hand, average fractal dimension of apical dendrite was 1.33 ± 0.06 for the superficial and 1.24 ± 0.04 for the deep cortical neurons, showing statistically significant difference between these two groups (p < 0.001). In conclusion, according to the fractal dimension values, apical dendrites of the superficial pyramidal neurons tend to show higher structural complexity compared to the deep ones. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Pyramidal neurons are principal cells of the mammalian cerebral neocortex that exhibit specific morphology based on the presence of apical dendrite and its arborization [1]. This essential feature of pyramidal cells provides structural framework for dendrite functioning, which includes receiving and conducting signals toward the soma, as well as summation and dynamic integration of synaptic inputs [2]. Moreover, morphological or spatial complexity of

∗ Corresponding author. Tel.: +381 113607146; fax: +381 113612567. E-mail addresses: [email protected] (N. Puˇskaˇs), [email protected] ´ [email protected] (I. Zaletel), s [email protected] (B.D. Stefanovic), ´ (D. Ristanovic). http://dx.doi.org/10.1016/j.neulet.2015.01.044 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

dendritic arborization determines the range of synaptic inputs that can be received by a neuron, and influences neuronal firing patterns [3,4]. Morphology of dendrites is now well-known [5,6]. However, complexity of dendritic arborizations is mainly described by the means of topological parameters, such as Sholl analysis and the number of dendritic branching points or dendritic tips [7,8]. On the other hand, in spite of available data [9–14], fractality of dendritic arborizations still awaits to be studied in many neuronal (sub) types including various pyramidal cells residing within different cortical layers and distinct cytoarchitectonic areas (e.g., the class of “short” pyramidal neurons) [5,15]. Fractal analysis is one of the most widely used methods to measure fractality, i.e., fractal dimension of geometrical objects, and has proven to be a good method to address the complexity of natural

N. Puˇskaˇs et al. / Neuroscience Letters 589 (2015) 88–91

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Fig. 1. Outcomes of two different Golgi procedures: impregnated superficial pyramidal neuron with classic, aldehyde Golgi–Kopsch technique (left); non-selective labeling of apical dendrites in cortical lamina I–III with silver-chloride Golgi-like method, and perikaryal Nissl substance stained in red (right).

objects such as neuronal and glial cells [16,17]. Since fractal dimension reflects the object complexity in a quantitative and numerical manner [18], fractal analysis is particularly suitable to quantify and compare the complexity of dendritic trees [19]. The most common used fractal analysis method is the box-counting method. It is considered as a basic technique to study fractality of dendritic arborizations [11]. Therefore, we have applied this method to compare apical dendritic arborizations of the superficial and the deep pyramidal neurons in the rat cerebral neocortex.

cell bodies positioned in lamina V–VI, whereas apical dendrites terminate in layers III or IV (Fig. 2). After taking pictures of selected neurons at numerous focal planes, complete cell images have been retrieved and further processed in the ImageJ 1.48 v software (NIH, Bethesda, USA; free download from http://rsbweb.nih.gov/ij), in order to obtain appropriate binary images of the apical dendritic arborizations (Fig. 3) [17]. Fractal dimension of these binary images was calculated with the same ImageJ program. 2.2. Characterization of apical dendrite arborizations

2. Materials and methods 2.1. Experimental procedure, selection and graphic processing of pyramidal neurons Coronal brain slices of male Wistar rats (200–250 g), fixed with buffered formaldehyde, were used in this study. Probe samples were subjected to the aldehyde Golgi–Kopsch technique as described earlier [17,20], whereas controls have been processed for silver-chloride Golgi-like impregnation with neutral red counterstaining [21], in order to show the overall organization of the cortical apical dendrites (Fig. 1). Successfully impregnated pyramidal neurons have been observed in all well-known “pyramidal” layers (lamina II, III, V and VI). A total of 30 pyramidal cells from the parieto-temporal and fronto-temporal, sensomotor neocortex have been selected and further processed (15 superficial and 15 deep cells). The two main criteria were introduced to define terms “superficial” or “deep”, and to select adequate pyramidal neurons: (i) the cell body position in respect to the cortical lamination, and (ii) cortical lamina in which apical dendrites terminate. According to such criteria, the superficial pyramidal neurons are those having a cell body located in lamina II–III, and apical dendrites terminating in layer I. On the other hand, the deep pyramidal neurons have

In addition to tracing apical dendrites from the proximal segment toward terminal branches, we have counted the number of dendritic branching points, terminal tips and dendrite segments in each of selected pyramidal neurons. According to Van Pelt and Schierwagen [4], a segment is defined as a portion of the dendrite extending between two ramification “nodes”, or between the node and tip. These topological parameters allow us the comparison of apical dendrite arborization homogeneity of superficially and deeply situated cells. 2.3. Measuring the total length of apical dendrites The skeletonized images of pyramidal neurons were used in order to measure the total length of apical dendrites and their branches. For this purpose, an ImageJ macro called measure skeleton length has been applied (the macro written by Volker Baecker, INSERM, 2010; http://mri.cnrs.fr/index.php?m=67&c=110). The same macro was used to measure the value of the apical dendrite perimeter that is a distance between the base of apical dendrite and its most distant tip. For each pyramidal neuron, the average segment length was calculated by dividing the values of total length and the number of segments.

Fig. 2. Representative examples of the superficial and the deep cortical pyramidal neurons. The figure shows apical dendrites of pyramidal cells that were selected to calculate their fractal dimension using modified box-counting technique.

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N. Puˇskaˇs et al. / Neuroscience Letters 589 (2015) 88–91

Fig. 3. Golgi-impregnated superficial cortical pyramidal neuron and steps in its graphic processing. A – digital photo of the neuron obtained by taking photos of the same histological section at different focal planes. Using ImageJ software and its Z project command, taken images were projected onto an image stack along the axis perpendicular to the image. (magnification ×400). B – binary image of the same pyramidal neuron after removal of dendritic spines, background and adequate graphic processing. C – image of the apical dendrite of the deep cortical pyramidal neuron after removing cell body and basal dendrites, as the final step of neuron processing prior to calculating fractal dimension.

2.4. Choice of the box sizes By default, the box sizes for counting the fractal dimension using ImageJ software are 2, 3, 4, 6, 8, 12, 16, 32, 64. In this paper, the modified box-counting method was used, with the box sizes as an increasing geometric progression 2n where n = 0, 1, 2. . .10, as described in detail by Ristanovic´ et al., [22]. 2.5. Statistical analysis Statistical analysis was performed with Student’s t test and Mann-Whitney’s U test, where appropriate. Results are represented as mean ± standard error of mean, and as median with interquartile range. For the p-values less than 0.05, the differences between the means of populations were considered statistically significant.

Table 1 The number of branching points, dendritic terminals, dendrite segments, total length, fractal dimension, average segment length and perimeter of apical dendrites in superficial and deep cortical pyramidal neurons Parameters

BP DT Segments TL ASL FD Perimeter

Type of cortical pyramids

p

Superficial

Deep

9.3 ± 1.6 10.3 ± 1.6 19.5 ± 3.1 707.8 ± 111.1 36.2 (29.6–44.7) 1.329 ± 0.014 170.3 (136.4–241.2)

8.3 ± 1.0 9.3 ± 1.0 17.3 ± 2.1 1047.7 ± 73.5 59.4 (52.6–86.3) 1.238 ± 0.010 486.6 (321.0–535.4)

0.6 0.6 0.6 0.02