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HIV-1 Tat protein: sufficient to cause HIV-Associated Neurological Deficits (HAND)?

This study investigates the role of HIV-1 Tat protein in causing HIV-associated neurological deficits (HAND) and its effects on learning and memory performance. The expression of Tat in the brain and its neurotoxic properties are examined using a transgenic mouse model.

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HIV-1 Tat protein: sufficient to cause HIV-Associated Neurological Deficits (HAND)?

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  1. HIV-1 Tat protein: sufficient to cause HIV-Associated Neurological Deficits (HAND)? Jay P. McLaughlin Department of Pharmacodynamics, University of Florida

  2. Background: Human Immunodeficiency Virus (HIV): changing death rates for all ages suggest transition • Retrovirus infection responsible for acquired immunodeficiency syndrome (AIDS) • Progressive failure of immune system allows life-threatening infections, cancer • Therapeutics now available have improved prognosis and lifespan

  3. Background: Psychiatric comorbidities remain common among HIV patients Sakamoto et al., www.slideshare.net/UCSDCFAR/test-forhivassocdementiainindia • Additional comorbid psychiatric disorders significantly impair quality of life: • Major Depression • Anxiety • Post-Traumatic Stress Disorder • Increased substance abuse: alcohol, cocaine, opioids

  4. Background: Persistence of HIV biological factors • Although HAART suppresses HIV infection to undetectable levels, it doesn’t eliminate latent HIV reservoirs (such as thought to be in CNS) • Virus continues to produce and secrete inflammatory cytokines and HIV Transactivator of Transcription (Tat) protein • Tat: HIV regulatory protein: vastly increases transcription of HIV genes. • Secreted intact and functional from infected cells; detected in brain and CSF of HAD patients. Serum Tat+ (50 HIV Subjects tested quarterly) CSF [Tat] in 3 of 8 HIV subjects tested once (86%) (70%) (38%) HIV patients Controls Mediouni et al., Infect. Disord. Drug Targets 12:81, 2012 Johnson et al., PNAS 110:13588, 2013

  5. Background: HIV-1 Tat induces a wide range of deleterious biological effects • Tat protein is neurotoxic; with even transient exposures detrimental GLT-1i Hyperglutamatergic State NMDA receptors Spermine Oxidase Oxidative Stress MAO MnSODi Mitochondrial Complex IVi Mitochondrial Impairment (No known cellular homologs) NAD+i SIRT1i • Tat mediated neurophysiological effects could lead to HIV cognitive dysfunction: • Exposure to Tat protein promotes loss of synapses and neuronal death • Tat in vitro suppresses LTP in hippocampal culture (Behnisch et al., 2004) • Psychiatric effects unclear, but minimal behavioral evidence implicates Tat: • Tat point mutation (Clade C) linked to HAD prevalence and neuron toxicity • Injecting Tat: ↑ deficits in working memory in 8-arm maze (Li et al., 2004) • Injecting Tat: ↑ depression-like behavior in mouse (Lawson et al., 2011)

  6. Model: Inducible Tat expression with the GT-tg bigenic mouse • To better understand the role of Tat in neuropathogenesis, dysfunction and behavioral disorders, need transgenic mouse • Traditional “knock-in” Tat mice show tumors, poor viability (Corallini et al., 1993) • New model: GT-tg transgenic mouse (Kim et al., Am J. Pathol. 162: 1693; 2003) • Gene for Tat protein (from Clade B) is inducible and brain-selective • Glial, GFAP-linked Tet-on system conditionally activates Tat synthesis • Majority of Tat expression triggered by Doxycycline (Dox) • Expression conditional, and only in GFAP-containing astrocytes • Expression of Tat produces histopathology as seen with NeuroAIDS • Avoids mechanical injury, confounds from direct injection of Tat to brain Dox (mg/ml) in drinking water for 8 days (Kim et al., 2003)

  7. Western blot characterization of doxycycline-mediated Tat expression in the GT-tg mouse brain • Results with Abcam polyclonal ab43014, lot #904506 (1:2000) (Carey et al., Behav Brain Res, 229: 2012). • Limitation: present antibodies for Tat uniformly terrible. * * * * * * 19 kDa 200 300 19 kDa 19 kDa 200 † 250 150 150 200 100 % Control labeling (± SEM) 150 100 % Control labeling (± SEM) % Control labeling (± SEM) 100 50 50 50 0 0 0 Dox (mg/kg/d, i.p. for 7 d) Days Dox (100 mg/kg/d, i.p.) 0 7 14 Saline, 7d; 1 3 5 7 14 25 50 Saline, 125 Saline, harvest 14d 7d Harvest, days after Dox after 14 d (100 mg/kg, i.p. for 7 d) Paris et al, Psychopharmacology (Berl), 231: 2014

