Dopaminergic dysfunction in neurodevelopmental disorders:
recent advances and synergistic technologies to aid basic
research
J. Elliott Robinson
and
Viviana Gradinaru
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,
USA
Abstract
Neurodevelopmental disorders (NDDs) represent a diverse group of syndromes characterized by
abnormal development of the central nervous system and whose symptomatology includes
cognitive, emotional, sensory, and motor impairments. The identification of causative genetic
defects has allowed for creation of transgenic NDD mouse models that have revealed
pathophysiological mechanisms of disease phenotypes in a neural circuit- and cell type-specific
manner. Mouse models of several syndromes, including Rett syndrome, Fragile X syndrome,
Angelman syndrome, Neurofibromatosis type 1, etc., exhibit abnormalities in the structure and
function of dopaminergic circuitry, which regulates motivation, motor behavior, sociability,
attention, and executive function. Recent advances in technologies for functional circuit mapping,
including tissue clearing, viral vector-based tracing methods, and optical readouts of neural
activity, have refined our knowledge of dopaminergic circuits in unperturbed states, yet these tools
have not been widely applied to NDD research. Here, we will review recent findings exploring
dopaminergic function in NDD models and discuss the promise of new tools to probe NDD
pathophysiology in these circuits.
Graphical abstract
Corresponding Author:
Viviana Gradinaru, viviana@caltech.edu.
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Conflicts of Interest: None
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Curr Opin Neurobiol
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Published in final edited form as:
Curr Opin Neurobiol
. 2018 February ; 48: 17–29. doi:10.1016/j.conb.2017.08.003.
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Introduction
In the last decade, the widespread adoption of technologies for functional circuit mapping in
animal models has greatly enhanced our ability to understand the input-output relationships
between populations of neurons and determine their function
in vivo
. These include
techniques for the visualization, reconstruction, and analysis of intact circuits across micro-
and macroscales. Examples include serial section electron microscopy [
1
,
2
], the Brainbow
toolkit [
3
,
4
] and intersectional labelling strategies [
5
,
6
], improved neuroinformatic tools for
neurite tracing [
7
], tissue clearing [
8
,
9
], light sheet microscopy [
10
,
11
], and serial two-
photon tomography [
12
,
13
]. Additionally, optogenetic [
14
] and chemogenetic [
15
] actuators,
genetically encoded indicators of neuronal activity [
16
,
17
], and advanced
in vivo
imaging
modalities [
18
–
23
] have allowed for the functional deconstruction of genetically defined
circuits in order to probe their contributions to complex behaviors. The development of viral
vectors that can deliver transgenes in a pathway- and cell type-specific manner [
24
–
28
] or
broadly transduce neurons across the CNS [
29
] have greatly facilitated efforts to
anatomically and functionally characterize complex neurobiological systems in both basal
and disease states.
New tools for ‘connectomic’ or circuit-centered research that can survey large scale
functional connectivity patterns are particularly well suited to the study of
neurodevelopmental disorders (NDDs), such as autism spectrum disorder (ASD), where
diverse genetic and environmental insults during neurodevelopment can vastly perturb
circuit architecture and physiology across brain areas [
30
,
31
]. While the neural substrates of
ASD symptomatology are multifaceted, mesencephalic dopamine systems, consisting of A8
retrorubral, A9 nigrostriatal, and A10 mesocorticolimbic projections [
32
], represent circuits
of interest given their potential contribution to several common ASD symptoms, including
perseverant interests, stereotyped movements, impaired attention and executive function, and
difficulty with social interactions [
33
]. Several recent studies implicate these circuits in
behavioral phenotypes observed in rodent NDD disease models, including Angelman
syndrome (AS), Rett syndrome (RS), fragile X syndrome (FXS), neurofibromatosis type 1
(NF1), etc. (Table 1), yet widespread adoption of new tools for functional circuit mapping
has yet to occur in these models. In this review, we will highlight common patterns of
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cellular and circuit level phenotypic variation across NDD mouse models and discuss the
promise of recent neurotechnological advances such as whole brain tissue clearing and gene
delivery by systemic viral vectors to further elucidate NDD pathophysiology in
dopaminergic circuits.
