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Synapse. Author manuscript; available in PMC 2013 Jul 8.
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PMCID: PMC3704183
NIHMSID: NIHMS329116

In Vivo Microdialysis in Awake, Freely Moving Rats Demonstrates HIV-1 Tat-Induced Alterations in Dopamine Transmission

Abstract

Individuals infected with human immunodeficiency virus (HIV) may develop neuropsychological impairment, and a modest percentage may progress to HIV-associated dementia (HAD). Research using human and nonhuman, in vitro and in vivo models, demonstrates that subcortical dopamine (DA) systems may be particularly vulnerable to HIV-induced neurodegeneration. The goal of the current investigation is to provide an understanding of the extent to which the HIV-1 protein Tat induces alterations in striatal DA transmission using in vivo brain microdialysis in awake, freely moving rats. The current study was designed to investigate Tat-induced neuronal dysfunction between 24-h and 48-h post-Tat administration, and demonstrates a reduction in evoked DA for the Tat-treated group relative to vehicle-treated group at 24 and 48 h. The Tat-induced reduction of DA overflow by 24 h suggests dysfunction of nerve terminals, and a compromised DA system in Tat-treated animals. Furthermore, the current study provides direct support for HIV-associated decline of DA function at a systemic level, helping to characterize the functional outcome of the relatively large amount of research on the molecular and behavioral levels of HIV-induced neurotoxicity. This initial study may provide additional characteristics of Tat-induced neuronal dysfunction to inform research on therapeutic intervention, and it provides a springboard for future in vivo research currently needed in the field.

Keywords: HIV, AIDS, Tat, DA, microdialysis, NeuroAIDS

INTRODUCTION

Of the estimated 30 to 40 million people infected with human immunodeficiency virus (HIV) worldwide in 2007 (UNAIDS/WHO, 2007), a proportion will likely develop neuropsychological (NP) impairment. Highly active antiretroviral therapy (HAART) is effectively increasing the lifespan of HIV-infected individuals, leading to an increased prevalence of NP impairment, which will continue to increase if central nervous system (CNS) directed therapy is not included in antiretroviral regimens.

The majority of published behavioral research has labeled the NP manifestations of HIV a subcortical dementia, affecting mostly dopamine (DA)-rich areas of the brain (for reviews see Berger et al., 2000; Ferris et al., 2008; Koutsilieri et al., 2002; Nath et al., 2000). Both in vitro and in vivo animal models support HIV (protein)-induced increases in oxidative damage and toxicity to DA neurons and glial cells (Aksenov et al., 2001, 2003; Bansal et al., 2000; Fitting et al., 2008a; Turchan et al., 2001), as well as behavioral alterations associated with this toxicity (Fitting et al., 2006; Fitting et al., 2008b; Glowa et al., 1992; Hill et al., 1993). Tat is currently the most investigated neurotoxic HIV protein; however, we are unaware of any published article which has demonstrated Tat-induced alterations in neurotransmission in awake, freely moving rats.

Therefore, the purpose of the current investigation is to study alterations in DA transmission induced by infusion of the HIV-protein Tat1–86 into the striatum of the rat; an area known to be heavily infiltrated by the virus in humans. In vivo brain microdialysis is ideally suited to this experimental approach. Hence, our goal was to utilize brain microdialysis to monitor Tat-induced alterations in striatal DA transmission in an in vivo rat model where Tat-induced neurotoxicity and oxidative stress have already been established using neuropathological and stereological techniques (Aksenov et al., 2001, 2003; Fitting et al., 2008).