  8. Five days’ exposure to Tat results in GT-tg mouse brain gray matter density reductions: ex vivo MRI Regions of significant loss: 1) sublenticular extended amygdala, 2) piriform cortex, 3) amygdala, 4) hypothalamus, 5) peri-/entorhinal cortex, 6) amygdala-hippocampal area Carey et al, Prog Neuropsychopharmacol Biol Psychiatry, 43: 2013

  9. Initial Magnetic Resonance Spectroscopy studies suggest Tat-induced neurochemical abnormalities in brains of GT-tg mice Medial Frontal Cortex, GT-tg mice 9.4T Proton Magnetic Resonance Spectroscopy 7-d exposure to Tat protein increases medial frontal cortex glutathione (GSH) Dr. Marc Kaufman, Translational Imaging Laboratory, McLean Hospital

  10. Hypothesis Global Hypothesis: Expression of HIV-1 Tat protein in brain is sufficient to produce the neuropsychiatric effects associated with HIV-1 infection Specific studies: • Study I: The expression of Tat in brain is sufficient to impair learning and memory performance in the (A) Barnes maze and (B) novel object recognition assays, as well as cognition in (C) the pre-pulse inhibition assay. • Study II: The expression of Tat in brain is sufficient to induce increases in (A) anxiety- and (B) depression-like behaviors. • Study III: Tat protein potentiates the psychostimulant and rewarding effects of the reinforcing substances such as morphine in the conditioned place preference (CPP) assay and two-bottle choice assay

  11. Study I: Experiment 1 Methods: The Barnes Maze • Tests: spatial learning and memory • Goal: Locate escape box under one of 40 hole using distal cues to navigate • Motivation: bright open arena & static noise • Testing schedule • Days 1-4: Acquisition, two 3-min trials/day, 15-min ITI • Day 4: Probe trial, 90 s • Day 5: Reversal learning, 4 trials • Measures • Latency • Errors • Probe trial success

  12. Uninduced 170 5 d 100 mg/kg Dox 7 d 100 mg/kg Dox 160 150 140 130 120 Latency to escape (s±SEM) 110 100 90 80 1 2 3 4 Day Study IA: Tat-induced GT-tg mice demonstrated longer latencies to find the escape hole than uninduced GT-tg littermates GT-tg bigenic mice Note: C57Bl/6J show strain Differences from uninduced GT-tg! Tat-induced GT-tg mice also: • Committed more errors during acquisition † * * * † • Showed significant deficits in finding the escape location during probe trial Carey et al, Behav Brain Res, 229:2012

  13. 180 160 Uninduced 5 d 100 mg/kg Dox 140 7 d 100 mg/kg Dox Latency to escape (s±SEM) 120 100 1 2 3 4 80 Trial Study IA: Tat-induced mice required more trials to learn the new escape location during a reversal learning task GT-tg bigenic mice † † * ^ * Carey et al, Behav Brain Res, 229:2012

  14. time spent on object B time spent on object A + B % Recognition Index = Study I: Experiment 2 Methods: The Novel Object Recognition Assay Familiarization/Acquisition Phases 10 min/each Testing Phase 10 min Object A 10 min 10 min Object B Phase 1 2 Dice Phase 2 2 Dice Phase 3 Object B = marble Methods from Carey et al., J Neuroscience 2009

  15. Study IB: Tat-induced impairment of novel object recognition is long-lasting C57BL/6J + Dox GT-tg bigenic mice (treated daily for 7 d) * * 80 Uninduced (saline treated) induced- tested 48 h post Dox 70 induced- tested 1 wk post Dox induced- tested 1 mo post Dox 60 % Recognition Index + SEM 50 40 30 20 Phase 1 Phase 2 Phase 3 Carey et al, Behav Brain Res, 229:2012

  16. tested 48h post induction tested 1wk post induction 80 70 60 50 40 30 Study IB: Tat-induced impairment of novel object recognition is dependent on the dose of Dox administered and duration of exposure * * % Recognition Index - Phase 3 25 50 100 1d 3d 5d 7d C57-Dox Uninduced GT-tg Dox for 7 d (mg/kg/d) 100 mg/kg Dox each day Carey et al, Behav Brain Res, 229: 2012