Elucidating abnormal patterns of dopaminergic connectivity in NDD models
Dopaminergic projection neurons are a heterogenous population whose function, activity,
neurotransmitter content, and pattern of connectivity varies with brain region and connection
target [
34
–
36
]. For example, efferents arising from the midbrain ventral tegmental area
(VTA) project throughout the extended amygdala [including the nucleus accumbens (NAc)],
hippocampus, and prefrontal cortex (the mesocorticolimbic pathway) and have been widely
studied for their role in cognition, reinforcement, and motivation [
37
,
38
], while
dopaminergic populations in the substantia nigra pars compacta (SNc) project primarily to
the dorsal striatum (nigrostriatal pathway) and are critical for the selection and execution of
motor programs and habitual behavior [
39
,
40
]. Other populations outside the mesencephalon
include those in the dorsal raphe nucleus (DRN)/ventral periaqueductal gray area (vPAG)
that affect social behavior, nociception, and arousal [
41
–
43
] and tuberoinfundibular
projections from the hypothalamic arcuate nucleus to the median eminence that regulate
prolactin release [
44
].
Mesencephalic dopaminergic neurons in mice arise from a pool of progenitors in the
midbrain floor plate under the control of numerous signaling molecules, including sonic
hedgehog, WNT1, engrailed 1 and 2, fibroblast growth factor-8, etc., undergo radial
migration to their final positions in either the VTA or SNc by embryonic day 13.5, and
exhibit extensive axonal outgrowth along the anteroposterior and dorsoventral axes with
synaptogenesis in downstream targets continuing into postnatal development [
45
].
Consequently loss of NDD-associated genes, such as
EN2
,
MECP2
,
CNTNAP2
, and
NF1
,
produce hypo- or hyperdopaminergic behavioral phenotypes, such as abnormal motor,
cognitive, or social behavior, in mouse models by perturbing neuronal maturation,
migration, or neurite outgrowth [
46
–
53
]. Efforts to understand these phenotypes would
benefit from a comprehensive ultrastructural understanding of how specific NDD-associated
genetic changes alter dopaminergic circuit architecture and function and inform new
therapeutic strategies, such as whole brain gene therapy or genome editing, to help
ameliorate NDD symptomatology.
Several recent viral vector-based labeling methods are likely to greatly enhance our
understanding of the input-output relationship between dopaminergic efferent and afferent
connections in NDD models (Table 2A). This toolkit includes a new adenoassociated viral
(AAV) vector for retrograde labeling (AAV2-retro) [
26
] (in addition to existing retrograde
labeling vectors [
24
,
25
,
27
]), intersectional strategies to target individual neuronal
projections and their inputs (INTERSECT [
5
], TRIO [
6
]), mGRASP for fluorescent labeling
of connections between synaptic partners [
54
,
55
], and a single cell projection mapping via
RNA barcoding (MAP-seq [
56
]). The recently developed brain-penetrant AAV PHP.eB can
efficiently deliver viral transgenes to the CNS after peripheral administration (Figure 1A–B)
[
29
,
57
], including Brainbow reagents [
58
] for multicolor labeling via stochastic expression
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of fluorescent proteins (XFPs) [
57
] and genetically encoded calcium indicators (GECIs;
[
59
,
60
]). This tool should also prove useful for non-invasive delivery of optogenetic [
14
] or
chemogenetic [
15
] tools, AAV-optimized CRISPR-Cas9 effectors for genome editing (e.g.
[
61
–
63
]), and therapeutic transgenes across large brain volumes. Additionally, PHP.eB can
deliver the cargo of interest co-administered with a titratable inducer vector for controlled
sparseness while maintaining high viral transgene copy number [
57
], which is beneficial for
effective neurite tracing with methods such as mGRASP [
55
] or Brainbow [
58
] (Figure 1C–
D). This method is also likely to benefit sensors that need sparse expression to reduce
background fluorescence [
64
–
66
].
