A Visible-Light-Sensitive Caged Serotonin

Article abstract of DOI:10.1021/acschemneuro.7b00083

Serotonin, or 5-hydroxytryptamine (5HT), is an important neurotransmitter in the nervous system of both vertebrates and invertebrates. Deficits in 5HT signaling are responsible for many disabling psychiatric conditions, and its molecular machinery is the target of many pharmaceuticals. We present a new 5HT phototrigger, the compound [Ru(bpy)₂(PMe₃)(5HT)]²⁺, where PMe₃ is trimethylphosphine. As with other ruthenium-bipyridyl based caged compounds, [Ru(bpy)₂(PMe₃)(5HT)]²⁺ presents activity in the visible region of the spectrum. We characterize and discuss the photochemical properties of the caged compound, and demonstrate its use by modulating the excitability of mouse prefrontal principal neurons.

Full text of DOI:10.1021/acschemneuro.7b00083

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Article
A visible light-sensitive caged Serotonin.
Ricardo Cabrera, Oscar Filevich, Beatriz Garcia Acosta, Jegath
Athilingam, Kevin J Bender, Kira E Poskanzer, and Roberto Etchenique
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 01 May 2017
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ACS Chemical Neuroscience
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A visible lightꢀsensitive caged Serotonin.
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Ricardo Cabrera , Oscar Filevich , Beatriz García-Acosta , Jegath Athilingam , Kevin J. Bender ,
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Kira E. Poskanzer and Roberto Etchenique *
1
Departamento de Química Inorgánica, Analítica y Química Física, INQUIMAE, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, CONICET, Ciudad Universitaria Pabellón 2,
AR1428EHA Buenos Aires, Argentina.
2
Ciclo Básico Común, Universidad de Buenos Aires.
3
Laboratorio de Neurobiología Molecular, BIOMED, Pontificia Universidad Católica Argentina,
CONICET, Buenos Aires, Argentina.
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UCSF Weill Institute for Neuroscience, Kavli Institute for Fundamental Neuroscience
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Department of Neurology, University of California, San Francisco
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Department of Biochemistry and Biophysics, University of California, San Francisco, 1550 4th St.,
San Francisco, CA 94143
AUTHOR CONTRIBUTIONS:
Ricardo Cabrera: Chemical synthesis, Data Analysis, Discussion, Writing.
Oscar Filevich: Data Analysis, Discussion, Writing.
Beatriz GarcíaꢀAcosta: Chemical synthesis, Data Analysis, Discussion, Writing.
Jegath Athilingam: Electrophysiological Experiments, Discussion, Writing.
Kevin J. Bender: Electrophysiological Experiments, Discussion, Writing.
Kira E. Poskanzer: Electrophysiological Experimental Design, Discussion, Writing.
Roberto Etchenique: Chemical setups, Data Analysis, Discussion, Writing.
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ABSTRACT
Serotonin, or 5ꢀhydroxytryptamine (5HT), is an important neurotransmitter in the nervous system of
both vertebrates and invertebrates. Deficits in 5HT signaling are responsible for many disabling
psychiatric conditions, and its molecular machinery is the target of many pharmaceuticals. We present a
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new 5HT phototrigger, the compound [Ru(bpy) (PMe )(5HT)] , where PMe is trimethylphosphine. As
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with other rutheniumꢀbipyridyl based caged compounds, [Ru(bpy) (PMe )(5HT)] presents activity in
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the visible region of the spectrum. We characterize and discuss the photochemical properties of the
caged compound, and demonstrate its use by modulating the excitability of mouse prefrontal principal
neurons.
KEYWORDS Ruthenium complex, Serotonin, Phototriggers, Neurophysiology.
1
. Introduction
A caged compound, or phototrigger, is a molecule reversibly bound to or "caged within" a protecting
group that restricts its interactions with cellꢀmembrane receptors, proteins, or other molecules. Entities
that have been caged include ions, [1] acids, [2] bases, [3] neurotransmitters, [4] fluorophores, [5,6]
smallꢀmolecule drugs, [7] gene inducers [8,9] and even nucleic acids [10] and proteins [11,12].
Phototriggers are lightꢀsensitive, and will release the caged molecule upon irradiation with light of the
proper wavelength, [13] thus removing the restrictions for the caged entity to interact normally with its
targets. As the uncaging spot size can be precisely determined by focusing a lowꢀpower laser, this
technique can have very high spatial and temporal resolution, making it of great advantage in biological
research, for it allows the delivery of molecules without mechanically acting on the preparation, a
problem usually associated with more invasive techniques like microinjection.
