HIV-1 Tat alters neuronal intrinsic excitability
Francesconi et al. BMC Res Notes
HIV-1 Tat alters neuronal intrinsic excitability
Walter Francesconi 0 2
Fulvia Berton 0 2
Maria Cecilia G. Marcondes 1 2
0 Department of Anatomy and Cell Biology, College of Medicine, University of Illinois Chicago , 6068 COMRB MC 512, Chicago, IL 60612 , USA
1 San Diego Biomedical Research Institute , 10865 Road to the Cure, San Diego, CA 92121 , USA
2 The Scripps Research Institute , 10550 North Torrey Pines Rd., La Jolla, CA 92037 , USA
Objective: In HIV+ individuals, the virus enters the central nervous system and invades innate immune cells, producing important changes that result in neurological deficits. We aimed to determine whether HIV plays a direct role in neuronal excitability. Of the HIV peptides, Tat is secreted and acts in other cells. In order to examine whether the HIV Tat can modify neuronal excitability, we exposed primary murine hippocampal neurons to that peptide, and tested its effects on the intrinsic membrane properties, 4 and 24 h after exposure. Results: The exposure of hippocampal pyramidal neurons to Tat for 4 h did not alter intrinsic membrane properties. However, we found a strong increase in intrinsic excitability, characterized by increase of the slope (Gain) of the inputoutput function, in cells treated with Tat for 24 h. Nevertheless, Tat treatment for 24 h did not alter the resting membrane potential, input resistance, rheobase and action potential threshold. Thus, neuronal adaptability to Tat exposure for 24 h is not applicable to basic neuronal properties. A restricted but significant effect on coupling the inputs to the outputs may have implications to our knowledge of Tat biophysical firing capability, and its involvement in neuronal hyperexcitability in neuroHIV.
neuroHIV; HIV Tat; Hippocampal neurons; Electrophysiology; Synaptic transmission
Neuronal plasticity and regeneration are interrelated
phenomena, tightly dependent on the normal
functioning of the Central Nervous System (CNS). Changes
in glia drastically affect neuronal capacity to recover
and rewire [
]. Microglia invade the CNS at prenatal
stages, increasing density during the first weeks of
postnatal life, reaching a maximum by P18, concomitant
to an intense synaptogenesis period [
express cytokines, neurotrophins [
], glutamate and
], which regulate synaptic properties.
Importantly, microglia are critical during Human
Immunodeficiency Virus (HIV) infection, because they get infected
by virus carried into the brain by macrophages at early
time points [
The consequences of HIV in the brain include high
incidence of neurological dysfunctions, even in the
post-antiretroviral age [
]. Of all HIV-1 peptides, Tat is
involved in viral transcription, and is unconventionally
secreted by infected targets [
], with consequences
to neighboring cells, including neurons. HIV-Tat has
been linked to impaired learning and memory, and gray
matter deficits [
], suggesting its involvement in the
development of HIV-associated neurological disorders
The hippocampus is a brain region involved in
cognition, and memory formation, organization, and
retrieval, where the main cell type is the excitatory
glutamatergic pyramidal neuron, integrating spatial,
contextual, and emotional information, while
transmitting all outputs to cellular targets throughout the
brain, in response to glutamate, a key
]. Pyramidal cells in the CA1 and
subiculum regions carry output by firing individual or high
frequency bursts of action potentials (AP), increasing
synaptic communication through evoking a
postsynaptic spike [
]. They also participate in plasticity
and place field development [
synaptic transmission in hippocampal neurons is
susceptible to changes, contributing to cognitive impairments.
Due to the abundance of these neurons, the
hippocampus is crucial for recalling when and where an
event occurred, or ‘episodic memory’ [
], one of the
first functions lost in HAND and in aging [
Importantly, we have demonstrated that HIV Tat
prevents long-term potentiation in the hippocampal CA1
region . Our goal was to further characterize the
neuronal response to HIV Tat in primary cultures.