MATERIALS AND METHODS

Immunohistochemistry

Immunohistochemistry was utilized to validate infusion of Tat in the Nucleus Accumbens (NAcc). Immunofluorescent images were acquired from 20-µm coronal sections of rat striatum (AP = +1.2) to validate that Tat1–86 was successfully infused into the rat NAcc, using 1:500 dilution of chicken polyclonal antibody to HIV-1 Tat (Abcam Inc., Cambridge, MA, USA). Goat antichicken Alexa Fluor 488 secondary antibody (Molecular Probes, Eugene, OR; 1:1500 dilution) was used to detect Tat primary antibody. The probe-tip portion (in NAcc) of fluorescent labeled sections for Tat-treated animals plus primary antibody, Tat-treated animals minus primary antibody (negative control), and vehicle control animals plus primary antibody (control) were magnified 100× and fluorescent signals were acquired (Nikon Eclipse E800 microscope).

Microdialysis and HPLC

Sixteen male Sprague-Dawley rats (300–350 g; Harlan Laboratories, Birmingham, AL, USA) were singlehoused on a 12-h light/dark cycle with food and water available ad libitum. All animals were handled and habituated daily for at least 1 week prior to surgery, and were randomly assigned to either the Tat condition or vehicle control condition so that groups possessed eight animals each. All animals were maintained according to the National Institutes of Health (NIH) guidelines in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of South Carolina, Columbia (assurance number A-3049-01).

All animals were anesthetized prior to surgery using sevoflurane gas, induced using 7% inhalant and maintained at 3% inhalant for the duration of the surgery. All animals received one microdialysis guide cannula (Bioanalytical Systems, Inc., West Lafayette, IN, BAS) in either the left or the right ventromedial striatum (NAcc); counterbalanced across group) using stereotaxic coordinates AP + 1.2 mm, L ± 2.0 mm, DV − 5.0 mm relative to Bregma, midline, and skull surface, respectively. Guide cannulas were fixed to the skull with skull screws and dental acrylic/cement. Animals were allowed 2–3 days to recover after surgery.

Once recovered from surgery, awake animals were administered an intrastriatal infusion of the recombinant HIV-1 protein Tat1–86 (20 µg in 5 µl; DIATHEVA, Fano, Italy), or vehicle control (5 µl) through the guide cannula. Neurochemical monitoring was performed over two sessions, with a 24-h lapse between sessions. The first microdialysis session was performed in all animals the day following Tat1–86 or vehicle infusion, to determine the response of striatal DA systems to local depolarization 24 h after infusion. Briefly, 2 h after probe insertion five baseline collections were obtained (15 min fractions) in which the probes were continuously perfused at 2.0 µl/min with artificial cerebrospinal fluid (aCSF; pH 6.5) composed of NaCl 150 mM, KCl 3.0 mM, CaCl2 1.7 mM, MgCl2 0.9 mM, d-glucose 4.9 mM. The probes were subsequently perfused with 100 mM KCl for one collection, then switched back to aCSF for two additional collections, for a total of eight fractions collected. Session 2 was identical to Session 1, and was included to assess the response of striatal DA systems to local depolarization 48 h after Tat1–86 or vehicle infusion.

All dialysates were analyzed by liquid chromatography with electrochemical detection. Separation of DA from metabolites was achieved by injecting 15 µl of each sample onto a C-18 analytical column (100 × 1 mm; 3 µm; BAS) using a mobile phase (pH 3.4) containing 14.5 mM NaH2PO4, 30 mM sodium citrate, 27 µM disodium EDTA, 10 mM diethylamine HCl, 2.2 mM 1-octane sulfonic acid, 4% acetonitrile, and 1% tetrahydrofuran at a flow rate of 100 µl/min. DA was detected by oxidation at a glassy carbon electrode with an applied potential of +650 mV vs. an Ag/AgCl reference electrode. Peaks corresponding to DA were quantified by comparison with a three point external standard curve bounding the expected range of DA values.

RESULTS

Probe placement validation (Fig. 1)—via histological assessment—confirmed that two animals had to be removed from the experiment due to misplaced probes. Immunohistochemistry validated that Tat1–86 was successfully infused into the NAcc (Fig. 2).