  17. Study IB: Prevention of Tat-induced novel object recognition deficits by co-administration of the Tat inhibitor, didehydro-Cortistatin A (dCA) Didehydro-Cortistatin A (dCA) Daily treatment for 5 d with: Vehicle (i.c.v.) / Saline, i.p. dCA (10 nmol, i.c.v.) / Saline, i.p. * Vehicle (i.c.v.) / Dox (100 mg/kg, i.p.) * * dCA (10 nmol, i.c.v.) / Dox (100 mg/kg, i.p.) 70 60 Mousseau et al, Cell Host & Microbe 12:2012 % Recognition Index + SEM 50 40 30 Phase I Phase II Phase III

  18. Study 1C: Exposure to Tat protein impairs prepulse inhibition of the acoustic startle response in GT-tg bigenic mice ♫!120 dB ♫!120 dB ♪! 74 dB Pulse Pulse Startle response Startle Response Inhibited Prepulse Intensity Intensity 100 ms Time Time * * * 80 Pretreatment: Indomethacin (10 mg/kg/d, i.p.) * 80 * * 70 74 dB 74 dB * 70 60 * * 78 dB %Startle Amplitude (% of BL) 78 dB * † 50 60 74 dB %Startle Amplitude (% of BL) † 78 dB * * 40 86 dB 50 86 dB † 86 dB 40 14 1 7 14 7 1 7 Saline (d, i.p) Doxycycline (100 mg/kg/d, i.p) Doxycycline (100 mg/kg/d, i.p) Saline (d, i.p) Days Administered Days Administered Paris et al., Behav Brain Res, 291: 2015

  19. Study I: Summary findings • Mice expressing Tat protein show deficits in spatial learning and memory performance in the Barnes maze • Mice expressing Tat protein demonstrate perseveration in learned spatial responses. • Mice expressing Tat protein demonstrate long-lasting impairment of novel object recognition • Effect reversed by daily treatment with Tat inhibitor, dCA • Exposure to Tat protein impaired paired-pulse inhibition in a exposure dependent manner, suggesting impaired cognition • Impairment reversed with indomethacin, but only if administered early

  20. Study II A: Experiment 1 Methods: Mouse models of anxiety-like behavior • Open field test: • Fewer entries into the center indicate anxiety-like behavior. • Mouse social interaction test: • Subject placed in corner of open field; weight matched novel mouse placed in the opposite corner, and interaction observed for 5 min. • Less time spent in social behavior indicates anxiety-like behavior. • Marble burying test: • More marbles buried indicates anxiety-like behavior. 30 min • Acoustic startle reflex (ASR): • Longer time spent “frozen” indicates anxiety-like behavior.

  21. Study II A: Increased exposure to Tat protein increases anxiety-like behavior in GT-tg mice Open field Social interaction Marble burying • C57BL/6J mice show no differences between saline and Dox treatment Paris et al, Psychopharmacology (Berl), 231: 2014

  22. Study II A: Anxiety-like effects resulting from exposure to Tat protein endure at least two weeks after induction Open field Social interaction Marble burying • Separate cohorts tested to be certain to avoid test decay • C57BL/6J mice show no differences between saline and Dox treatment Paris et al, Psychopharmacology (Berl), 231: 2014

  23. Study II A: Prevention of Tat-induced anxiety-like behavior in the open field assay following daily co-administration of didehydro-Cortistatin A (dCA) Daily treatment for 5 d with: 35 Vehicle (i.c.v.) / Saline, i.p. † dCA (10 nmol, i.c.v.) / Saline, i.p. Vehicle (i.c.v.) / Dox (100 mg/kg, i.p.) 30 dCA (10 nmol, i.c.v.) / Dox (100 mg/kg, i.p.) 25 Time in Center (s + SEM) * 20 15 0

  24. Study IIA: Exposure to Tat protein impairs the acoustic startle reflex in an exposure-dependent manner reserved by indomethacin treatment * 450 * 450 * * 400 400 * 350 350 300 † Acoustic Startle Reflex (± SEM) 300 Acoustic Startle Reflex (± SEM) 250 250 200 200 150 150 0 - + + - Dox (100 mg/kg, 1d) 1 7 0 - + + - Dox (100 mg/kg/d, 7d) 5 3 1 14 - - + + Indomethacin (10 mg/kg) Days of Days of Doxycycline Saline (i.p.) (100 mg/kg, i.p.) Paris et al, Behav Brain Res, 291: 2015