Neuronal membrane properties are characterized by
means of the input–output response function, giving
the rate of AP discharge as a function of the injected
current strength. The linear relationship between
neuronal input and output is defined by the rheobase
(minimum synaptic input that generates an AP), and
by the slope (gain). The gain control is a central
feature of neural information processing [
in gain control, associated with alterations in the
conductance of voltage-gated channels such as the A-type
(IA), the delayed-rectifier K+ (Id) and L-type
voltagegated Ca2+ (IKCa) channels, are critical in several
pathophysiological conditions. An increase in IA, Id
and IKCa reduces neuronal gain. In contrast, increases
in the slow voltage-gated Ca2+ channel conductance
(GCaS) increase neuronal gain. The
hyperpolarization-activated inward channel (Ih) is ‘gain neutral’. The
functional relevance of such changes include
protecting neurons from over-excitation during ischemia,
infection, or aging, and for making neurons more
excitable during associative learning [
We used the slope of the fitted linear function (gain,
I–O slope) as a quantitative measure of biophysical
firing capabilities under DC step stimulation [
examine whether HIV-1 Tat can modulate
hippocampal neuron properties, explaining changes in memory
functions experienced by HIV+ subjects. In neuronal
primary cultures we modeled Tat exposure, and tested
its effects on excitability.
In cell line studies, Tat internalization by neurons
was detected at 4 h [
]. Effects on molecular
functions and morphology were detected at 24 h [
preceding neurotoxicity at 48 h . The ability of Tat
to modify neuronal excitability in the primary
hippocampal neuron culture system was tested on whole
cell patch electrophysiological testing paradigms, at 4
and 24-h time points. We found a significant effect of
Tat at 24 h after exposure.
Animal use was approved by Institutional Animal Care
and Use Committees of The Scripps Research Institute
(TSRI) and San Diego Biomedical Research Institute. In
three independent experiments, two pregnant C57Bl/6
females, 5–8 weeks old, were purchased from TSRI
Department of Animal Resources. E17 pups [
] (~ 7/
experiment) were sacrificed by CO2 inhalation.
Hippocampi were dissected in Ca2+/Mg2+-free,
HEPESbuffered Hank’s balanced salt solution (HBSS), pH7.45,
dissociated through flame-narrowed Pasteur pipettes of
decreasing aperture, and resuspended in DMEM without
glutamine, 10% fetal bovine serum and
penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively). Cells
were plated (120,000/dish) onto 25 mm round Matrigel
(200 μl, 0.2 mg/ml; BD Biosciences) pre-coated cover
glass glued to cover a 19-mm-diameter opening drilled
through the bottom of a 35 mm Petri dish. Neurons were
grown in 10% CO2, at 37 °C, and fed on days 1 and 6,
by exchanging 75% of the media with DMEM
containing 10% horse serum and penicillin/streptomycin. In all
experiments, pyramidal-shaped neurons behaved as such
Recombinant HIV-1 Tat (Clade B) was from the National
Institutes of Health AIDS Research and Reference
Reagent Program, Division of AIDS, National Institute of
Allergy and Infectious Diseases. In control experiments,
Tat was heat-inactivated at 85 °C for 30 min. The cells
were incubated with Tat 10 ng/ml (6.4 nM). Tat (or
inactivated Tat) was added to the media 4 or 24 h prior to
recordings. Coded cultures were removed from 37 °C,
and a 95% O2/5% CO2 injector was placed in wells, under
a differential interference contrast microscope (Leica).
Electrophysiology was performed in randomized
cultures, in a blinded manner.
Whole‑cell patch clamp recordings and intracellular stimulation
Patch clamp recordings and intracellular stimulation
were performed and captured using Multiclamp 700B
amplifier (Axon Instruments). Stimulus waveforms were
generated using data acquisition software DASYLab11.0
(National Instruments) in Windows computer equipped
with National Instruments PCI-MIO-16-E4 board. We
used rectangular hyperpolarizing and depolarizing
current pulses as stimuli for physiological
characterization. Specifically, 350 ms current pulses starting from
− 200 pA, were incremented by 10 pA. Voltage responses
were analyzed using software developed by Delphi, 2009,
using the following parameters: Resting membrane
potential, Resting input resistance, Rheobase, AP
Threshold, AP amplitude and duration, AP after
hyperpolarization, and the I-O function (gain). The spontaneous
excitatory postsynaptic currents (sEPSCs) were recorded
in voltage clamp mode at − 70 mV holding potential. The
spontaneous inhibitory postsynaptic current (sIPSC)
was recorded in voltage clamp mode at − 40 mV holding
Using Prism5 (GraphPad Software Inc, La Jolla, CA), we
examined deviations from normality in the data using
Kolmogorov–Smirnov test. Slopes were tested using
Pearson’s correlation coefficient (r) (p < 0.01), and linear
regression. The mean of input–output relationship [
was compared by ANOVA, and Bonferroni’s post hoc
test (p < 0.05). Individual parameters between controls
and Tat 24 h were compared using Student’s t test.
