Dopaminergic dysfunction in neurodevelopmental disorders: recent advances and synergistic technologies to aid basic research

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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Author manuscript

Curr Opin Neurobiol

. Author manuscript; available in PMC 2019 February 01.

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 [

], the Brainbow

toolkit [

] and intersectional labelling strategies [

], improved neuroinformatic tools for

neurite tracing [

], tissue clearing [

], light sheet microscopy [

], and serial two-

photon tomography [

]. Additionally, optogenetic [

] and chemogenetic [

] actuators,

genetically encoded indicators of neuronal activity [

], and advanced

in vivo

imaging

modalities [

–

] 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 [

–

] or

broadly transduce neurons across the CNS [

] 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 [

]. While the neural substrates of

ASD symptomatology are multifaceted, mesencephalic dopamine systems, consisting of A8

retrorubral, A9 nigrostriatal, and A10 mesocorticolimbic projections [

], 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 [

]. 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 [

–

]. 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 [

], 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 [

]. 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 [

–

] and tuberoinfundibular

projections from the hypothalamic arcuate nucleus to the median eminence that regulate

prolactin release [

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 [

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 [

–

]. 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) [

] (in addition to existing retrograde

labeling vectors [

]), intersectional strategies to target individual neuronal

projections and their inputs (INTERSECT [

], TRIO [

]), mGRASP for fluorescent labeling

of connections between synaptic partners [

], and a single cell projection mapping via

RNA barcoding (MAP-seq [

]). The recently developed brain-penetrant AAV PHP.eB can

efficiently deliver viral transgenes to the CNS after peripheral administration (Figure 1A–B)

[

], including Brainbow reagents [

] for multicolor labeling via stochastic expression

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of fluorescent proteins (XFPs) [

] and genetically encoded calcium indicators (GECIs;

[

]). This tool should also prove useful for non-invasive delivery of optogenetic [

] or

chemogenetic [

] tools, AAV-optimized CRISPR-Cas9 effectors for genome editing (e.g.

[

–

]), 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 [

], which is beneficial for

effective neurite tracing with methods such as mGRASP [

] or Brainbow [

] (Figure 1C–

D). This method is also likely to benefit sensors that need sparse expression to reduce

background fluorescence [

–

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 [

] or

high-speed volumetric serial two-photon (STP) tomography [

] (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) [

]. Several tissue clearing strategies have

been recently described or refined (Table 2C); these include immersion clearing with high

refractive index (RI) solutions (SeeDB [

], FRUIT [

], RIMS [

]), clearing via

hyperhydration (Sca/eS [

], CUBIC [

]), hydrogel embedding followed by detergent

delipidation (CLARITY [

], PARS [

], PACT [

]), and solvent-based clearing

methods (uDISCO [

]). 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 [

–

], ePACT [

], and MAP [

]). Hydrogel-based methods preserve endogenous

fluorescence while maintaining compatibility with tools for proteomic analysis [

–

], RNA profiling (smFISH or smHCR probes [

]), and time-stamped

fluorescent readouts of neuronal activity (e.g. ArcTRAP [

]).

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 [

identify an anatomically distinct projection to the posterior striatum [

] that preferentially

encodes novel cue information rather than reward prediction errors [

], and refine our

knowledge of cholinergic inputs to the SNc and VTA [

]. 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 [

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 [

]. 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 [

] for a review) and some non-syndromic ASD cases [

], 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 [

]. 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 [

]. Altered neurotransmitter

content or release from VTA or SNc neurons in downstream targets has been reported in

mouse models of AS [

100

101

], NF1 [

], and RS [

102

] and in

Cntnap2

knockout

mice [

]. 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

[

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 [

]) provide cell type-specific fluorescent readouts of neuron activity during

behavior that is stable over months of testing and is scalable [

]. 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 [

]. 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 [

]. 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 [

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 [

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 [

], EDC-CLARITY [

], and Ex-FISH

[

] 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 [

] 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 [

], 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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