  25. Study II: Exposure to Tat protein increases activation of Iba1-labeled microglia and GFAP content in frontal cortex of GT-tg bigenic mice in a time-dependent manner Morphology rating (Davis et al., 1994): 1 = resting 2 = early activated 3 = activated/reactive 4 = reactive Vehicle Doxycycline * 50 2.75 Agranular Insular Cortex * Anterior Cingulate Cortex 2.50 45 Prelimbic Cortex * ^ 2.25 40 2.00 35 Frontal Cortex GFAP (ng/mg ± SEM) Morphology Rating (±SEM) 1.75 30 1.50 25 1.25 1.00 20 0 1 3 5 7 1 3 5 7 Days of Doxycycline Days of Doxycycline Saline Saline (100 mg/kg/d, i.p.) (100 mg/kg/d, i.p.) (i.p., for 7d) (i.p., for 7d) Paris et al, Behav Brain Res, 291: 2015

  26. Study II B: Exposure to Tat protein decreases consumption of saccharine, but not water, in a two-bottle choice test • Saccharine consumption test: • Train individually-housed mice 4 days with two bottles of water, • replace one with saccharine (0.2%) for testing • Decreased saccharine consumption indicates anhedonia and depression-like behavior. Average Saccharine Consumption (ml/d ± SEM Bottle A: water Bottle B: Saccharine (0.2%)

  27. Study II B: Exposure to Tat protein increases depression-like behavior in the tail-suspension test in a persistent, exposure-dependent manner • Tail-suspension test: suspend by tail for 5 min • Increased time immobile indicates depression-like behavior. Saline, 0.9% (d, i.p.): Doxycycline (100 mg/kg/d, i.p.): 1 d 7 d 1 d 7 d 7 d * * 130 120 * 110 * 100 * 90 Time Immobile (s±SEM) 80 70 GT-tg mice , 60 G-tg mice (no Tat) 50 Tat Level Normalized 1 7 14 28 Testing, days after treatment

  28. Study IIB: Co-treatment with methylsulfonylmethane (MSM) mitigates Tat-induced depression-like behavior in the tail-suspension test Saline, 0.9% (7 d, i.p.): Doxycycline (100 mg/kg/d, i.p.): MSM (100 mg/kg/d, i.p.), MSM (100 mg/kg/d, i.p.), then saline, 0.9% (i.p.) then Doxycycline (100 mg/kg, i.p.) 140 * 130 * 120 * ‡ 110 * 100 Time Immobile (s±SEM) † † † 90 † 80 * * 70 60 † 50 1 7 28 14 Testing (days after treatment)

  29. Study II: Summary findings • Mice expressing Tat protein demonstrate increased anxiety- and depression-like behavior in a dose- and duration-dependent manner • Anxiety- and depression-like effects of Tat exposure persist for at least two weeks after completion of doxycycline treatment… and beyond point Tat protein is detected in brain • May signify progression from signaling to neurodegenerative effects? • Duration of impaired startle reflex varies with duration of Tat exposure • Matches pattern of inflammation measured by Iba-1 labeled microglia and GFAP in brain • ASR deficits caused by brief exposure to Tat are reversed by indomethacin treatment, but this is ineffective after 7 days’ Tat induction. • Tat-induced immobility in the tail-suspension test was mitigated by co-treatment with methylsulfonylmethane

  30. Day: 1 2 3 4 = Place preference test (30 min) Study III Method: Conditioned place preference = Cocaine place conditioning, then saline place conditioning (30 min each, 4 h apart)

  31. Day: 1 2 3 4 = Place preference test (30 min) Study III: Tat expression potentiates the rewarding effects of morphine in the conditioned place preference (CPP) assay Vehicle Doxycycline * 600 (100 mg/kg/d, 7 days) 500 400 = Morphine place conditioning, then saline place conditioning (40 min each, 4 h apart) 300 morphine-paired chamber (s±SEM) Difference in time spent in 200 (Mice pretreated 7 d with saline or Dox (@ 100 mg/kg/d) 100 C57BL/6J GT-tg bigenic 0 G-tg mice mice mice (no Tat gene)

