Anxiety-like behavior of mice produced by conditional central expression of the HIV-1 regulatory protein, Tat

Abstract

Rationale: Human immunodeficiency virus (HIV) infection is associated with substantial increases in generalized anxiety. The HIV regulatory protein, transactivator of transcription (Tat), has been implicated in the neuropathogenesis related to HIV-1 infection. However, direct examination of the effect of Tat on behavioral measures of anxiety has not been demonstrated.

Objective: To identify whether expression of the Tat1-86 protein exerts dose-dependent and persistent anxiety-like effects in a whole animal model, the GT-tg bigenic mouse.

Methods: GT-tg mice and C57BL/6J controls were administered doxycycline in a dose- (0, 50, 100, or 125 mg/kg, i.p., for 7 days) or duration- (100 mg/kg, i.p., for 0, 1, 3, 5, or 14 days) dependent manner to induce Tat1-86 in brain. Mice were assessed for anxiety-like behavior in an open field, social interaction, or marble burying task 0, 7, and/or 14 days later. Central expression of Tat1-86 protein was verified with Western blot analyses.

Results: Doxycycline produced no effects on C57BL/6J controls that lacked the Tat1-86 transgene. Among GT-tg mice, doxycycline (100 mg/kg for 3, 5, or 7 days) significantly increased anxiety-like behavior in all tasks, commensurate with enhanced Western blot labeling of Tat1-86 protein in brain, displaying optimal effects with the 7-day regimen. Greater exposure to doxycycline (either 125 mg/kg for 7 days or 100 mg/kg for 14 days) impaired locomotor behavior; whereas lower dosing (below 100 mg/kg) produced only transient increases in anxiety-like behavior.

Conclusions: Expression of HIV-1-Tat1-86 in GT-tg mouse brain produces exposure-dependent, persistent increases in anxiety-like behavior.

Fig. 1

Doxycycline-induced Tat_1-86 protein expression in GT-tg mouse whole-brain. The β-actin antibody labeled a single band (upper panels) corresponding to the weight of the β-actin protein of similar intensity across all samples. By contrast, the Tat antibody labeled a number of proteins non-selectively (lower panels), but only demonstrated a difference in labeling intensity at the 19 kDa band that corresponded to the (a) dose and (c) duration of doxycycline, (e) administered for less than 14 days after treatment. Summary graphs of the quantified whole brain Western blots were plotted as a percent of control (saline-treated GT-tg) labeling. Doxycycline treatment resulted in an increase in Tat-antibody labeling in GT-tg mice dependent on the duration of pretreatment. Labeling intensity differences corresponded to the dose (b) or duration (d) of doxycycline administered, or harvest date after induction (f) at 19 kDa, which has been suggested to be the observed weight of expressed Tat protein in the GT-tg mouse. Symbols indicate significant difference from saline controls (*) or Dox-treated mice that were assessed immediately (0 days) after treatment end (†), p < 0.05.

Fig. 2

(a) GT-tg bigenic mice were administered saline or a dose of doxycycline (Dox) for 7 days. As depicted, mice were repeat-tested 0, 7, and 14 days after treatment and were assessed for anxiety-like response in an (b) open field (n = 24 / grp), (c) social interaction (n = 20 / grp), or (d) marble burying (n = 24 / grp) test at those times. † indicates significant difference from post-treatment day 0, demonstrating habituation due to repeated testing. On respective post-treatment days, symbols indicate significant difference from saline control (*), Dox 25 mg/kg group (ˆ), or all other groups (‡), p < 0.05.

Fig. 3

(a) GT-tg bigenic mice were administered saline or a given duration of doxycycline (Dox; 100 mg/kg). As depicted, mice were repeat-tested 0, 7, or 14 days after treatment and were assessed for anxiety-like response in an (b) open field (n = 24 / grp), (c) social interaction (n = 20 / grp), or (d) marble burying (n = 24 / grp) test at those times. † indicates significant difference from post-treatment day 0, demonstrating habituation due to repeated testing. On respective post-treatment days, symbols indicate significant difference from saline control (*), Dox 1-day group (@), or Dox 3-day group (∥), p < 0.05.

Fig. 4

(a) To obviate effects of habituation from repeated testing, GT-tg bigenic mice were administered saline or doxycycline (Dox; 100 mg/kg) for 7 days and were tested only once: either 0, 7, or 14 days following treatment. At those times, mice were assessed for anxiety-like response in an (b) open field (n = 24 / grp), (c) social interaction (n = 16 / grp), or (d) marble burying (n = 24 / grp). * indicates significant difference from saline administration, p < 0.05.