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Most common caged compounds comprise an organic protecting group, usually a nitrobenzyl [14] or
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nitroindonilyl [15] which are active in the UV region and therefore can damage the biological tissue.
Therefore, new kinds of organicꢀbased protecting groups have been developed, such as coumarine
derivatives, which can extend the photoactivity to the violet region, thus reducing cell damage. [16]
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As we have shown in recent years, rutheniumꢀbipyridyl coordination complexes yield excellent
phototriggers suitable for neurophysiology studies. They present unique properties, including
nanosecond photoreaction kinetics through heterolytic photocleavage with no secondary reaction
products, [7,17] and sensitivity to visible light in the blue and green regions. [3,6,7,18ꢀ20] As an
important advantage over other photoprotective groups, Ruꢀbipyridyl phototriggers can be used in a twoꢀ
photon regime. [17, 21ꢀ24] Ruthenium phototriggers were also used for general delivery of drugs,
toward application in therapy. Turro and Bonnet have shown the versatility of Ru(II) polypyridines for
caging nitriles and thioethers.[25ꢀ27] A similar strategy using the photoredox properties of the Ru(II)
polypyridyl chemistry was used by Ford and Santana da Silva to release nitric oxide (NO) by visible
light, both directly or mediated by energy transfer processes from molecular antennas.[28ꢀ29]
Ruthenium phototriggers are very stable at room temperature, both as a solid or in physiological
solutions when kept in the dark, minimizing dark leakage of the caged biomolecule, one of the most
common drawbacks of nitrobenzyls and similar caging groups.
Serotonin is one of the most important neuromodulators in a very wide range of animal phyla. It
modulates basic behaviours such as feeding and mating, [30] and imbalances in its physiology lead to
debilitating psychiatric conditions in humans, such as obsessive compulsive disorder, panic disorder, or
major depression. [31] For this reason, the serotonergic system is the target of many pharmaceuticals
such as tricyclic antidepressants (TCAs) selective serotonin reuptake inhibitors (SSRIs) and
monoaminooxidase inhibitors (MAOIs), and even many recreational drugs. [32] Discerning the effects
of 5HT on its various receptor and cellular subtypes and understanding its interactions with other
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systems of neurotransmitters would be greatly aided by a caged 5HT. As serotonin does not exhibit a
carboxylate group, most strategies used to cage neuroactive aminoacids cannot be extended directly to
cage this amine. Currently, two of the commercially available caged serotonins are NPECꢀ5HT and
BHQꢀOꢀ5HT, which use the phenol group of the 5HT for anchoring the photoremovable protecting
group and are active under UV light (365 nm). While the first shows negligible absorption in twoꢀ
photon regime [33] and has a very slow response time in the hundreds of milliseconds, [34] BHQꢀOꢀ
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HT can also be used at 740 nm (2P) with good quantum yields. [35,36] Other caged 5HT compounds
have been described, showing dissimilar performances. [36ꢀ40]
We report the synthesis and chemical properties of the complex [Ru(bpy) (PMe )(5HT)] , which
+
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releases 5HT upon absorption of visible light, and demonstrate its use by modulating the excitability of
mouse prefrontal principal cells that have previously been shown to be regulated by 5HT. [41]
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. Results and discussion
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The complex [Ru(bpy) (PMe )(5HT)] is yellowꢀorange in color. Its water solubility is well above
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the tens of millimolar range. No decomposition was detected in samples stored in the dark for months at
room temperature either dry or in solution. Water solutions of this complex present a metal to ligand
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ꢀ1
charge transfer (MLCT) band with a molar extinction coefficient (ε) of 6718 M cm and an absorption
maximum in the visible range at 447 nm. Its synthesis is a simple oneꢀpot reaction and the photolysis is
fast and clean, as depicted in Scheme 1.
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Scheme 1. Coordination reaction between serotonin and [Ru(bpy) (PMe )(H O)] (forward) and the
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clean, one step photorelease reaction (reverse) releasing serotonin upon light absorption.