The utility of viral vector-based mapping tools has been improved by microscopic
techniques for rapid imaging of large samples, such as light sheet microscopy [
11
,
67
,
68
] or
high-speed volumetric serial two-photon (STP) tomography [
12
] (Table 2B), and tissue
clearing protocols that render biological samples optically transparent for analysis of intact
circuits in whole brains or thick slices (Figure 2) [
8
,
9
]. Several tissue clearing strategies have
been recently described or refined (Table 2C); these include immersion clearing with high
refractive index (RI) solutions (SeeDB [
69
], FRUIT [
70
], RIMS [
71
]), clearing via
hyperhydration (Sca/eS [
72
], CUBIC [
73
,
74
]), hydrogel embedding followed by detergent
delipidation (CLARITY [
75
,
76
], PARS [
77
,
78
], PACT [
77
,
78
]), and solvent-based clearing
methods (uDISCO [
79
]). Clearing methods that build upon water-absorbent CLARITY
hydrogels to create hyperabsorbant hydrogels have also been implemented to facilitate high
resolution imaging of small structures, such as individual dendrites or neurites (ExM [
80
–
82
], ePACT [
78
], and MAP [
83
]). Hydrogel-based methods preserve endogenous
fluorescence while maintaining compatibility with tools for proteomic analysis [
76
,
78
,
82
–
84
], RNA profiling (smFISH or smHCR probes [
71
,
81
,
85
,
86
]), and time-stamped
fluorescent readouts of neuronal activity (e.g. ArcTRAP [
87
,
88
]).
Several recent studies have successfully integrated these technologies to probe the structure
of dopaminergic and related circuits in healthy mice. For example, retrograde labeling, tissue
clearing, and LSM have been used to parse SNc subcircuit connectivity and function [
89
],
identify an anatomically distinct projection to the posterior striatum [
90
] that preferentially
encodes novel cue information rather than reward prediction errors [
91
], and refine our
knowledge of cholinergic inputs to the SNc and VTA [
92
]. An input-output analysis of VTA
connections using TRIO uncovered a novel projection from the anterior cingulate cortex to
the lateral NAc that produces behavioral reinforcement using an optogenetic intracranial
self-stimulation paradigm [
93
].
Bridging the gap between synaptic function and neural circuit dynamics in
NDD models
One common feature amongst NDDs is that the causative genes (e.g.
FMR1
in FXS,
UBE3A
in AS,
MECP2
in RS,
NF1
in NF1,
EN1
and
EN2
,
SHANK
genes, etc.) affect
synapse formation, maintenance, and plasticity in rodent models [
94
,
95
]. As such, there
have been considerable efforts to characterize synapse function in dopaminergic circuits in
these mice. For example, reduction in SHANK-3, an excitatory synapse scaffolding protein
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whose loss of function is associated with Phelan McDermid Syndrome (also called 22q13
deletion syndrome; see [
96
] for a review) and some non-syndromic ASD cases [
97
], via
delivery of a short hairpin RNA (shRNA) into the VTA impairs maturation of excitatory
synapses and reduces dopaminergic neuron excitability and social preference via increased
inhibitory tone [
98
]. Mice modeling 15q11–13 Duplication Syndrome (where Ube3A
protein levels are increased three-fold) exhibit a loss of sociability due to downregulation of
the glutamatergic synapse organizer CBLN1 in the VTA [
99
]. Altered neurotransmitter
content or release from VTA or SNc neurons in downstream targets has been reported in
mouse models of AS [
100
,
101
], NF1 [
46
,
47
], and RS [
52
,
102
] and in
Cntnap2
knockout
mice [
53
]. While there is a large body of research delineating the role of synaptic or
microcircuit deficits in NDD models [
103
–
106
], less is known about how those changes
alter population dynamics or neuron ensemble activity to produce behavioral phenotypes;
improvements in optical tools to monitor neural activity across multiple spatial scales
[
59
,
107
–
109
] should help bridge this divide.