We examined whether dysfunctional properties are
detectable in hippocampal neurons exposed to HIV-1 Tat
for 4 and 24 h. We measured electrophysiological
properties in the pyramidal subset, in comparison to cultures
treated with inactivated Tat. In both time-points, control
cultures showed similar behaviors and thus
measurements were pooled.
Figure 1 summarizes the parameters characterizing
neuronal subsets. Baseline recordings showed that
hippocampal neuronal subsets in primary cultures exhibit
expected behaviors, and are a valid system for
studying the direct effects of HIV peptides such as Tat, or
neuroimmune factors. In control cultures, pyramidal
Glutamatergic neurons (Fig. 1, red lines) differed from
GABAergic interneurons (Fig. 1, blue lines), in recording
patterns during whole cell patch-clamp assays.
Pyramidal neurons displayed characteristic voltage sag during
hyperpolarizing pulse, and strong adaptation (Fig. 1a,
upper trace), while interneurons fired at higher frequency
without signs of adaptation (Fig. 1a, lower trace).
Regarding AP evoked by a short depolarizing current pulse,
pyramidal neurons showed depolarizing potential during
the repolarization phase (Fig. 1b, upper trace), while in
interneurons, AP was followed by a fast
after-hyperpolarization (fAHP) (Fig. 1b, lower trace). The AP width was
shorter in interneurons (Fig. 1c, blue line) compared to
pyramidal neurons (Fig. 1c, red line). All parameters were
according to predicted results for these subsets.
Using this system, we tested the hypothesis that Tat
can directly affect neuronal performance, detectable by
increase or decrease in pulse current intensity necessary
to elicit an AP. We have focused on glutamatergic
pyramidal neurons, due to their excitatory role in circuitry
architecture, and due to their consistent pattern of
Tatelicited changes, compared to control neurons. All
cultures showed a linear relationship between the injected
current and the number of spikes, determined by
Pearson’s coefficient (Control r2 = 0.9969, p = 0.0031; Tat 4 h
r2 = 0.9989, p = 0.0011; Tat 24 h r2 = 0.9827, p = 0.0004).
Compared to control cultures, Tat did not alter neuronal
behaviors at 4 h. However, a strong increase of the gain
function was seen at 24 h (Fig. 2). The slope comparison
by linear regression revealed a significant difference, with
F = 16.4446, DFn = 2, DFd = 8, and p = 0.001465, and
with a 0.15% chance of randomly choosing data points
exhibiting these differences. The comparison of the mean
of the slopes input–output (I/O) relationship within the
tested interval showed a significant difference between
Control and Tat 24 h (p = 0.002, Bonferroni’s p < 0.05).
Yet, pyramidal neurons treated with Tat did not alter the
RMP, input resistance, rheobase (or the minimal
depolarizing current input that generates an AP), and AP
threshold at 24 h (Table 1).
We found that Tat critically affects neuronal intrinsic
excitability in cultured hippocampal pyramidal neurons,
as a result of a lower reactive threshold to current. This
effect was not observed at 4 h after HIV-1 Tat exposure,
but only at the 24 h time point.