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Figure 1 shows the HꢀNMR spectra of the complex (top) and of its partial photolysis (bottom). In the
first, unphotolyzed spectrum, the aromatic region shows the expected 8 doublets and 8 triplets that are
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characteristic of the bipyridines in a cis octahedral complex of the form [Ru(bpy) (L )(L )] , and the
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four aromatic signals of 5HT. In the aliphatic region (Figure 1b), besides the PMe signal at 0.92 ppm
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the two aliphatic signals for the amine and methylenes group of coordinated 5HT can be seen between
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.60 and 1.71 ppm. The fact that proton signals of amine group can be seen in a D O solution at 3.57
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ppm and 2.65 ppm and the shift and splitting of the methylene signals indicates the coordination of
serotonin to the metal atom through its amine nitrogen. Also visible in the first spectrum are the set of
signals corresponding to the trans configuration of the complex, although in much smaller proportion.
The subtle differences in the photochemical behaviour of the cis and trans configurations of the
complex are negligible in the scope of this work, and no further distinction between these isomers will
be made, although a full characterization of both isomers should be needed for any biomedical uses.
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δ / ppm
Figure 1. 1HꢀNMR spectra of a 5 mg/mL solution of [Ru(bpy) (PMe )(5HT)]Cl in D O before
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irradiation (upper trace); and after 60 s irradiation with 525 nm Green LED inside the NMR tube (lower
trace). Note that after irradiation appear the triplets a and b, characteristics of free serotonin.
Once irradiated inside the NMR tube for 60 seconds, the photolysis progressed around 40%. The
signals of the bipyridines in the original complex appear diminished, while a new set of signals
corresponding to the aquoꢀcomplex are evident. In the aliphatic region, the two characteristic triplets of
the methylenes of free 5HT appear at 3.17 ppm and 2.98 ppm (Figure 1, signals a and b). Furthermore,
amine protons are rapidly exchanged for deuterium and these signals are not present in the free 5HT.
The signal from PMe protons is now shifted to higher fields and corresponds to that of the newly
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formed aquo complex.
To measure the quantum efficiency of photolysis, a water solution of the complex was irradiated with
a 8.56 mW, 405 nm (violet) diode laser at pH = 7 and T = 24 °C. Figure 2 shows the UVꢀVis spectra
during the progression of the photolysis reaction. As is deduced from the presence of an isosbestic point
at 434 nm, the reaction yields just one absorbing product and proceeds to completion in around 12
minutes. The spectrum of the photoproduct corresponds to that of the aquo complex
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+
[
Ru(bpy) (PMe )(H O)] accordingly with the known photochemistry of the complexes of the form
2 3 2
n+
[Ru(bpy) (PMe )(L)] in water at neutral pH. [17, 22, 23] At pH>12 a broader and redꢀshifted
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absorption spectrum is obtained, which corresponds in this case to the hydroxo complex
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Figure 2: Subset of UVꢀVis spectra of [Ru(bpy) (PMe )(5HT)] acquired in aqueous solution at pH=7
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while irradiating with a 405 nm laser diode. The arrows indicate photolysis progress.
The progression of the photoreaction is shown in Figure 3 as the amount of photoreleased 5HT versus
the total number of photons absorbed. Although the quantum efficiency of the phototrigger can be
directly obtained from the initial slope of this graph, a more precise value was obtained by fitting the
complete curve, as described below.
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Figure 3: Progression of the UVꢀVis spectra during a photolysis reaction against absorbed photons. The
continuous line is the theoretical fit according to equation 1. The initial slope of the curve represents the
quantum yield φPD = 0.034.
Given the power of the irradiation beam, its optical path, and the amount of complex in the solution, it
is possible to calculate the differential amount of product as:
dnP
dt
AbsR
AbsT
−Abs
T
=
Ibeam
⋅
(
1−10
)
⋅
⋅
φPD
(1)
where n is the number of the moles of uncaged product, I
is the intensity of the incident light in
p
beam
Einsteins/s, AbsT and Abs are the total solution's absorbance and the reactant's absorbance,
R
respectively, and φPD is the quantum yield of photouncaging. The expression has nonlinear factors, and
adjusting the value of φPD by iterating a finite differences algorithm over the entire obtained spectra
converges to the unique solution for the integrated expression of equation 1.
