*For correspondence:
viviana@caltech.edu
Competing interests:
The
authors declare that no
competing interests exist.
Funding:
See page 24
Received:
02 June 2019
Accepted:
21 September 2019
Published:
23 September 2019
Reviewing editor:
Inna Slutsky,
Tel Aviv University, Israel
Copyright Robinson et al. This
article is distributed under the
terms of the
Creative Commons
Attribution License,
which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Optical dopamine monitoring with
dLight1 reveals mesolimbic phenotypes in
a mouse model of neurofibromatosis
type 1
J Elliott Robinson
1
, Gerard M Coughlin
1
, Acacia M Hori
1
, Jounhong Ryan Cho
1
,
Elisha D Mackey
1
, Zeynep Turan
1
, Tommaso Patriarchi
2
, Lin Tian
2
,
Viviana Gradinaru
1
*
1
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, United States;
2
Department of Biochemistry and Molecular Medicine,
University of California, Davis, Davis, United States
Abstract
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose
neurodevelopmental symptoms include impaired executive function, attention, and spatial learning
and could be due to perturbed mesolimbic dopaminergic circuitry. However, these circuits have
never been directly assayed in vivo. We employed the genetically encoded optical dopamine
sensor dLight1 to monitor dopaminergic neurotransmission in the ventral striatum of NF1 mice
during motivated behavior. Additionally, we developed novel systemic AAV vectors to facilitate
morphological reconstruction of dopaminergic populations in cleared tissue. We found that NF1
mice exhibit reduced spontaneous dopaminergic neurotransmission that was associated with
excitation/inhibition imbalance in the ventral tegmental area and abnormal neuronal morphology.
NF1 mice also had more robust dopaminergic and behavioral responses to salient visual stimuli,
which were independent of learning, and rescued by optogenetic inhibition of non-dopaminergic
neurons in the VTA. Overall, these studies provide a first in vivo characterization of dopaminergic
circuit function in the context of NF1 and reveal novel pathophysiological mechanisms.
DOI: https://doi.org/10.7554/eLife.48983.001
Introduction
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder of neural crest-derived tissues
that affects approximately 1 in 3500 individuals worldwide and is caused by loss of one functional
copy of the
NF1
gene on chromosome 17 (
Wallace et al., 1990
). Neurofibromin, the protein prod-
uct of
NF1
, inhibits Ras-dependent cellular growth and proliferation (
Basu et al., 1992
) and enhan-
ces cAMP signaling pathways (
Tong et al., 2002
). The clinical features of NF1 include pigmentary
lesions, neoplasia (e.g. cutaneous and plexiform neurofibromas, optic gliomas, malignant peripheral
nerve sheath tumors), cognitive and learning disabilities, peripheral neuropathy, musculoskeletal
abnormalities, and gross and fine motor delays (
Cimino and Gutmann, 2018
;
Gutmann et al.,
2012
). Cognitive dysfunction is a significant source of lifetime morbidity, as up to 70% of affected
individuals experience impaired executive functioning, speech and language delays, attention defi-
cits, hyperactivity, and/or impulsivity (
Hyman et al., 2005
). Furthermore, approximately one third of
patients with NF1 meet DSM-V criteria for attention deficit hyperactivity disorder (ADHD)
(
Hyman et al., 2005
;
Miguel et al., 2015
). Despite the societal burden of NF1-associated cognitive
sequelae, their etiology has not been fully elucidated.
Robinson
etal
. eLife 2019;8:e48983.
DOI: https://doi.org/10.7554/eLife.48983
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RESEARCH ARTICLE
Although homozygous genetic disruption of the
Nf1
gene is embryonic lethal in mice (
Silva et al.,
1997
), cognitive deficits in NF1 have been successfully modeled in several transgenic and condi-
tional knockout mouse lines (
Silva et al., 1997
;
Zhu et al., 2001
;
Hegedus et al., 2007
;
Cui et al.,
2008
;
Brown et al., 2010a
;
Anastasaki et al., 2015
;
Omrani et al., 2015
;
Li et al., 2016
;
Xie et al.,
2016
). Heterozygous knockout mice (
Nf1
+/-
) exhibit impaired spatial learning (
Costa et al., 2001
;
Silva et al., 1997
), which is Ras/ERK-dependent (
Costa et al., 2002
), rescued by the Ras inhibitor
lovastatin (
Li et al., 2005
), and may be due to increased inhibitory GABA tone (
Costa et al., 2002
).
