Neuroscience
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21691
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Browsing Neuroscience by Author "Contreras, Jo"
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Item Androgen Receptors in the Middle Aged Male Rat Brain: Influence of Testosterone Deprivation on Expression(2017-03-14) Contreras, Jo; Cunningham, Rebecca; Fort*, Callie; Cuellar*, Elric; Lopez, Gladys; Metzger, Daniel; Oppong-Gyebi, Anthony; Schreihofer, Derek; Smith, Charity B.S.Purpose: 1) To determine whether long-term testosterone deprivation (LTTD) alters the levels and/or distribution of androgen receptors in the middle-aged male rat brain and 2) to determine whether testosterone replacement after LTTD influences androgen receptor levels. Methods: Twelve-month old male Fischer 344 rats were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing testosterone (STTD). Additional groups were castrated for 10 weeks before being treated with testosterone (LTTD+T) or cholesterol (LTTD). Four weeks later, rats were euthanized and brains were collected for immunoblotting and immunohistochemistry (IHC) for androgen receptors (AR) using antibodies targeting the N-terminus or C-terminus of the protein. The cerebral cortex, hippocampus, thalamus, hypothalamus were examined, and testes were used as positive control tissue. Results: Contrary to expectations, the full-length AR (116 kDa) was barely detectable in the hippocampus and cerebral cortex by immunoblotting with antibodies directed to either end of the AR. Rather, smaller fragments were readily detected. Examination of the size of these fragments (~30, 37, 50, and 80 kDa) and consultation of the literature for the human AR, suggested that they represented calpain-dependent cleavage fragments. A series of control experiments was performed in an attempt to extract the full-length AR using rat testes as a positive control tissue. Protease inhibitors, EDTA, and the AR agonist dihydrotestosterone failed to reduce the appearance of fragments. Interestingly, the pattern of fragments from the hippocampus (80 [greater than] 37 kDa) differed from that from the cortex (37 [greater than] 80 kDa) suggesting differential processing. IHC of coronal brain sections though the forebrain revealed nuclear AR staining consistent with full-length AR in regions of high expression, including the hypothalamus. In agreement with immunoblotting AR staining in the cortex and hippocampus appeared to be cytoplasmic, rather than nuclear. No significant differences were observed between treatment groups. Conclusions: These data suggest that AR protein in some areas of the middle-aged male rat brain is rapidly degraded into fragments with altered localization and potential for transcriptional activity and/or signaling functions. Although no differences in expression were apparent between treatment groups, the differential processing of AR in the rat brain is a novel finding warranting further investigation.Item Influence of Testosterone Deprivation and Replacement on Cognition and Oxidative Stress in Middle-Aged Male Rats(2017-03-14) Contreras, Jo; Metzger, Daniel; Oppong-Gyebi, Anthony; Kasanga, Ella; Vann, Philip; Sumien, Nathalie; Cunningham, Rebecca; Schreihofer, Derek; Smith, Charity B.S.Purpose: Data from aged men suggests a negative correlation between testosterone levels and cognitive function, including the development of mild cognitive impairment and Alzheimer’s disease. The purpose of this study was to 1) determine whether long-term testosterone deprivation (LTTD) impairs cognition and increases oxidative stress in the middle-aged male rat brain and 2) determine whether testosterone (T) replacement after LTTD can reverse these effects. Methods: Twelve-month old male Fischer 344 rats (13 per group) were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing T (short-term T deprivation; STTD). Additional groups were castrated for 10 weeks before being treated with T (long-term T deprivation; LTTD+T) or cholesterol (LTTD). Rats underwent cognitive testing with the Morris water maze (MWM). A 4-day acquisition phase was used for rats to learn the location of a hidden platform. A retention day was used to determine whether rats remembered the platform location after it was removed. A 2-day reversal trial in which the platform was moved to a new location was used to examine mental flexibility. These tests require both hippocampal and cortical areas of the brain. Following MWM rats were euthanized and brains were collected for immunoblotting for markers of cell death (Spectrin) and oxidative stress responses (NFkB, COX2, NOX2) in the hippocampus and cerebral cortex. Plasma advanced oxidative protein products (AOPP) were used as a peripheral marker of oxidative stress. Total testosterone was measured by ELISA. Results: Castration reduced total testosterone to 40% of intact levels whereas testosterone implants increased levels back to those of intact males. Overall, intact rats performed significantly worse on the MWM than STTD and LTTD with or without T replacement. We saw no significant changes in blood AOPP among treatment groups. Similarly, there were no significant differences in the expression of oxidative stress regulated genes or Spectrin cleavage in the hippocampus. Cortical measurements are on-going. Conclusions: These data suggest that castration with or without T replacement improves cognitive function in middle-aged rats, but does not significantly alter oxidative stress in the brain or periphery. These data support the safety profile of testosterone replacement to physiological levels and do not recapitulate correlative data observed in men.