Validation of Doublecortin Hippocampal Localization

Doublecortin plays an important role in immature neurons by helping to structure microtubules. Because it is highly prevalent in immature neurons, sites of neurogenesis, such as the hippocampus and olfactory bulbs, are frequently targeted to analyze doublecortin expression. To elucidate where doublecortin is localized in the hippocampus, a previously tested protocol was used to test its effectiveness in staining for doublecortin. As hypothesized, doublecortin was found to be located in the granule cell layer of the dentate gyrus. Additionally, the protocol proved to be effective in identifying doublecortin-labeled cells, adding support to its localization in the hippocampus. Introduction Doublecortin is a neuronal migration protein; it functions to bind, bundle, and stabilize microtubules (Francis et al. 1999). Due to its nearly exclusive expression in developing neurons, doublecortin has been used increasingly as a marker for neurogenesis (Couillard-Despres et al. 2005). Clinically, mutated doublecortin is part of an x-linked lissencephaly in males, due to the lack of migration of immature neurons. In females, mutated doublecortin leads to double cortex syndrome, marked by two bands of misplaced neurons within the subcortical white region of the brain (Gleeson et al. 1998). In regards to doublecortin’s expression, Nacher et al. (2001) found strong doublecortin immunoreactivity in the dentate gyrus region of the hippocampus of young adult rats, with many granule neurons located in the hilar border of the granule layer demonstrating labeling. Brown et al. (2003) also found doublecortin to be prominently expressed in the dentate gyrus region in adult rats. Similarly, Rao and Shetty (2004) confirmed doublecortin’s localization in the dentate gyrus, finding newly generated doublecortin cells in the dentate gyrus of a male 4-month-old rat. These findings provide support to the hypothesis that doublecortin expression is restricted to the granule cell layer of the dentate gyrus region of the hippocampus, Figure 1. In the following experiment, an immunohistochemistry protocol supplied by Denny et al. (2012) will be validated for its overall effectiveness in staining for doublecortin. As demonstrated by Denny, the anti-doublecortin stain detected neurons expressing doublecortin and did not stain for any neurons that failed to express doublecortin or that had undergone x-irradiation. This proves the protocol to be an effective measure for detecting doublecortin positive neurons, which will be confirmed in the following experiment, specifically looking for localization in the dentate gyrus region. The following study will provide support for doublecortin’s hippocampal localization, as well as test the validity of the proposed protocol for replication. Determining subcellular localization is important for understanding protein function and is a critical step for genome annotation (Scott et al. 2005). With this knowledge, improved target identification during drug research may be facilitated, which may help find treatments for lissencephaly and double cortex syndrome, both of which have limited viable treatment options. Methods Collection of tissues: Three adult male F344XBN rats were sacrificed between 28-32 months of age. Brain samples were collected through a transcardianal perfusion process using a ketamine/xylazine mixture to anesthetize the rats. Then, 4% paraformehdlehyde was pushed through the cardiovascular system. Brains were incubated overnight in paraformehdlehyde and 40um coronal sections were collected using a vibratome. Every 12th section was saved and placed in cryoprotectant. Immunohistochemistry: Doublecortin immunohistochemistry was carried out by washing sections in 1x PBST three times for ten minutes and then quenched in 0.3% hydrogen peroxide in 1x PBS at room temperature for 15 minutes. Sections were washed in 1x PBST three times for ten minutes again and then blocked in 10% normal donkey serum in 1x PBST for two hours at room temperature. Sections were incubated in primary antibody (Goat antidoublecortin, 1:500 dilution, Santa Cruz, # SC.8066) for 48 hours at 4°C in 10% normal donkey serum. Sections were then washed in 1x PBS three times for 15 minutes at room temperature. Then, sections were incubated in secondary antibody (Jackson Immuno # 705-065-003, 1:500 dilution) for two hours at room temperature in 1x PBS. Sections were washed in 1x PBS three times for ten minutes and incubated in an ABC Elite Kit for one hour at room temperature. Sections were washed in 1x PBS three times for ten minutes and incubated overnight in 1x PBS. Then, sections were washed in 1x PBS three times for ten minutes and incubated for nine minutes in diaminobenzidine. Tissue was washed in 1x PBS three times for ten minutes and Nissl stained. Nissl staining was carried out by exposing sections to ddH2O for 30 seconds followed by incubation in 0.1% cresyl violet for 3.5 minutes. Sections were incubated in ddH2O for 30 seconds, then through an alcohol gradient of 70%, 95%, and 100% ethanol for 90 seconds, 15 seconds, and 30 seconds respectively. Sections were rehydrated in ddH2O for 30 seconds. Rat AET3AD was the only rat to undergo the Nissl staining on the first day. The next day, the remaining two rats were Nissl stained. Rat AET8LAD was exposed to the cresyl violet for four minutes, while rat AET9AD was exposed to the cresyl violet for 4 minutes and 15 seconds. Tissue was incubated in 1x PBS over the weekend. Four days later, sections were then mounted to slides and left to dry. Slides were then treated with Xylene for four minutes before being coverslipped with Permount. Results The diagram in Figure 1 depicts the main regions of a hippocampus. Staining was expected in the cell bodies of granule cells in the granule cell layer of the dentate gyrus (Figure 1). However, possible doublecortin staining was expected in the axonal projections of granule cells, which extend particularly to the CA3 region of the hippocampus. Analysis of doublecortin stained hippocampal sections showed positive staining in the granule cell layer of the dentate gyrus (Figure 2). However, quantification of the staining was not completed in this study. Visually, the cells appeared to be lightly stained. Upon closer inspection under the microscope, analysis verified that some limited staining was present and revealed the degree of the light staining. The limited staining made identifying doublecortin stained cells very difficult. Nevertheless, some stained granule cells in the granule cell layer (Figure 1) were found using microscopy imaging (Figure 2). Figure 1. Hippocampus diagram. The diagram shows the outer molecular layer (mo) in lavender color, and the inner granule cell layer (sg) in magenta color, in the dentate gyrus (DG). The CA1 and CA3 regions are also shown. Primary Article