Journal: Frontiers in Neural Circuits

Article Title: Functional Neuronal Topography: A Statistical Approach to Micro Mapping Neuronal Location

doi: 10.3389/fncir.2018.00084

Figure Lengend Snippet: Steps for tissue sampling and measurement from behavioral data. (A) Run behavioral models . Any expression of a chosen behavior can be used as a model. In our example we have used Auditory Pavlovian fear conditioning. Behavioral testing was conducted with adult male Sprague-Dawley rats in acoustic classical fear conditioning chambers. A 0.6 mA foot shock with duration of 500 ms was paired with a tone of 5 kHz and 75 dB, 20 s in duration to produce an associative fear memory. (B) Perform immunocytochemistry . Avidin–biotin peroxidase complex method is demonstrated here. Sections from the lateral amygdala (LA) were labeled for Arc, scanned using a slide scanner and cropped at 2x magnification. Enlarged inset square shows Arc + neurons in the dorsolateral portion of the LA at 20x magnification. Inverted gray scale images of fluorescent immunocytochemistry would also be suitable. (C) Choose suitable anatomical marker to be used as an anchor . The caudate putamen and lateral ventricle are two examples of anatomical landmarks, that we have used previously, and can be differentiated in serial sections for section alignment by Feret length within the ventile or between anatomical landmarks. Photomicrographs show three consecutive 60 μm sections across the rostrocaudal axis of the rat brain, depicting 2.76, 2.70, and 2.64 mm anterior to Bregma in the medial prefrontal cortex (mPFC). Feret diameter is shown – red arrow. Brain sections at Bregma coordinates –3.32, –3.36, and –3.40 mm posterior from Bregma were used to align the LA (Source: see ). The maximum Feret length of the caudate putamen in the prefrontal cortex was shown to be statistically different across Bregma coordinates, animals and conditions. (D) Establish section alignment . The Rat Brain Atlas is an important tool to assist alignment of sections. Schematic diagrams are shown depicting the regions of interest. The dorsolateral portion of the lateral amygdala (LAd), the ventromedial portion of the lateral amygdala (LAvm) and the ventrolateral portion of the amygdala (LAvl) are shown in three serial sections caudal from bregma –3.36 mm. The prelimbic (PL) and infralimbic (IL) cortex are represented by three serial sections caudal from bregma 2.52 mm. Brain Atlas diagrams are adapted from .

Article Snippet: Example: The sample data set consisted of fear conditioned adult male Sprague-Dawley rats ( RRID:RGD_5508397 ) ( n = 40) that underwent behavioral procedures in standard Pavlovian fear conditioning chambers (Coulbourn Instruments, Allentown, PA, United States) (see Figure ).

Techniques: Sampling, Expressing, Immunocytochemistry, Avidin-Biotin Assay, Labeling, Marker

Journal: Frontiers in Neural Circuits

Article Title: Functional Neuronal Topography: A Statistical Approach to Micro Mapping Neuronal Location

doi: 10.3389/fncir.2018.00084

Figure Lengend Snippet: Steps for producing and analyzing topographical density maps. (A) Create topographic density map . A neuronal topographic density mean map is produced by transferring binned data from Excel to Sigma Plot (or equivalent software) (X data = x coordinates, Y data = y coordinates). Density maps can be created for each sub region. A coefficient of variance map can be prepared by dividing the standard deviation by the mean across all samples in one condition. Difference maps can also be created between conditions. The data matrix from Origin Pro (or equivalent) is transferred to a spreadsheet. This procedure is followed for each animal from a single condition/group. An average across all sheets produces the data for a mean density map. The standard deviation is calculated and divided by the mean, producing the data required for the coefficient of variance (CV) map. Example – topographic density (mean and CV) maps shown for Bregma –3.36, pMAPK + neurons in the ventrolateral portion of the LA of rats that underwent extinction training ( n = 7). (B) Align density map with contour and brain sections . To enhance visualization of specific neuronal subsets, density maps can be inserted into the contours or superimposed over brain sections. Density maps may be edited to change the styles, colors, font sizes, labels etc., providing alternatives conducive to individual requirements. Information regarding cell layers can be determined from visualizing the distribution of activated neurons as shown in the pMAPK labeling of the mPFC of rats that have undergone auditory fear conditioning ( n = 7): mean map generated in Sigma Plot (or equivalent), map placed into contour, map overlaid on rat brain section. (C) Quantitative analysis of variance between conditions. A variety of statistical analysis can be performed to compare binned data such as Bonferroni correction, principal component analysis (PCA), false discovery rate (FDR), multiple discriminant analysis and mixed model ANOVA. Example of mean maps for the expression of pMAPK in the LA provides visual comparison between auditory fear conditioned ( n = 6) and naïve ( n = 7) rats. pMAPK + ranks comparing extinction ( n = 7) and no extinction ( n = 5) groups within the ventrolateral portion of the LA p = 0.0022 ( t -test, Mann–Whitney rank and SEM).

Article Snippet: Example: The sample data set consisted of fear conditioned adult male Sprague-Dawley rats ( RRID:RGD_5508397 ) ( n = 40) that underwent behavioral procedures in standard Pavlovian fear conditioning chambers (Coulbourn Instruments, Allentown, PA, United States) (see Figure ).

Techniques: Produced, Transferring, Software, Standard Deviation, Labeling, Generated, Expressing, Comparison, MANN-WHITNEY