Understanding how networks of interconnected neurons encode and translate relevant
environmental stimuli into a motivated behavior requires a high throughput readout of
neuron firing with single-cell resolution. Metal electrodes or electrode arrays are a robust
tool to measure spiking with high temporal precision and can be coupled with optogenetic
tools to manipulate activity or infer cell identity (i.e. opto-tagging, [
110
]). Opto-tagging has
been used to monitor diverse populations across the CNS (e.g. cortical interneurons
[
111
,
112
], AgRP neurons in the arcuate nucleus [
113
], dopaminergic neurons in the VTA
[
114
], etc.), yet this technique is limited in the number of neurons it can sample and may not
be suitable for all populations due to the challenges in efficiently and accurately opto-
tagging (e.g. cortical pyramidal neurons), as well as genetically similar populations that are
too sparse or dense to be reliably identified. In contrast, optogenetic stimulation of
dopaminergic circuitry during blood oxygen level dependent contrast (BOLD) fMRI
imaging can approximate mesocorticolimbic or nigrostriatal network activity in rodents
[
115
–
117
], yet this technique lacks both cellular resolution and temporally precise
neurophysiological readouts.
Alternatively, genetically encoded calcium indicators (GECIs; e.g. the GCaMP6 family of
proteins [
16
]) provide cell type-specific fluorescent readouts of neuron activity during
behavior that is stable over months of testing and is scalable [
21
]. Using two-photon
mesoscopes with wide field of view objectives [
108
] or random access scanning strategies
[
107
] to image through large cranial windows in head-fixed mice, researchers can record the
calcium dynamics of hundreds to thousands of neurons at once. Bulk calcium signals can
also be measured across superficial cortical areas using a wide field fluorescence
macroscope featuring a 12 mm field of view, which has been used to assess global
representations of motivated behavior in multiple cell types [
59
]. While these tools have
been optimized for relatively superficial (< 1mm deep) structures, several tools should help
extend the depth of non-invasive optical access, such as three-photon microscopy [
118
], the
implementation of axially elongated Bessel foci [
119
], photoacoustic tomography [
120
], and
guidestar-assisted wavefront engineering techniques to limit optical scattering [
121
], such as
time reversal of ultrasound encoded light (TRUE) [
122
,
123
].
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Several recent technologies have provided optical access to deep brain areas in behaving
mice for activity measurements in bulk or with single-cell resolution. For bulk
measurements, fiber photometry [
124
] and TEMPO [
125
] allow for quantification of
calcium or voltage sensor dynamics, respectively, using implanted optical fibers in order to
correlate activity of genetically defined populations with behavioral events. Calcium
imaging via implanted gradient index microendoscopes (GRIN lenses) provides single cell
resolution at depths >4mm below the skull surface [
126
]. While two-photon GRIN lens
imaging is most commonly performed in head-fixed mice [
127
], several strategies such as 2-
photon fiberscopes [
128
,
129
] and miniaturized head-mounted 2-photon microscopes
[
130
,
131
] have been developed for freely moving behavior. Head-mounted miniaturized
epifluorescence microscopes [
132
] are also available and have been more widely adopted for
use in behaving animals. Single cell calcium dynamics have been imaged via GRIN lens in
the VTA [
133
], SNc [
134
], and interconnected regions, including the dorsal striatum [
134
],
lateral hypothalamus [
134
,
135
], medial preoptic area [
136
], medial prefrontal cortex
[
137
,
138
], bed nucleus of the stria terminalis [
133
], hippocampus [
139
,
140
], etc.
Additionally, chronic imaging windows have permitted monitoring of sparsely labelled SNc
axons in the dorsal striatum, which revealed distinct temporal and spatial encoding of reward
and motor signals [
39
]. While several groups employ cortical two-photon calcium imaging
in NDD models, including RS [
141
] and FXS [
142
] mice, analysis of deeper structures has
not been reported to date.
Considerations and future outlook
The identification of causative genetic defects in neurodevelopmental syndromes and
subsequent creation of transgenic mouse models has greatly enhanced our understanding of
the developmental perturbations that produce synaptic, cellular, and behavioral phenotypes
in these mice. While several recent studies examining dopaminergic circuitry have
uncovered pathophysiological mechanisms underlying aberrant social interactions, positive
reinforcement, stereotyped behavior, etc., few studies have employed new technologies for
functional circuit mapping in NDD models. This may be due to several factors; first, given
that phenotype expression is dependent on genetic background in many mouse models, such
as NF1 [
143
], it will be important to continue identifying and developing minimal gene
regulatory elements (promoters, enhancers, miRNA binding sites) that can be
accommodated within well-tolerated viral capsids for cell type-specific targeting without the
need to cross mice to Cre or Flp driver lines. Several cell type-specific promoters have been
developed to target different cell populations in the CNS, including catecholaminergic
(tyrosine hydroxylase promoter), serotonergic (FEV), Purkinje (PCP2) [
144
], and forebrain
GABAergic (mDlx5/6) neurons [
145
], although they vary in leakiness and promoter size,
which can limit packageable transgene size due to AAV carrying capacity of 4.7 kb [
146
].