  32. Study III: Tat-induced potentiation of morphine-CPP correlates with magnitude of Tat induction in GT-tg mice * * 600 600 Uninduced (Saline, i.p./d) 500 500 Tat-induced (Dox,100mg/kg/d) 400 400 300 300 morphine-paired chamber (s±SEM) Difference in time spent in Difference in time spent in the morphine-paired side (s±SEM) 200 200 100 100 0 0 0 25 50 100 7 1 3 5 7 Doxycycline (mg/kg/d) Days treated

  33. Study III: Daily pretreatment with indomethacin and didehydro-Cortistatin A (dCA) prevents Tat-induced potentiation of morphine-conditioned place preference Dox +Treatment Day: 1 2 3 4 5 6 7 8 9 10 11 12 600 Uninduced (Uses Saline/ d @ i.p. for 7 d) * 500 Tat-Induced (Uses Dox @100 mg/kg/d for 7 d, i.p.) 400 300 morphine-paired chamber (s±SEM) Difference in time spent in † † 200 100 - - - 0 + Indomethacin - - - (1 mg/kg/d, i.p.) Vitaliano Nanoparticle delivery? + dCA (30 nmol/d, i.c.v.) Pretreatment (each day, 30 min prior to Dox)

  34. Vehicle Doxycycline (100 mg/kg/d, 7 days) Study III: Exposure to Tat protein also potentiates cocaine- and ethanol-CPP in GT-tg bigenic mice 800 † * * Vehicle 600 600 Doxycycline (100 mg/kg/d, 450 400 5 days) * * * 300 * * * 200 150 ethanol-paired chamber (s±SEM) Difference in time spent in cocaine-paired chamber (s±SEM) Difference in time spent in 0 0 -150 -300 -200 C57BL/6J GT-tg C57BL/6J GT-tg C57BL/6J GT-tg C57BL/6J GT-tg Pre-CPP Post-CPP Pre-CPP Post-CPP Paris et al, Neuropsychopharmacology, 39:2014 McLaughlin et al, Current HIV Research, 12:2014

  35. * 600 500 400 300 Difference in time spent in cocaine-paired chamber (s±SEM) n.s. 200 100 0 Study III: Acute induction of Tat subsequently potentiates established cocaine-CPP during re-exposure to cocaine Veh Dox Day: 1 2 3 4 5 6 7 8 9 10 11 12 CPP after: 2 cycles place conditioning 3 cycles place conditioning - - - + Doxycycline (100 mg/kg/d for 7d) Paris et al, Neuropsychopharmacology, 39:2014

  36. Study III: Tat protein reinstates extinguished morphine-CPP in an exposure-dependent manner Preference test, 30 min Saline treatment (7 d) Morphine conditioning, 40 min Doxycycline (100 mg/kg/d; 1, 3, 5 or 7d) Week: 1 2 3 4 5 Vehicle conditioning, 40 min Saline or Dox, 1-7 d GT-tg bigenic mice: (Uninduced) (Tat induced) ‡ 500 * 400 300 * 200 morphine-paired chamber (s±SEM) Difference in time spent in † 100 † † † 0 -100 Post CPP Pre Post 1 3 5 7 Sal + 21 d Extinction CPP CPP (7d) Days Dox Reinstatement test

  37. Exp. IIIB: Exposure to Tat protein increases consumption of morphine in the two-bottle choice assay Consumption Consumption Consumption Prior to induction During induction treatment: After induction treatment ( , Saline (d/i.p.) Tat- mice Mice equivalent) Doxycycline Tat+ mice * * (100 mg/kg/d, i.p.) * 4.0 * * * * 3.5 3.0 Tat- GT-tg mice 2.5 Morphine Fluid consumed (mL/d±SEM) Quinine 2.0 Tat+ GT-tg mice 1.5 Morphine Quinine 1.0 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Bottle A: Quinine (0.25 mg/ml) Day Bottle B: Morphine (0.4 mg/ml)