Fig. 1
Schematic representation of probe placements. Anterior/posterior coordinates located between +1.0 mm and +1.3 mm relative to Bregma.
Fig. 2
Validation of successful infusion of Tat1–86 into the rat NAcc. (A) Immunofluorescent image (100×; probe-tip) of Tat antibody (fluorescent green) present in Tat-treated animal striatal section. (B) Immunofluorescent image (100×; ...

There was no baseline difference in basal DA levels between the two groups for either session. Within both sessions, local depolarization by 100 mM KCl produced a robust increase in DA release for vehicle-treated animals (P < 0.0001) that was significantly attenuated in Tat-treated animals. For Session 1 (24 h post-Tat infusion), K+-evoked DA levels were significantly attenuated by 30% in Tat-treated animals (P < 0.05; Fig. 3), and a nonsignificant trend toward Tat-induced basal decreases were noteworthy. For Session 2 (48 h post-Tat infusion), K+-evoked DA levels were further reduced in Tat-treated animals by 55% (P < 0.05; Fig. 4). Time × treatment interactions were present for both the sessions as well (P < 0.0001; Figs. 3 and and4),4), indicating a dynamic Tat-induced reduction in DA response to K+-perfusion.

Fig. 3
Effect of intrastriatal infusion of Tat or vehicle control on raw striatal DA levels (A) and percent of baseline (B) within Session 1. A significant Tat-induced reduction in K+-evoked DA levels was present 24 h after Tat infusion. Note: *P < 0.05 ...
Fig. 4
Effect of intrastriatal infusion of Tat or vehicle control on raw striatal DA levels (A) and percent of baseline (B) within Session 2. A significant Tat-induced reduction in K+-evoked DA levels was present 48 h after Tat infusion. Note: *P < 0.05 ...

DISCUSSION

Previous research in our laboratory has validated neuro-pathological and stereological indices of neurotoxicity and oxidative stress in the model utilized in the current investigation (Aksenov et al., 2001, 2003; Fitting et al., 2008a). To date, we are unaware of any publications in the HIV literature utilizing microdialysis in awake, freely moving rats; and are aware of only one publication demonstrating Tat + methamphetamine reductions in DA overflow in anesthetized rats (Cass et al., 2003). Thus, the current investigation extends previous research in our laboratory and among colleagues to demonstrate Tat-induced reductions in brain neurotransmitter function, using a dose of Tat well within the range of doses utilized in the literature (e.g., Bansal et al., 2000; Cass et al., 2003; Fitting et al., 2006; Theodore et al., 2006).

The significant Tat-induced reduction in DA overflow in Sessions 1 (24 h) and 2 (48 h) is a likely consequence of Tat neurodegeneration of nerve terminals as microdialysis is sensitive to reductions in the number of functional nerve terminals (Parsons et al., 1991; Smith and Justice, 1994). Interestingly, the 24-h effect size corresponds well with investigations of Tat-induced neurotoxicity after 24 h, where neuronal death effect sizes of roughly 25 to 30% can be noted (Aksenov et al., 2003, 2006).

The current investigation is timely in that it may provide initial data as to an early time course for acute Tat-induced alterations in DA transmission, thereby investigating the direct influence of Tat on systemic DA function across multiple days. Understanding the acute effects of Tat on DA transmission is of utmost importance for further development of therapeutic interventions for Tat-induced molecular alterations that precede degenerating synaptic communication and nerve terminals. It would be more beneficial to prevent toxicity in the acute stages than to compensate for toxicity over the long term. Thus, describing the acute effects of Tat, from the intracellular alterations to the level of synaptic communication, will provide a stronger foundation to inform therapeutics. If synaptic communication is altered by Tat as demonstrated in the current investigation, one could judge the efficacy of a novel therapy not only by its ability to mitigate neurotoxicity but also by its restorative effects on DA transmission. Furthermore, it is possible that acute Tat-induced alterations in synaptic communication occur throughout the course of HIV infection of the CNS, as more areas of the brain succumb to viral load and more cells are exposed to the Tat protein.

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