In cell line studies, conflicting results relate to the
diversity of models. In a neuro-epithelial-like stem (NES)
cell line from human fetal hindbrain, Tat at 10 times
lower doses than in our study caused deep changes in
gene expression and cytoskeletal structure at 24 h, and a
reduction of output excitability at 48 h [
], likely due to
In primary cultures, Tat-induced changes are subset,
dose and time-depend. For instance, in rat dorsal root
1.9 ± 0.7
5.9 ± 0.6
ganglion small diameter capsaicin-sensory neurons, Tat
at 20 nM greatly enhanced excitability, suggesting a direct
role in pain [
]. On the other hand, studies in rat
hippocampal neurons show Tat-induced biphasic changes
in NMDA-evoked increases in intracellular Ca2+, with
consequences to spontaneous activity [
]. Tat (at
5-fold higher concentrations than in our study) acutely
reduced spontaneous spike frequencies while increasing
AP bursts amplitude and duration, followed by
attenuation, and adaptation at 24 h. These changes were
hypothesized to result from aberrant network activity, attributed
to changes in NMDA-gated intracellular Ca2+, mediated
by Src kinase and NO signaling [
]. Our results
complement those, suggesting that neuronal adaptability to
lower Tat concentrations may be relative, or not
applicable to all aspects of neuronal function. Our findings are
in agreement with the excitatory effect of Tat on cultured
human fetal neurons, and rat hippocampal slices [
The neuronal ability to receive and transmit
information depends on neurotransmitter concentrations in
presynaptic terminals, numbers and intrinsic properties
of postsynaptic receptors on dendritic trees, and
receiving synaptic inputs, which depend on the type of
voltagedependent membrane ionic channels. These channels,
upon inputs and AP, allow ionic movement, changing
the excitability. We observed that Tat increases neuronal
excitability, or the slope of the input–output relationship.
Tat may enhance firing via Ca2+ influx [
prolonging Ca2+ potentials mediated by L-channels.
Importantly, neuronal voltage-gated K+ channels (Kv) are
involved in memory processes [
], and in acquired
neuronal channelopathies observed in HIV-associated
neurocognitive disorders [
]. Further studies must
determine what conductance is affected by Tat exposure,
and whether these findings apply to neuroHIV models
in vivo. If so, these may have consequences to how HIV
in the brain affects perception, reactivity to sensory
stimulation, and memory, in part explaining HIV-associated
HIV-Tat acts on hippocampal pyramidal neurons by
lowering the current pulse intensity threshold that
elicits an action potential response, and increases gain
slope 24 h following exposure, indicating an enhanced
intrinsic neuronal excitability.
This study was in isolated neurons in culture, not
subjected to neuroimmune changes, or active infection,
differing from the HIV-infected brain. Yet, it is a
system for the examination of direct effects of HIV and its
peptides, and that can accommodate complexities from
neuroimmune cells. More studies are need for
identifying mechanisms by which Tat affects excitability,
without affecting other functions.
Tat: trans-activator of Transcription; CNS: central Nervous System; P18:
prenatal day 18; NO: nitric oxide; HIV: human immunodeficiency virus; HAND:
HIVassociated neurocognitive disorders; CA1: cornu ammonis layer 1; AP: action
potential; RPM: resting membrane potential; IA: A-type voltage gated channel;
Id: delayed rectifier K+ gated channel; IkCa: L-type voltage-gated Ca2+
channel; GCaS: voltage-gated Ca2+ channel conductance; Ih: inward channel; I-O:
input: output; DC: direct current; Hr: hour; Hrs: hours; E17: embryonic day 17;
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBSS: Hank’s
balanced salt solution; SDBRI: San Diego Biomedical Research Institute; TSRI: The
Scripps Research Institute; DMEM: Dulbecco’s modified Eagle’s medium; AIDS:
acquired immunodeficiency syndrome; pA: pico-amperes; mS: millisecond;
sIPSC: inhibitory post synaptic current; mV: millivolts; ANOVA: analysis of
variance; fAHP: fast after hyperpolarization; NES: neuro-epithelial-like stem cell;
NMDA: N-methyl-d-aspartate; Kv: voltage-gated K+ channels.
MCGM: study concept and design, data analysis and interpretation,
manuscript writing, editing and submission; WF and FB: study concept and design,
performance of electrophysiology techniques, data analysis and
interpretation, manuscript writing. All authors read and approved the final manuscript.
The authors wish to thank Dr. Sandra Encalada, The Scripps Research Institute,
La Jolla, CA, for providing the neuronal cultures used in this study, and Dr.
Bruno Conti for the space and amazing support. We also thank Daniel Ryan
(SDBRI) for critically reviewing our manuscript.
The authors declare that they have no competing of interests.
Availability of data and materials
Data are all contained within the paper. The datasets from the analyses are
available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
All animal use was reviewed and approved by the Institutional Animal Care
and Use Committee of The Scripps Research Institute and of the San Diego
Biomedical Research Institute.
The authors declare that this experiment was partially funded by the
R01DA036164 to MCGM.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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