Cyclic voltammetry was used to further characterize the synthesized complex. Figure 4 shows a cyclic
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voltammogram of [Ru(bpy) PMe (5HT)] recorded in acetonitrile. Ferrocene was used as internal
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reference. The Ru(III)/Ru(II) couple is observed at 1.07V vs. NHE (0.67 V vs. Fc/Fc ). Other two
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irreversible processes are evident at higher potentials and correspond to the oxidation of the coordinated
amine, a behaviour also found in all other similar compounds. [17]
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Figure 4. Cyclic voltammogram of [Ru(bpy) PMe (5HT)] recorded in acetonitrile at 100 mV/s on Pt
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wire electrode in CH CN containing 100 mM TBAPF as supporting electrolyte.
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To confirm the biocompatibility of this complex and evaluate its performance as a serotonin
phototrigger, a series of electrophysiology experiments in acute brain slices were performed.
In the mouse prefrontal cortex, separate populations of pyramidal neurons that project either
subcortically to the pons (corticopontine, CPn) or to the contralateral cortex (commissural, COM)
differentially express Hyperpolarizationꢀactivated cyclic nucleotide–gated (HCN) channels. HCN
channel expression can be assessed electrophysiologically by hyperpolarizing neurons in current clamp
recordings. HCN channels activate slowly over 10s of ms in response to neuronal hyperpolarization.
Thus, cells with high HCN expression levels (e.g., CPn neurons) respond to hyperpolarization with a
characteristic “sag” during and “rebound” following current pulses as the voltage overshoots steadyꢀ
state membrane potential in each direction. This overshoot is indicative of the delayed activation and
closure of HCN channels. [42] COM neurons express lower levels of HCN and display little to no sag,
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while CPn neurons express high levels of HCN and display significant sag. 5HT has differential effects
on firing properties in these pyramidal neuron classes. [41] In CPn neurons, 5HT reduces neuronal
excitability through 5HT1Aꢀdependent signaling. In COM neurons, this 5HT1Aꢀdependent reduction in
excitability is often followed by a slower, 5HT2Aꢀdependent increase in activity. [41] As such, 5HT
uncaging should result in both reductions and delayed increases in these two cell classes.
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To determine whether uncaging of [Ru(bpy) (PMe )(5HT)] could alter pyramidal cell firing
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rates, we made recordings from COM and CPn neurons in mouse prefrontal cortex. When the cagedꢀ
HT was added to the slice in the dark, we observed no change in electrophysiological measures
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including resting membrane potential and input resistance, suggesting that the RuBiꢀ5HT is not toxic in
its caged form.
Consistent with previous work, firing rate was lowered by 5HT uncaging in a CPn neuron. [41] A
neighboring COM neuron exhibited a characteristic biphasic modulation of firing rate after
[
Ru(bpy) (PMe )(5HT)] uncaging with an initial depression of firing rate followed by a brief increase in
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firing (Fig 5E). In a subsequent experiment, we selectively blocked the delayed excitation with the
HT2A receptor antagonist (1 µM MDL 100907). This experimental design thus provided an internal
5
control that 5HT photolysis occurred (by the generation of the initial decrease in firing rate) while also
demonstrating that changes in firing rate were indeed due to activation of 5HT receptors (by blocking
the increase in firing rate with MDL 100907).
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Figure 5. A) Schematic indicating location of recorded neurons in coronal slice from mPFC of mouse.
B) Twoꢀphoton zꢀstack image of recorded neuron filled with Alexaꢀ594. C) Voltage responses of
putative CPn neuron to hyperpolarizing and depolarizing current steps. DꢀF) A depolarizing current step
was delivered to the CPn neuron to elicit spiking. Top: Firing of CPn neuron at baseline (D), using a
pulse of blue light (470nm, 5ms, 3.6mW) to release 5HT (E), and with 5HT uncaging in the presence of
a specific 5HT2A receptor antagonist (1 µM MDL100907, F). Bottom: Peristimulus time histograms of
firing rate of the neuron in 200ms bins. G) Voltage responses of putative COM neuron to
hyperpolarizing and depolarizing current steps. HꢀI) Top: Firing of COM neuron at baseline (H) and
with uncaging of 5HT (I). Bottom: Peristimulus time histograms of firing rate of the neuron in 200ms
bins.
