Intra-cornu ammonis 1 administration of the human immunodeficiency virus-1 protein trans-activator of transcription exacerbates the ethanol withdrawal syndrome in rodents and activates N-methyl-D-aspartate glutamate receptors to produce persisting spatial learning deficits

Abstract

Human immunodeficiency virus-1 (HIV-1) infection may produce neurological deficits, such as cognitive decline, that may be worsened by concurrent ethanol (EtOH) abuse. Among the many biochemical cascades likely mediating HIV-1-associated neuronal injury is enhancement of N-methyl-d-aspartate (NMDA) receptor function and progression to excitotoxicity, an effect that may be directly or indirectly related to accumulation in brain of the HIV-1 trans-activator of transcription (Tat) factor. The present studies were designed to examine the hypothesis that binge-like EtOH pre-exposure would enhance effects of Tat on NMDA receptor function. These studies employed a modified in vivo binge EtOH exposure regimen designed to produce peak blood EtOH levels (BEL) of <200 mg/dl in adult male rats and were designed to examine effects of intra-hippocampal injection of Tat (0.5 microl/500 pM/2 min) on EtOH withdrawal-related behavior, spatial learning, and histological measures. Unilateral cannulae were implanted into the cornu ammonis 1 (CA1) pyramidal cell layer of animals prior to beginning a 4-day binge EtOH regimen. EtOH was administered via intragastric intubation ( approximately 3.0-5.0 g/kg) with dose determined by behavioral ratings of intoxication daily for 4 days (at 08:00, 16:00, and 24:00 h). EtOH withdrawal behaviors were monitored 12 h after the last administration of EtOH. Morris water maze learning was assessed during the following 4 days, at which times brains were harvested for autoradiographic measurement of NMDA receptor density and neuroinflammation. Maximal BELs of 187.69 mg/dl were observed 60 min after EtOH administration on day 2 of the regimen. In contrast, peak BELs of approximately 100 mg/dl were observed 60 min after EtOH administration on day 4 of the regimen, suggesting development of metabolic tolerance. Significant behavioral abnormalities were observed in EtOH withdrawn animals, including tremor and seizures. Intra-CA1 region injection of Tat significantly potentiated EtOH withdrawal behavioral abnormalities, an effect that was reduced by MK-801 pre-exposure. While EtOH withdrawn animals showed learning similar to control animals, EtOH withdrawn animals that received intra-CA1 Tat injection demonstrated persisting deficits in spatial learning on days 3 and 4 of training, effects that were markedly reduced by administration of the competitive NMDA receptor antagonist MK-801 30 min prior to Tat injection. No changes in [(3)H]MK-801 binding were observed. Binding density of [(3)H]PK11195, a ligand for peripheral benzodiazepine receptors expressed on activated microglia, was elevated proximal to cannula tracks in all animals, but was not altered by EtOH or Tat exposure. These findings suggest that EtOH abuse and/or dependence in HIV-positive individuals may promote HIV-1-associated cognitive deficits by altering NMDA receptor function in the absence of microglial activation or neuroinflammation.

Figure 1

Representative image of the distribution of thionin (0.2%) injected through an intra-CA1 pyramidal cell layer cannula at coordinates AP: −3.6; DV: 2.8; ML: 2.0/−2.0 relative to bregma and ventral to the juncture of the parietal, retrosplenial, and occipital cortices.

Figure 2

Mean daily EtOH doses and blood EtOH concentrations in EtOH-treated animals at 0900, 0930, and 1500 hrs on Days 2 and 4 of binge EtOH treatment. No significant differences in mean EtOH doses were observed, though blood EtOH levels were significantly reduced by Day 4, as compared to Day 2 of the EtOH dosing regimen. * = P < 0.05 vs measurements taken at same time on Day 2.

Figure 3

EtOH withdrawal-related behavioral abnormalities, including tremor, tail rigidity, splayed paw and seizure, were significantly exacerbated by intra-CA1 Tat injection (0.5µl of a 500 pM Tat solution/ 2 minutes) and attenuated by MK-801 pre-treatment (0.075mg/kg, i.p.). *=P<0.05 vs control and Tat-injected animals. **=P<0.05 vs all other groups.

Figure 4

Latency to find the hidden platform in the Morris water maze task. Animals that were pre-exposed to the binge EtOH regimen (ending 24 hr prior to Acquisition Day 1) and received intra-CA1 region Tat injection had significantly greater latencies to find the platform than all other groups, most notably, on training days 3 and 4. *= P <0.05 vs all other groups. This learning impairment was prevented by pre-treatment with the non-competitive NMDA receptor antagonist MK-801 (0.075mg/kg, i.p.).

Figure 5

Effects of intra-CA1 region cannula implantation on binding density of the peripheral benzodiazepine binding site ligand [³H]PK11195 in the hippocampus. [³H]PK11195 binding was markedly increased as a result of cannulae implantation but was not altered by Tat or EtOH treatment. *= P <0.05 vs all other groups.

Figure 6

Representative false-color images of hippocampal [³H]PK11195 binding in EtOH-naïve and withdrawn (EWD) animals, including those administered MK-801 (0.075mg/kg, i.p.; TEM) following water maze training.