*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.
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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
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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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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
60 mV in
Nf1
+/+
and
Nf1
+/-
putative dopaminergic neurons. Rheobase (
right
;
unpaired t-test; t
48
= 4.05, p<0.001) but not action potential threshold (I; t
48
= 1.93, p=0.06) or width (J; t
48
= 0.39, p=0.70) was increased in
Nf1
+/-
(n = 29) putative dopaminergic neurons compared to
Nf1
+/+
(n = 21). *denotes p<0.05 vs
Nf1
+/+
. Data presented as mean
±
SEM.
DOI: https://doi.org/10.7554/eLife.48983.003
The following figure supplements are available for figure 1:
Figure supplement 1.
Raw fluorescent photometry signals.
DOI: https://doi.org/10.7554/eLife.48983.004
Figure supplement 2.
Striatal catecholamine content and tyrosine hydroxylase immunofluorescence.
DOI: https://doi.org/10.7554/eLife.48983.005
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mouse r.o.;
Figure 1F
,
right
) to label dopaminergic neurons. This allowed for visual identification
during patch clamp experiments. GFP-positive cells were considered to be dopaminergic if their
action potential duration was >1 ms, a previously validated threshold to distinguish dopaminergic
from GABAergic neurons in the VTA (
Chieng et al., 2011
). We found that putative dopaminergic
neurons in
Nf1
+/-
midbrain slices exhibited lower spontaneous whole-cell firing rates (
Figure 1G
)
and required more rheobase current to elicit a spike when compared to
Nf1
+/+
neurons (
Figure 1H
).
This finding supports the hypothesis that phenotypic differences in baseline dLight1.2 event metrics
are activity-dependent. Action potential threshold, duration, amplitude, and after hyperpolarization
magnitude did not differ between genotypes (
Figure 1I–J
,
Table 1
).
Morphological characterization of VTA dopaminergic neurons in
Nf1
+/+
and
Nf1
+/-
mice
During whole-cell recordings, we also observed that
Nf1
+/-
putative dopaminergic neurons exhibit
increased input resistance (R
m
) and decreased membrane capacitance (C
m
) compared to
Nf1
+/+
lit-
termates (
Figure 2A
) without a change in other membrane properties (
Table 2
). This finding was
robust across experiments (
Table 2
). Because increased R
m
could be indicative of reduced soma vol-
ume (
Torres-Torrelo et al., 2014
), we manually traced over two thousand TH-positive dopaminergic
somata in the VTA per genotype (
Figure 2B
). We found that cross-sectional area, major axis length,
and minor axis length were reduced in
Nf1
+/-
mice (
Figure 2C–D
,
Figure 2—figure supplement 1
).
Proportionality was maintained, however, as the soma aspect ratio was equivalent between geno-
types (
Figure 2—figure supplement 1
). TH immunofluorescence and total neuron counts in the VTA
did not differ between
Nf1
+/-
and
Nf1
+/+
dopaminergic neurons (
Figure 2D
). No phenotypic differ-
ences were observed in the adjacent substantia nigra pars compacta (
Figure 2—figure supplement
1
). These findings indicate that relative differences in soma size were VTA-specific and could have
contributed to changes in passive membrane properties.
Dendritic complexity also contributes to cell input resistance (
Bekkers and Hausser, 2007
;
S
ˇ
is
ˇ
kova
́
et al., 2014
), so we modified the two-component, systemic AAV-based method VAST (
Vec-
tor-
Assisted
Spectral
Tracing) (
Chan et al., 2017
) to create
Th
-VAST. This tool facilitates anatomical
reconstruction of dendritic arbors by providing recombinase-independent, sparse, multicolor label-
ing of catecholaminergic neurons. VAST achieves hue diversity via stochastic expression of three tet-
racycline response element (TRE)-regulated fluorescent proteins (XFPs; mRuby2, mNeonGreen, and
mTurquoise2) following systemic delivery with AAV-PHP.eB. Sparseness is subsequently tuned by
titration of a co-delivered, tet-off transactivator (tTA) inducer vector (
Chan et al., 2017
). In
Th
-VAST,
tTA expression is targeted to catecholaminergic neurons via use of the
Th
promoter, and retro-
orbital delivery of the XFP cocktail (AAV-PHP.eB-TRE-XFP; 1
10
12
vg/mouse total) and the inducer
vector (AAV-PHP.eB-
Th
-tTA; 1
10
11
vg/mouse) produced dense multicolor labeling of
Th
neurons
Table 1.