Additionally, the neurofibromin C-terminus is a positive regulator of G-protein-stimulated adenylyl
cyclase activity (
Hannan et al., 2006
;
Tong et al., 2002
), and cAMP deficiency in NF1 knockout
models causes altered in vitro neuronal morphology and growth, visual learning deficits, and
changes in cortical architecture in mice (
Brown et al., 2012
;
Brown et al., 2010b
;
Hegedus et al.,
2007
;
Wolman et al., 2014
). Attenuated dopaminergic neurotransmission in mesolimbic and nigros-
triatal circuits are putative mechanisms underlying attentional, learning, and motivational deficits
observed in NF1 model mice (
Diggs-Andrews and Gutmann, 2013
). Mesolimbic reward circuits
involve the convergence of dopaminergic projections from the midbrain ventral tegmental area
(VTA) with glutamatergic inputs from cortical and subcortical regions on medium spiny neurons in
the nucleus accumbens (NAc). These circuits facilitate the translation of relevant internal and external
stimuli into motivated behaviors (
Wise, 2005
) and have been implicated in the pathophysiology of
ADHD and other disorders of impulse control (
Li et al., 2006
;
Purper-Ouakil et al., 2011
).
In the optic glioma mouse model of NF1 (OPG, a conditional
Nf1
knockout in astrocytes on an
Nf1
+/-
background), reduced striatal dopamine is associated with motor, exploratory, spatial learn-
ing, and attentional abnormalities (
Brown et al., 2010a
;
Diggs-Andrews et al., 2013
;
Anastasaki et al., 2015
), which are ameliorated by treatment with the catecholamine re-uptake
eLife digest
About one in 3,500 people have a genetic disorder called neurofibromatosis type
1, often shortened to NF1, making it one of the most common inherited diseases. People with NF1
may have benign and cancerous tumors throughout the body, learning disabilities, developmental
delays, curvature of the spine and bone abnormalities. Children with NF1 often experience
difficulties with attention, hyperactivity, speech and language delays and impulsivity. They may also
have autism spectrum disorder, or display symptoms associated with this condition.
Studies in mice with a genetic mutation that mimics NF1 suggest that abnormal development in
cells in the middle of the brain may cause the cognitive symptoms. These midbrain neurons produce
a chemical called dopamine and send it throughout the brain. Dopamine is essential for
concentration and it is involved in how the brain processes pleasurable experiences.
Now, Robinson et al. show that, at rest, the NF1 model mice release dopamine less often than
typical mice. This happens because, when there are no stimuli to respond to, neighboring cells slow
down the activity of dopamine-producing neurons in NF1 model mice.
In the experiments, both NF1 model mice and typical mice were taught to associate
environmental cues with rewards or punishments. Robinson et al. then measured the release of
dopamine in the mice using a sensor called dLight1, which produces different intensities of
fluorescent light depending on the amount of dopamine present. This revealed that the NF1 model
mice produced more dopamine in response to visual cues and had enhanced behavioral responses
to these stimuli. For example, when a looming disc that mimics predators approached them from
above, the NF1 model mice tried to hide in an exaggerated way compared to the typical mice.
Previously, it had been shown that this type of behavior is due to the activity of the dopamine-
producing neurons’ neighboring cells, which Robinson et al. found is greater in NF1 model mice.
Next, Robinson et al. stopped neighboring cells from interfering with the dopamine-producing
neurons in NF1 model mice. This restored dopamine release to normal levels at rest, and stopped
the mice from overreacting to the looming disc. The experiments help explain how the NF1 model
mice process visual information. Further study of the role dopamine plays in cognitive symptoms in
people with NF1 may help scientists develop treatments for the condition.
DOI: https://doi.org/10.7554/eLife.48983.002
Robinson
etal
. eLife 2019;8:e48983.