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Conclusions
We have developed [Ru(bpy) (PMe )(5HT)] , a ruthenium bipyridilꢀbased 5HT phototrigger which
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has its absorption maximum well into the visible range. Due to its absorption spectrum, 5HT can be
released by irradiating with readily available lowꢀpower visible wavelength laser diodes at 405, 445 and
even 532 nm. The photoprocess is very clean, showing no sign of other photoproducts besides free
serotonin and the corresponding aquo complexes, as is expected from the known photochemistry of this
kind of compound. Its redox potential lays into the expected values for a complex of this family, and is
highly related with the position of the MLCT band [43]. However, its quantum efficiency of
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photorelease is somewhat lower that expected. The analog complexes [Ru(bpy) PMe (Dopa)] (Dopa =
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dopamine)[23] and [Ru(bpy) PMe (GABA)] present the same absorption [44] but three times more
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activity than [Ru(bpy) PMe (5HT)] , suggesting that some degree of recaptation after uncaging may be
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occurring. On the other hand, the sensitivity of this new caged serotonin is high enough to allow clean
and fast uncaging in biological models, as is demonstrated by confirming neuronal characterization
experiments while uncaging with a 5 ms 470nm LED light source.
This phototrigger can be used with harmless visible light instead of UV light as is needed with the
more traditional organic protecting groups. The water solubility of its chloride salt
[
Ru(bpy) (PMe )(5HT)]Cl is around 200 mM, much higher than needed for biological experiments, and
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the excellent stability of its solutions at neutral pH has been confirmed.
METHODS
Syntheses
All chemicals were purchased from SigmaꢀAldrich and used as received without further purification.
Ru(bpy) Cl was synthesized according to literature [45] and from this [Ru(bpy) (PMe )(Cl)]PF was
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obtained as previously described. [17]
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Syntheses of [Ru(bpy) (PMe )(5HT)]Cl : 207 mg (320 µmol) of [Ru(bpy) (PMe )(Cl)]PF were
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ꢀ
dissolved in 2 ml acetone and 10 ml water. The mixture was stirred with Cl ꢀloaded Dowex ion
exchange resin during 15–30 minutes. After evaporation of acetone the resultant solution of
[
Ru(bpy) (PMe )(Cl)]Cl was heated at 70 °C to obtain the aquo complex [Ru(bpy) (PMe )(H O)]Cl.
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Then 320 mg (1500 µmol) of serotonin hydrochloride salt were introduced under N atmosphere in a
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Schlenk flask and the pH was raised to 9ꢀ10 by adding 1400 µL of NaOH 1 M. When the formation of
serotonin complex was confirmed by UVꢀvisible spectroscopy, the mixture was cooled in an ice bath
and HCl 1 M was added to lower the pH and prevent possible oxidation of serotonin by air during the
subsequent procedures. The solution was filtered and after the addition of 1500 µL of KPF 0.5 M the
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complex [Ru(bpy) (PMe )(5HT)](PF ) precipitates as an orange powder. Anal. Calcd. for C, 41.47; H,
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3.90; N, 8.79. Found: C, 41.2; H, 4.1; N, 8.5. For the purification of the serotonin complex,
hexafluorophosphate anion was interchanged for chloride using Dowexꢀ22 ion exchange resin. The
complex was reprecipitated with KPF 0.5 M at pH 3, the anion interchanged again and pH adjusted
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between 7–8. The lyophilized complex [Ru(bpy) (PMe )(5HT)]Cl was characterized by NMR
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spectroscopy. HꢀNMR (D O, K CO ) δ, ppm = 8.78 (1H, d, bpy), 8.57 (1H, d, bpy), 8.36 (1H, d, bpy),
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.13 (1H, d, bpy), 8.10 (1H, t, bpy), 7.99 (1H, d, bpy), 7.94 (1H, d, bpy), 7.83 (1H, t, bpy), 7.79 (1H, t,
bpy), 7.69 (1H, t, bpy), 7.64 (1H, t, bpy), 7.36 (1H, t, bpy), 7.31 (1H, d, bpy), 7.29 (1H, d, bpy), 7,19
1H, d, J=8.5 Hz, 5HT), 7.13 (1H, t, bpy), 6.95 (1H, t, bpy), 6.89 (1H, s, 5HT), 6.63 (1H, dd, J=8.5 Hz
(
and J=2 Hz, 5HT), 6.21 (1H, d, J=2 Hz, 5HT), 3.57 (1H, t, 5HT), 2.65 (1H, t, 5HT), 2.61ꢀ2.47 (2H, m,
5HT), 2.42 (1H, m, 5HT), 1.76 (1H, m, 5HT), 0.92 (9H, d, PMe3).