Action potential features across patch clamp electrophysiology experiments.
Property
Experiment
p
+/+: Mean
±
SEM, n
+/-: Mean
±
SEM, n
Rheobase
Baseline characterization
<0.001
124.1
±
8.65 pA, n = 21
171.7
±
7.779 pA, n = 29
AP Threshold
Baseline characterization
0.059
39.32
±
1.266 mV, n = 21
36.45
±
0.8708 mV, n = 29
AP Duration
Baseline characterization
0.695
3.671
±
0.2525 ms, n = 21
3.562
±
0.1499 ms, n = 29
AP Height
Baseline characterization
0.555
60.89
±
1.607 mV, n = 21
59.42
±
1.749 mV, n = 29
AP AHP
Baseline characterization
0.897
15.43
±
1.19 mV, n = 21
14.88
±
1.046 mV, n = 29
Firing Rate
Baseline characterization
0.016
2.633
±
0.2464 Hz, n = 12
1.703
±
0.244 Hz, n = 18
Rheobase
Picrotoxin rescue
<0.001
131.5
±
7.537 pA, n = 25
89.14
±
6.413 pA, n = 20
AP Threshold
Picrotoxin rescue
0.456
36.92
±
1.193 mV, n = 25
38.33
±
1.472 mV, n = 20
AP Duration
Picrotoxin rescue
0.610
4.156
±
0.1589 ms, n = 25
4.03
±
0.1891 ms, n = 20
AP Height
Picrotoxin rescue
0.946
56.16
±
2.021 mV, n = 25
55.99
±
1.151 mV, n = 20
AP AHP
Picrotoxin rescue
0.168
13.84
±
1.125 mV, n = 25
11.78
±
0.844 mV, n = 20
Firing Rate
Picrotoxin rescue
0.714
2.434
±
0.208 Hz, n = 16
2.535
±
0.1596 Hz, n = 13
DOI: https://doi.org/10.7554/eLife.48983.006
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200
250
300
350
400
100
150
200
250
300
200
250
300
350
400
+/+
+/-
0
100
200
300
400
500
Passive Membrane Properties
+/+
+/-
0
20
40
60
80
*
*
D
+/+
+/-
+/+
+/-
Mean Pixel Intensity (a.u.)
Cells Per Section
*
Cell Counts
TH Expression
+/+
+/-
C
0
200
400 600
0.2
0.4
0.6
0.8
1
Cumulative Probability
+/+
+/-
p < 0.001
R
m
(MOhm)
C
m
(pF)
Cross-sectional Area (
P
m
2
)
A
B
0
100
200
300
400
0
2
4
6
Sholl Analysis
Distance from Soma (
P
m)
Intersections
+/+
+/-
+/+
+/-
0
500
1000
1500
2000
2500
Total Length (
P
m)
Th
tTA
WPRE
XFP
TRE
WPRE
Th
-VAST: Vector-Assisted Spectral Tracing of Catecholaminergic Neurons
AAV-PHP.eB
+/+
+/-
1
3
5
Dopaminergic Neuron Reconstructions
VTA
Soma Area (
P
m
2
)
Average Soma
Size
E
F
G
H
VTA
0
SNc
AAV-PHP.eB-
Th
-tTA:
1 x 10
11
vg/mouse
AAV-PHP.eB-
Th
-tTA:
6 x 10
9
vg/mouse
+/+
+/-
VTA
SNc
TH
Neurite Length
Figure 2.