DOI: https://doi.org/10.7554/eLife.48983
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Research article
Neuroscience
inhibitor methylphenidate or the dopamine precursor L-DOPA (
Brown et al., 2010a
). Despite these
efforts, dopaminergic neurotransmission has never been investigated in NF1 models in vivo. In order
to address this gap in the understanding of NF1, we utilized the new, ultra-fast, genetically encoded
dopamine sensor dLight1 (
Patriarchi et al., 2018
) to monitor dopamine dynamics in the lateral
nucleus accumbens (LNAc) during motivated behavior in 129T2/SvEmsJ::C57Bl/6NTac F1 hybrid
Nf1
wildtype (
Nf1
+/+
) and heterozygous knockout (
Nf1
+/-
) mice. This hybrid background produces more
robust behavioral phenotypes than those on a pure C57Bl/6 background (
Cui et al., 2008
;
Li et al.,
2005
;
Shilyansky et al., 2010
). Novel dopaminergic phenotypes were further parsed with patch
clamp electrophysiology and optogenetics. Because previous morphological analysis has largely
been restricted to neuronal cultures (
Brown et al., 2010a
;
Anastasaki et al., 2015
), we comprehen-
sively characterized dopaminergic neuron structure in situ in
Nf1
+/+
and
Nf1
+/-
mice using tissue
clearing, tracing methods, and the novel systemic AAV-based tool
Th
-VAST (catecholaminergic neu-
ron-targeted vector-assisted spectral tracing). These efforts revealed distinct dopaminergic pheno-
types, identified putative mechanisms governing their expression, and explored how
Nf1
haploinsufficiency moderates the motivational salience of relevant environment stimuli.
Results
In vivo optical monitoring of dopaminergic neurotransmission using
dLight1.2
In order to investigate dopamine dynamics in freely behaving
Nf1
+/+
and
Nf1
+/-
mice, we utilized the
genetically encoded, fluorescent dopamine sensor dLight1.2 (
Patriarchi et al., 2018
), which allows
for sub-micromolar detection of extracellular dopamine concentrations with sub-second resolution
and negligible sensitivity to other monoamines, GABA, and glutamate (
Corre et al., 2018
;
Patriarchi et al., 2018
). Fluorescent dopamine signals in the LNAc were monitored with fiber pho-
tometry (
Gunaydin et al., 2014
); this terminal field region was chosen because its afferent ventral
tegmental dopaminergic inputs exhibit a high diversity of responses to both rewarding and aversive
stimuli and stimulus-predictive cues (
de Jong et al., 2019
;
Lammel et al., 2011
). To facilitate optical
dopamine measurements, an adeno-associated viral vector (AAV9-hSyn-dLight1.2) was stereotaxi-
cally injected into the LNAc to express dLight1.2 in neurons, followed by implantation of a 400
m
m
optical fiber (
Figure 1A
) for sensor excitation and emitted photon collection via a custom photome-
try system (
Cho et al., 2017
) (
Figure 1B
).
After surgical recovery, we measured baseline differences in spontaneous dopaminergic neuro-
transmission by monitoring dLight1.2 signals (
Figure 1C
,
Figure 1—figure supplement 1
) in the
LNAc during 5-min epochs in which mice sat in a dark, sound-attenuating chamber. Peak analysis
was performed to identify local trace prominences (
Figure 1D
) and revealed that the dopamine tran-
sient event rate was reduced in
Nf1
+/-
mice compared to
Nf1
+/+
littermates (
Figure 1E
). Baseline
(median) fluorescence, peak amplitude, and full width at half maximal intensity (FWHM) was equiva-
lent between genotypes. Because reduced LNAc dopamine content and afferent terminal TH
expression have been observed in OPG mice (
Brown et al., 2010a
;
Diggs-Andrews et al., 2013
),
we measured monoamine and monoamine metabolite levels in the NAc using high-performance liq-
uid chromatography. We failed to detect differences in dopamine (DA), serotonin (5-HT), norepi-
nephrine (NE), or their metabolites between genotypes (
Figure 1—figure supplement 2
).
Additionally, there was no difference in dopaminergic terminal tyrosine hydroxylase expression
across striatal sub-compartments (
Figure 1—figure supplement 2
). These findings suggest that
basal differences in dLight1.2 event rate are not due to changes in dopaminergic terminal density or
dopamine synthetic capacity.