Spectroscopic Measurements and Photolysis
The optical bench used for UVꢀVis measurements consists, briefly, of a set of collinear lasers directed
towards a fourꢀfaceted cuvette, stirred and thermostated at 25 °C. The solution’s absorbance is
monitored perpendicularly to the laser path by an OceanOptics PC2000 diodeꢀarray spectrophotometer
run by OOIChem sofware.
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Quantum yield measurements of photouncaging were performed recording full absorption spectra
while the photoreaction occurs. Then, the quantum yield of photolysis (φPD) was adjusted as a parameter
in order to fit the equation 1 to the measured spectra.
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NMR spectra were obtained with a 500 MHz Bruker AMꢀ500. The compound was photolyzed inside
the unopened NMR test tube with an ownꢀdesigned illuminator (see supporting information, Figure S2)
using an array of high power LEDs centered at 450 ±20 nm FWHM.
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Voltammetry
Cyclic voltammetry was recorded at 100 mV/s scan rate in acetonitrile solution and
tetrabutylammonium salt as supporting electrolyte, using a threeꢀelectrode potentiostat based on an
operational amplifier TL071 in currentꢀvoltage configuration. A Pt wire with a diameter of 500 ꢀm was
used as a working electrode and acquisition software was written in QuickBasic 4.5. Ferrocene was used
as internal reference.
Electrophysiology. Slice Preparation.
Coronal brain slices (250 µm) containing medial prefrontal cortex were made from a P45 C57BL/6
male mouse. Slicing solution was chilled to 4°C and contained (in mM): 234 sucrose, 26 NaHCO , 11
3
glucose, 10 MgSO , 2.5 KCl, 1.25 NaH PO , 0.5 CaCl , bubbled with 5% CO / 95% O . Slices were
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incubated in artificial cerebrospinal fluid (aCSF) at 32°C for 30 minutes and then at room temperature
until recording. ACSF contained (in mM): 123 NaCl, 26 NaHCO , 11 glucose, 3 KCl, 2 CaCl , 1.25
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NaH PO , 1 MgCl , also bubbled with 5% CO / 95% O .
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Serotonin Uncaging & Patch Clamp Electrophysiology
[
Ru(bpy) (PMe )(5HT)]Cl was applied as a powder to the aCSF bath solution (100 µM) and was
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photolysed by 1 photon excitation with a single 5 ms light flash of a 470 nm LED (~3.6 mW).
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Neurons were visualized using DODT contrast microscopy on an upright microscope (Bruker).
Recordings were made using a Multiclamp 700B (Molecular Devices) amplifier and acquired with
IgorPro. Patch pipettes (2ꢀ5 Mꢁ tip resistance, Schott 8250) were filled with the following (in mM): 130
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KGluconate, 10 KCl, 10 HEPES, 10mM Trisꢀphosphocreatine, 1.1 EGTA, 2 MgCl , 2 MgATP, 0.3
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Na GTP, and 0.1 Alexaꢀ594. All recordings were made at 32ꢀ34°C. Series resistance ranged from 10ꢀ15
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Mꢁ and pipette capacitance was compensated.
Current pulses were applied through the somatic patch pipette for electrophysiological
characterization (ꢀ300 and +150 pA in 250 ms duration). The neuron that displayed voltage sag at step
onset and rebound at step offset when hyperpolarized (indicative of high expression of HCN channels)
was identified as a putative corticopontine (CPn) projecting neuron [41, 42, 46]. The neuron lacking
significant sag and rebound was identified as a putative commissural (COM) projecting neuron [41, 42,
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7]. A 9 s positive current step was then applied to elicit spiking during uncaging trials (100 and 75 pA
in CPn and COM cells, respectively). 1 µM MDL100907 (stock in DMSO), a potent 5HT2A receptor
antagonist, was applied before uncaging for some experiments.
ACKNOWLEDGEMENTS
This research was supported by the National Agency for Science and Technology Promotion,
CONICET, and the University of Buenos Aires (R.E); NIH R01NS099254, NSF 1604544, and the Brain
Research Foundation (K.E.P.); NIH R01 DA035913 (K.J.B.), and NRSA F31 MH111219ꢀ01 (J.A.). R.
E. is a member of CONICET.
AUTHOR INFORMATION
rober@qi.fcen.uba.ar
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