Immediate Early Gene (IEG) mapping of spinal cord neurons activated by cystometry induced micturition in rat

Janet Keast, Ph.D.
,
Peregrine Osborne, Ph.D.
,
Nicole Wiedmann, Ph.D.
,
Agnes Wong W, Ph.D.

Spatial mapping of cystometry-activated neurons in the sacral spinal cord in awake male and female rats.

Updated on September 30, 2020 (Version 1)

Corresponding Contributor:

Janet Keast
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Dataset Overview

Study purpose: The lower urinary tract (LUT) is controlled by an external neural circuit, of which the spinal component is only partly described. To address this we used immunohistochemical immediate-early gene (IEG) markers to map neuronal activity in lumbosacral spinal cord after stimulating intense micturition in awake, unrestrained female and male rats.

Data collection: The dataset contains microscopic images, neuron counts, and cystometrograms obtained after stimulating intense micturition in rats. Standard cytometry parameters were digitally recorded (PowerLab 4/26, ADInstruments, NSW, Australia). After stimulation, animals were anesthetized, fixed and the spinal cord was removed. Frozen sections (40 μm) were then cut in the transverse plane and collected as four 1:4 series (160 μm between sections) spanning the spinal cord segments L5–S2. Five sections per spinal segment were labeled with each antibody combination of interest. For each antibody combination, spinal cord sections (L5–S2; five sections per segmental level) were anatomically ordered from 1 to 20. Entire transverse sections were imaged (tile scanned at 12 Bit, pixel scaling 0.645 μm × 0.645 μm) using a Zeiss AxioImager M2 (Zeiss, Oberkochan, Germany). In each section, positive neurons were counted across spinal cord regions, defined by the boundaries described in (Watson, Paxinos, & Kayalioglu, 2009), and the sacral preganglionic nucleus (SPN) outlined in (Forrest, Payne, Keast, & Osborne, 2015).

Primary conclusion: The outcome of analyses using this data are reported in Wiedmann et al. 2020. We identified sex differences in the segmental and regional activation of lumbosacral spinal cord following stimulated micturition in awake rats. This included net activation of c-Fos+ neurons in dorsal horn of females versus inhibition in males; and stronger female activation of two autonomic motor regions: sacral spinal nucleus (SPN, detected in TH+ catecholamine neurons) and spinal nucleus of the dorsal commissure (SDComm).


Curator's Notes

Experimental Design: Animals (total 24 rats) were allocated to four groups defined by the between-subject factors: Experimental group (Cystometry | Control) and Sex (Female | Male). Micturition cycles were detected by recording bladder pressure and quantified by measuring standard urodynamic parameters. Rats in group Control experienced the same habituation, handling, and exposure to the experimental apparatus but did not undergo cystometry surgery or recording. After completing in-vivo experiments, all rats were anesthetized, sacrified, and fixed. Samples of lumbosacral spinal cord were removed and processed for immunohistochemical activity mapping. This was done using ordered 1:4 series of 40 µm transverse sections through lumbosacral segments: L5, L6, S1 and S2 (5 sections per segment).

The response measures were numbers of neurons coexpressing combinations of two immunohistochemical markers: c-Fos(immediate-early gene, IEG, neuronal activity marker) and TH (tyrosine hydroxlase, catecholamine neuron marker), c-Fos and Pax2 (inhibitory spinal neuron marker), and c-Fos and EGR-1 (alternate IEG marker also known as Zif268). Neuron counts were grouped using three nested within-subject factors: Segment (lumbosacral spinal cord segments L5 | L6 | S1 | S2), Section (ordered series of 20 sections, 5 per segment), and Region (11 spinal cord regions identified by Watson et al., 2009). Neurons were counted using ImageJ FIJI Cell Counter plugin, where a marker (denoting an xy coordinate) was designated for each positive cell. To avoid double‐counting neurons, every fourth section was analyzed for each antibody combination (160 μm between sections).

Completeness: The dataset is a part of the larger study: "Peripheral connectome of the rat lower urinary tract"

Subjects & Samples: Adult male (n=12) and female (n=12) Sprague‐Dawley rats (Biomedical Sciences Animal Facility, University of Melbourne) aged 8–9 weeks were used in this study. Standard urodynamic parameters were recorded on live, awake, and unrestrained animals. Samples of the spinal cord were collected from fixed tissues in a series of 40 µm transverse sections through lumbosacral segments: L5, L6, S1, and S2 (5 sections per segment).

Primary vs. Derived: Data in the primary folder are divided by subject, then by the type of experimental output: microscopic images-in .czi format, cellcount- in .XLM format, and cystometry in .adicht file format. Image data (JPEG2000 and OME-TIFF) in the derivative folder was derived from primary images (.czi). The primary images were converted with 40:1 compression to JPEG2000 (.jp2) by MBF Bioscience for web streaming and visualization on the SPARC Data Portal. The primary images were also converted with lossless compression to OME-TIFF (.tif) by MBF Bioscience. Microscopy image metadata is included in the file header of all .jp2 and .tif in the derivative folder, as indicated on the manifest.R17-164-TissueMakerStack.jpx, R18-312-TissueMakerStack.jpx, and R19-422-TissueMakerStack.jpx were created in TissueMaker (RRID:SCR_017322) by aligning individual primary czi image planes together into a three-dimensional image stack. JPX image stacks have 40:1 lossy compression. JPX image stacks were converted to OME-TIFF format (lossless) by MBF Bioscience.

Code Availability: Not Applicable

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Publishing history

September 30, 2020
Originally Published
September 30, 2020 (Version 1)
Last Updated

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