In order to further parse differences in spontaneous dopaminergic transient activity, we per-
formed whole-cell patch clamp electrophysiological recordings in acute midbrain slices that con-
tained the lateral ventral tegmental area (
Figure 1F
), which is the main source of dopaminergic
projections to the LNAc (
Lammel et al., 2011
). Because the dependence of
Nf1
+/-
phenotypes on
genetic background precludes crossing with cell-type-specific reporter or Cre recombinase lines, we
used a blood-brain barrier penetrant, systemic adeno-associated viral vector (AAV-PHP.eB)
(
Chan et al., 2017
) containing a green fluorescent protein (GFP) transgene under control of the rat
tyrosine hydroxylase promoter (
Oh et al., 2009
) (AAV-PHP.eB-
Th
-GFP; 1
10
11
viral genomes/
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. eLife 2019;8:e48983.
DOI: https://doi.org/10.7554/eLife.48983
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Research article
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Tyrosine Hydroxylase
AAV9-hSyn-dLight1.2
490nm
LED
405nm
LED
Data Acquisition
& LED Control
Dichroic
Filter
Focusing
Lens
LED
Driver
10%
'
F/F
50 s
+/+
+/-
A
B
C
E
+/+ +/-
Event
Ampiltude
Fluorescence (z-score)
+/+ +/-
Event
FWHM
Time (s)
+/+ +/-
Event
Rate
Frequency (Hz)
+/+ +/-
Median
'
F/F
Fluorescence (
'
F/F)
Photo-
receiver
+/+
+/-
20 mV
1 s
G
+/+
+/-
0
1
2
3
4
5
Spontaneous Firing
Firing Rate (Hz)
*
0
5
10
15
20
-4
-2
0
2
4
6
8
10
12
Fluorescence (z-score)
Time (s)
dLight1.2
Peak
Prominence
FWHM
Baseline Analysis
VTA
AAV-PHP.eB-
Th
-GFP
4X
VTA
4X
10 mV
100 ms
+/+
+/-
500 pA
H
+/+
+/-
0
100
200
300
Excitability
Rheobase (pA)
*
D
F
+/+
+/-
-50
-40
-30
-20
-10
0
AP Threshold
Voltage (mV)
+/+
+/-
0
2
4
6
8
AP Width
Duration (ms)
I
J
DIC
-1.5
-1.0
-0.5
0.0
0.5
1.0
*
0.0
0.2
0.4
0.6
0.8
2.5
3.0
3.5
4.0
4.5
0.4
0.5
0.6
0.7
0.8
Figure 1.
Assessment of basal dopaminergic function in vivo with dLight1.2 and ex vivo patch clamp electrophysiology. (
A
) Illustration showing location
of stereotaxic injection of the AAV9-hSyn-dLight1.2 viral vector and photometry fiber implantation (
left
). Representative histological image (
right
, scale:
300
m
m) showing the fiber tip location and expression of dLight1.2 (stained for GFP, green) and dopaminergic terminal tyrosine hydroxylase (TH, Red).
(
B
) Schematic of fiber photometry system used for dLight1.2 (490 nm) and isosbestic (405 nm; reference signal) excitation and emission signal detection
in freely moving mice. (
C
) Representative dLight1.2 traces in
Nf1
+/+
and
Nf1
+/-
mice. (
D
) Representative trace and analysis features for baseline peak
detection. (
E
) Peak analysis of baseline dLight1.2 recordings revealed that Nf1
+/-
mice (n = 33) exhibit reduced transient frequency (unpaired t-test;
t
50
= 3.06, p=0.004) but not median fluorescence (unpaired t-test; t
50
= 1.01, p=0.32), transient amplitude (unpaired t-test; t
50
= 0.83, p=0.41), or full
width at half maximal amplitude (FWHM; unpaired t-test; t
50
= 0.43, p=0.67) when compared to Nf1
+/+
littermates (n = 19). (
F
) 4X differential
interference contrast (DIC) image (
left
) of an acute horizontal midbrain slice containing the ventral tegmental area (VTA) and 4X epifluorescence image
(
right
) with GFP-labeled catecholaminergic neurons following systemic delivery of AAV-PHP.eB-
Th
-GFP (1
10
11
v.g./mouse). (
G
) Representative traces
showing spontaneous whole-cell firing of putative VTA dopaminergic neurons (
left
). Spontaneous firing rates (
right
) were lower (unpaired t-test;
t
28
= 2.58, p=0.0 w) in
Nf1
+/-
putative dopaminergic neurons (n = 18) compared to
Nf1
+/+
neurons (n = 12). (
H
) Representative electrophysiological
traces (
left
) showing evoked firing by a 1 pA/ms ramp current from