  38. Conclusions • Study 1: Effect of Tat expression on learning and memory and cognition: • Spatial learning (i.e., acquisition) was impaired in the Barnes maze • Reversal learning also impaired • Novel object recognition performance was impaired in Tat-induced mice for up to one month in an exposure-dependent manner • Pretreatment with Tat-inhibitor dCA prevented impairment of NOR • Pre-pulse inhibition was impaired in Tat-induced mice • Study 2: Effects of Tat expression on disorders of mood: • Tat expression produces anxiety-like behavior in exposure-dependent manner • Low or high exposure to Tat amplified the acoustic startle reflex • Low or high Tat exposure activated microglia and increased GFAP • Indomethacin mitigated effects of brief, but not prolonged, Tat exposure • Tat exposure increased the time spent immobile in the mouse tail-suspension test • Persistence of depression-like effects dependent on duration of Tat exposure • Methylsulfonylmethane mitigated depression-like effects of Tat exposure

  39. Conclusions • Study 3: Effects of Tat expression on morphine reward: • Exposure to Tat protein potentiates the locomotor effects of morphine • Effect consistent with earlier cocaine results (Paris et al., 2014) • Effects of Tat expression on morphine reward: • Tat expression potentiates morphine CPP in exposure-dependent manner • Effect not seen in saline treated GT-tg mice, or Dox-treated G-tg or C57BL/6J mice • Increases place conditioning effect of additional (normally inconsequential) exposure to cocaine • Causes reinstatement of extinguished morphine-seeking behavior • Increases voluntary consumption of morphine… • Together, these results suggest that exposure to Tat protein is sufficient to: • Promote disorders of mood associated with NeuroAIDS and HAD • Play a role in the development of cognitive impairment • Potentiate the rewarding effect of abused substances, and produce relapse to drug-seeking behavior in abstinent subjects.

  40. Acknowledgements University of Texas Health Sciences Center: Dr. Johnny J. He McLean Hospital/ Harvard University: Dr. Marc Kaufman, Dr. Gordana Vitaliano ExQor: Franco Vitaliano Northeastern University: (Dr.) Amanda Carey Stacey Gomes Andrew Heusser Chris Shay Elizabeth Sypek Scripps-FL: Dr. Susana Valente Dr. Sonia Mediouni VCU: Dr. Jason Paris UF: Shainnel Eans, Jessica Medina, Heather Stacy, Dr. Kristen Hymel, Thomas Cirino Dr. Habibeh Khoshbouei and Doug Miller Drs. Jane Aldrich and Rob Huigens TPIMS: Michelle Ganno-Sherwood Elisa Mizrachi, Harminder Singh Special thanks to Yu (Woody) Lin (NIDA) and Jeymohan Joseph (NIMH) Support: F31 NS064972-01 (NINDS), 1R01 MH085607 (NIMH), 1R01 DA039044 (NIDA), 1R43 MH108481 (NIMH) and the State of Florida

  41. Experiment III: Exposure to Tat protein reduces consumption of water in the two-bottle choice assay Consumption Consumption Consumption Prior to induction During induction treatment: After induction treatment ( , Saline (d/i.p.) Tat- mice Mice equivalent) Doxycycline Tat+ mice 4.0 (100 mg/kg/d, i.p.) 3.5 3.0 * * 2.5 * Substance consumed (mL/d±SEM) * Tat- GT-tg mice 2.0 Trace Morphine * Quinine 1.5 Tat+ GT-tg mice Trace Morphine 1.0 Quinine 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Day Bottle A: Quinine (0.25 mg/ml) Bottle B: Trace Morphine (0.13 μg/ml)

  42. Exp. IIIB: In mice lacking Tat, exposure to Doxycycline briefly reduces consumption of morphine in the two-bottle choice assay Consumption Consumption Consumption During treatment: Prior to treatment After treatment ( , Saline (d/i.p.) Saline treated mice Mice equivalent) Doxycycline Dox treated mice (100 mg/kg/d, i.p.) 4.0 3.5 3.0 G-tg mice (no Tat gene) 2.5 Saline treatment * Morphine 2.0 Fluid consumed (mL/d±SEM) * Quinine * 1.5 Dox treatment Morphine 1.0 Quinine 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Bottle A: Quinine (0.25 mg/ml) Day Bottle B: Morphine (0.4 mg/ml)

  43. Day: 1 2 3 4 = Place preference test (30 min) Study III: Doxycycline-induced (100 mg/kg/d, i.p.; 7 d) expression of Tat protein has no effect on saline conditioned place preference Preconditioning preference 600 Postconditioning preference 450 = Saline place conditioning, then saline place conditioning (30 min each, 4 h apart) 300 n.s. 150 Difference in time spent on initial saline-paired side (s±SEM) 0 -150 -300 C57BL/6J Uninduced Tat-induced GT-tg bigenic mice

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