Morphological analysis of ventral tegmental dopaminergic neurons in
Nf1
+/+
and
Nf1
+/-
mice. (
A
) Whole-cell recordings revealed that
Nf1
+/-
putative dopaminergic neurons (n = 29) had increased input resistance (R
m
;
left
; unpaired t-test; t
48
= 2.97, p=0.005) and decreased capacitance (C
m
;
right
; t
48
= 2.54, p=0.01) compared to
Nf1
+/+
neurons (n = 21). (
B
) Representative ventral midbrain images containing the ventral tegmental area (VTA)
and substantia nigra pars compacta (SNc) stained for tyrosine hydroxylase (TH, scale: 300
m
m); TH-positive neurons in the VTA (
inset
, scale: 100
m
m). (
C
)
The cumulative probability distribution of the cross sectional area of manually traced
Nf1
+/+
(n = 2344) and
Nf1
+/-
(n = 2586) VTA dopaminergic neuron
somata (two-sample Kolmogorov-Smirnov test; D = 0.18, p<0.001). (
D
) Average VTA dopaminergic soma area (
left
; n
+/+
= 17, n
+/-
= 15; unpaired t-test;
t
30
= 4.65, p<0.001), TH immunofluorescence (
middle
; t
30
= 0.25, p=0.90), and number of neurons/histological section (
right
; t
30
= 0.15, p=0.88) per
mouse. (
E
)
Th
-VAST (
left
) produced multicolor labeling of dopaminergic neurons in the VTA (
middle
, scale: 300
m
m;
right
, scale: 100
m
m). (
F
) Dense (
left
,
scale: 20
m
m) or sparse multi-color labeling (
right
, scale: 20
m
m) was achieved via retro-orbital injection of either 1
10
11
or 6
10
9
vg/mouse AAV-
PHP.eB-
Th
-tTA, respectively, and 1
10
12
total vg/mouse of the XFP cocktail (AAV-PHP.eB-TREx7-mRuby2, -mNeonGreen, or -mTurquoise2). (
G
)
Representative dopaminergic neuron reconstructions following neurite tracing (scale: 20
m
m). (
H
) Sholl analysis failed to detect a difference in dendritic
complexity (
left
; two-way repeated measures ANOVA; F
80,2160
= 0.052, p
distance x genotype
>0.99; F
80,2160
= 63.9, p
distance
<0.001; F
1,27
= 0.25,
p
genotype
= 0.63) or total neurite length (
right
; unpaired t-test; t
27
= 0.18, p=0.86) between genotypes (n
+/+
= 13, n
+/-
= 16 for +/- group). * denotes
p<0.05 vs
Nf1
+/+
. Data presented as mean
±
SEM.
DOI: https://doi.org/10.7554/eLife.48983.007
The following figure supplements are available for figure 2:
Figure supplement 1.
Additional data: histological analysis.
DOI: https://doi.org/10.7554/eLife.48983.008
Figure supplement 2.
Additional data:
Th
-VAST.
DOI: https://doi.org/10.7554/eLife.48983.009
Robinson
etal
. eLife 2019;8:e48983.
DOI: https://doi.org/10.7554/eLife.48983
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in the VTA and SNc (
Figure 2E–F
,
Figure 2—figure supplement 2
). Compared to 1
10
12
vg/
mouse AAV-PHP.eB-
Th
-GFP (
Chan et al., 2017
), the specificity of
Th
-VAST vectors was lower in the
VTA (58.7% vs 81%) and SNc (74.2% vs 81%) (
Figure 2—figure supplement 2
) despite good XFP
restriction to these areas. This likely occurred because induction of XFP expression requires very low
levels of tTA, and a sub-population of VTA projection neurons have hybrid
Th
-GABAergic pheno-
types (
Root et al., 2014
;
Stuber et al., 2015
). As such, spectral tracing was only performed when
Th
-VAST-labeled neurons were unequivocally tyrosine hydroxylase-positive.
Using a lower inducer vector dose (6
10
9
vg/mouse) to provide sparse labeling (
Figure 2F
,
right
), we repeated
Th
-VAST in
Nf1
+/-
and
Nf1
+/+
mice. Following two weeks of expression, we pre-
pared and optically cleared (using RIMS) (
Yang et al., 2014
) 300
m
m horizontal VTA sections that
had been immunostained for TH to confirm post hoc that
Th
-VAST-labeled neurons were dopami-
nergic. After tracing in Imaris (
Figure 2G
), Sholl analysis was performed to quantify dendritic branch-
ing by detecting neurite intersections with concentric 5
m
m shells originating from the soma. No
difference in dendritic complexity or total neurite length was observed between genotypes
(
Figure 2H
), which suggests that, although
Nf1
+/-
dopaminergic neurons have smaller somata than
Nf1
+/+
neurons, they have similar neurite morphology.