Second, many of these techniques require specialized equipment, reagents, or expertise that
makes implementation challenging. Several helpful imaging, tissue clearing, and data
analysis protocols have recently been published [
54
,
76
,
78
,
133
,
147
–
149
] that can help guide
potential users.
To effectively integrate measures of neural activity with comprehensive dopaminergic
connectomes in mouse models obtained with such tools for precise structural and functional
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analysis of intact circuits, several advances will be required. First, we will need better
computational methods for automated detection, segmentation, and tracing of individual
fluorescently labelled neurons in whole cleared brains. This task is currently labor intensive
and works poorly for neurons with complex morphology, such as catecholaminergic neurons
with large axonal arbors that traverse several mm of brain tissue. Recent successes in
overcoming these challenges include the reconstruction of single projection neurons in the
claustrum, which branch extensively throughout the entire cerebrum [
150
]. Second, we will
need improved tools for converting neural activity states into fluorescent labels that can be
superimposed upon neuronal reconstructions. Several technologies show promise, such as
CaMPARI [
151
] and iTANGO [
152
], which provide light timestamped indicators of
intracellular calcium or dopaminergic neurotransmission, respectively. Hybridization chain
reaction (HCR) probes for single-cell, multiplexed RNA detection have been validated for
hydrogel-based clearing methods [
86
,
153
] and could allow for medium throughput
identification of projection- or activity-dependent changes in gene activity in mutant and
wildtype mice. At this time, only PACT/PARS [
71
], EDC-CLARITY [
86
], and Ex-FISH
[
81
] have been demonstrated to be compatible with RNA profiling, yet clearing methods are
advancing rapidly and will likely be useful for a broader range of applications in the future.
When examining the role of functional circuit mapping technologies in elucidating
dopaminergic connectivity in NDD models, one cannot neglect the ontogeny of these
circuits. Several methods have been used to clear mouse embryos at various stages of
development (reviewed by [
154
]), yet it is difficult to employ viral vector-based tracing and
labeling techniques in the developing mouse. It is thus of great interest to identify AAVs that
cross the blood-placenta barrier and selectively target the developing embryonic nervous
system. AAV selection platforms, such as CREATE (Cre recombination–based AAV
targeted evolution), which has been used to develop vectors that efficiently target the central
(PHP.B/PHP.eB) or peripheral (PHP.S) nervous systems [
29
,
57
] when given systemically,
could yield new vectors for
in utero
transgene delivery. Additionally, tools for large volume
functional imaging of developing organisms, such as a two-beam light sheet microscope
with adaptive optics and automated cell tracking [
11
], have been applied to early embryonic
mice [
155
,
156
], yet new methods to maintain optical access within the amnion will be
necessary to image and track post-implantation fetal cells.
Going forward, we anticipate that continued technological advances can yield progressively
more precise and comprehensive functional and connectomic maps of dopaminergic
circuitry across development. As these tools become more widely adopted by NDD
researchers, we will likely gain newfound understanding of how functional and structural
abnormalities synergize to produce behavioral and cognitive phenotypes in mouse models
and reveal putative mechanisms of disease symptomatology in human populations.
Ultimately these discoveries can inform the creation of behavioral and pharmacological
therapies that target circuit- or cell-type specific mechanisms of disease in order to benefit
the health of affected children and adults.
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Acknowledgments
We would like to acknowledge support from the Children’s Tumor Foundation (Young Investigator Award
2016-01-006 to JER), as well as the Heritage Medical Research Institute (VG) and the Tianqiao and Chrissy Chen
Institute for Neuroscience at Caltech. We would like to thank Jennifer Treweek, Benjamin Deverman, Ken Chan,
Min Jang, Alon Greenbaum, and Ryan Cho for histological images used in the manuscript figures.
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