Orlando I. Torres-Rodríguez ¹ , Karina Ruiz-Rivera ², James T. Porter ¹

1 Ponce Health Sciences University/Ponce Research Institute

2 Recinto Universitario de Mayagüez

The single prolonged stress (SPS) model was designed to induce post-traumatic stress disorder (PTSD)-like behaviors, which has been associated with increased inflammation in rodents.

Consistent with this, SPS-exposed rats display a higher expression of the ionized calcium-binding adaptor molecule 1 (Iba-1), a microglial marker in the ventral hippocampus (VH), suggesting increased VH microglial activity in SPS-induced behavioral impairments. Although previous reports have linked microglial-mediated inflammation and impaired behaviors, the effect of microglial depletion in SPS-induced behavioral impairments is not well understood. Along this line, we depleted microglial cells to study the role of microglial activity in SPS-induced PTSD-related behaviors. We anticipated that microglial depletion would prevent SPS-induced PTSD-related behaviors. Microglial depletion was achieved by adding 290mg/kg PLX3397 to the diet prior to behavioral procedures. Animals were randomly divided into (3) groups (NO-SPS, SPS-only, and SPS-PLX3397). NO-SPS and SPS-only groups were fed with a control diet (AIN-76A) throughout the experiment, while the SPS-PLX3397 group received PLX3397 in the diet. A combination of SPS with fear conditioning (COND) and extinction (EXT) was implemented to assess fear acquisition, extinction, and extinction recall (EXT-RT) in male and female Sprague-Dawley rats. We also tested anxiety-like behaviors by exposing the animals to a 10-min open field test (OFT). Behavioral results showed that SPS-only male rats displayed higher freezing during the COND phase when compared to SPS-PLX3397 and NO-SPS groups. However, none of the female rats presented differences in freezing responses during COND. This suggests that microglial depletion prevents an SPS-induced enhanced fear acquisition in males but not in female rats. Consistent with this, SPS-only male rats froze more during the EXT, and EXT-RT phases when compared to NO-SPS and SPS-PLX3397 groups; thus, suggesting an SPS-induced impaired fear extinction learning and extinction recall. On the other hand, none of the female groups presented differences in freezing responses during the EXT or the EXT-RT; consequently, proposing that microglial depletion only prevents SPS-induced impaired fear extinction learning and recall in males, but not in female rats.

OFT results did not display any differences among male and female groups when comparing time spent in the center. This implies that microglial depletion does not affect anxiety-like behaviors in male or female rats. Furthermore, immunofluorescence staining of Iba-1 (IF) indicates that SPSPLX3397 male rats did not exhibit differences in the number of Iba-1 cells in VH, but a lower Iba-1 expression in VH was found when compared to SPS-only and NO-SPS male rats. In contrast, SPSPLX3397 female rats showed fewer Iba-1 positive cells, but no differences in Iba-1 expression in the VH when compared with other female groups, validating the microglial depleting capacity of the PLX3397. Altogether this data reveals that microglial depletion prevents stress-induced PTSDrelated behaviors in males, but not in female rats., which suggests that the development of stressinduced PTSD-related behaviors might be via different mechanisms in males than in female rats.

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