Nf1
+/-
putative dopaminergic neurons exhibit excitation/inhibition
imbalance in the ventral tegmental area
The observation that
Nf1
+/-
dopaminergic neurons have reduced cross-sectional areas but higher
rheobase requirement was unexpected, given that smaller neurons tend to be more excitable
(
Torres-Torrelo et al., 2014
). In order to parse these differences, we first assayed dopaminergic I
h
currents, which contribute to the stability of spontaneous firing rates (
Neuhoff et al., 2002
;
Seutin et al., 2001
) and are attenuated in hippocampal interneurons in NF1 model mice
(
Omrani et al., 2015
). I
h
was determined by quantifying the sag current produced by a series of
hyperpolarizing voltage steps from
60 mV to
130 mV in voltage clamp (
Figure 3A
). We found
that
Nf1
+/-
dopaminergic neurons had smaller I
h
current amplitudes (
Figure 3A
,
Figure 3—figure
supplement 1
) without a change in voltage dependence (
Figure 3B
; determined by tail current anal-
ysis) relative to
Nf1
+/+
littermates. Differences in I
h
current amplitudes were not significant when nor-
malized to the cell capacitance to account for cell size (
Figure 3C
,
Figure 3—figure supplement 1
),
and maximum I
h
current amplitude was significantly correlated with C
m
across all animals and within
genotypes (
Figure 3D
). Since reduced cAMP production, which has been associated with
Nf1
+/-
neu-
ronal phenotypes in vitro (
Brown et al., 2012
), could attenuate the I
h
current, we repeated I
h
meas-
urements in
Nf1
+/-
slices in the presence of the adenylyl cyclase activator forskolin. Addition of 20
m
M forskolin to the bath solution did not significantly affect I
h
magnitude or voltage dependence in
Nf1
+/-
putative dopaminergic neurons (
Figure 3—figure supplement 1
). Thus, changes in I
h
Table 2.
Passive membrane properties across patch clamp electrophysiology experiments.
Property
Experiment
p
+/+: Mean
±
SEM, n
+/-: Mean
±
SEM, n
C
m
Baseline characterization
0.014
47.35
±
2.032 pF, n = 21
41.27
±
2.026 pF, n = 29
R
m
Baseline characterization
0.005
172.4
±
10.94 M
W
, n = 21
235.7
±
16.32 M
W
, n = 29
R
s
Baseline characterization
0.966
17.86
±
1.73 pF M
W
, n = 21
17.95
±
1.257 M
W
, n = 29
Holding
Baseline characterization
0.658
74.26
±
10.95 pA, n = 21
81.01
±
10.18 pA, n = 29
C
m
I
h
measurement
0.047
51.94
±
4.45 pF, n = 14
42.53
±
2.351 pF, n = 24
R
m
I
h
measurement
0.009
170.8
±
11.96 M
W
, n = 14
222.4
±
12.49 M
W
, n = 24
R
s
I
h
measurement
0.528
17.78
±
1.478 M
W
, n = 14
19.1
±
1.334 M
W
, n = 24
Holding
I
h
measurement
0.457
61.15
±
8.657 pA, n = 14
52.11
±
7.642 pA, n = 24
C
m
Picrotoxin rescue
0.004
47.74
±
2.276 pF, n = 29
36.62
±
2.956 pF, n = 20
R
m
Picrotoxin rescue
0.001
181
±
8.464 M
W
, n = 29
239
±
14.94 M
W
, n = 20
R
s
Picrotoxin rescue
0.670
17.23
±
1.054 M
W
, n = 29
18.04
±
1.648 M
W
, n = 20
Holding
Picrotoxin rescue
0.611
56.77
±
5.88 pA, n = 29
61.64
±
7.639 pA, n = 20
DOI: https://doi.org/10.7554/eLife.48983.010
Robinson
etal
. eLife 2019;8:e48983.
DOI: https://doi.org/10.7554/eLife.48983
7 of 30
Research article
Neuroscience