Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons

Abstract

HIV-1 Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current-or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 μM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists CNQX (10 μM) and AP-5 (50 μM), and were markedly reduced in a Ca 2+ -free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca 2+ imaging studies showed that Tat30-86 produced three types of Ca 2+ responses: 1) a fast and transitory increase, 2) Ca 2+ oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca 2+ response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons. Keywords calcium imaging; electrophysiology; postsynaptic currentsInfection of the central nervous system (CNS) with the human immunodeficiency virus 1 (HIV-1) may lead to cognitive, motor and behavioral dysfunctions, collectively termed HIVCorresponding author: G. Cristina Brailoiu, M.D., Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140 USA, Tel: 215-707-7705, Fax: 215-707-7068, E-mail: gbrailou@temple.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Kaul et al., 2001; Gonzales-Scara...

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

“…Measurement of Membrane Potential-The relative changes in membrane potential of single cardiomyocytes were evaluated using bis-(1,3-dibutylbarbituric acid) trimethine oxonol, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a slow-response, voltage-sensitive dye, as described previously (32)(33)(34). Upon membrane hyperpolarization, the dye concentrates in the cell membrane, leading to a decrease in fluorescence intensity, whereas depolarization induces the sequestration of the dye into the cytosol, resulting in an increase of the fluorescence intensity (35).…”

Acidic NAADP-sensitive Calcium Stores in the Endothelium

“…Measurement of Membrane Potential-Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC 4 (3), Invitrogen), a slow response voltage-sensitive dye, was used to assess relative changes in membrane potential of single cells, as described previously (26). Upon membrane hyperpolarization, the dye concentrates in the cell membrane, leading to a decrease of fluorescence intensity, whereas depolarization results in a sequestration of the dye into cytosol and is associated with an increase in the fluorescence intensity (27).…”

HIV-1 Transgenic Rat Prefrontal Cortex Hyper-Excitability is Enhanced by Cocaine Self-Administration

“…Several viral proteins are neurotoxic, including the envelope glycoprotein, gp120, an essential mediator of virus entry into host cells, and the transactivator of transcription (Tat), an activator of transcription of the viral genome (Jones et al, 1998;Haughey and Mattson, 2002). Tat excessively increases Ca 2+ influx in neurons, including pyramidal neurons (PN) of the medial prefrontal (mPFC) (Brailoiu et al, 2008;Napier et al, 2014), by prolonging Ca 2+ potentials mediated by L-type Ca 2+ channels (Lchannels) that profoundly enhances firing (Napier et al, 2014). In vitro studies have demonstrated that neurotoxic effects of gp120 and Tat can be enhanced by other viral proteins (Nath et al, 2000) and cocaine (Aksenov et al, 2006).…”