Optical Imaging of Dopamine Dynamics and Decoding its Role in Arousal and Salience

Dopamine (DA) is a key neuromodulator in the brain that can exert a profound impact on brain physiology and cognitive functions. There is consensus that DA plays critical roles in reward prediction error, reinforcement learning, and motor control, and that dysregulation of DA signaling is common in many neuropsychiatric diseases, such as Parkinson’s disease, drug addiction, and depression. Although the tools to study the functional roles of DA have considerably expanded with novel genetic tools and optical imaging methods, we are still limited in our ability to record or visualize DA release in vivo with long-term stability and high spatiotemporal resolution. This is an unmet need in DA research, as DA release at the post-synaptic sites can be decoupled from DA cell body firing due to local circuit interaction and influence from other afferent activities. In parallel, there is growing evidence that DA is functionally heterogeneous beyond its classically described roles for reward and movement, based on its anatomical location, projection target, electrophysiological properties, and response patterns to stimuli with motivational valence. Pharmacological and genetic studies have provided indirect evidence that DA can promote strong behavioral arousal and signal salience, but the precise neural substrates for these functions remain largely unknown. Towards this end, my thesis work has been focused on 1) developing and characterizing optical tools to visualize DA release in vivo and 2) utilizing such optical and genetic tools to study the overlooked, sparse DA populations in the dorsal midbrain, demonstrating that they are functionally unique DA cells for broadcasting arousal and salience signals to the forebrain targets. As neuromodulatory systems exert profound influences on brain function, understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. Towards this goal, in Chapter II, my colleagues and I developed dLight1, an intensity-based genetically encoded DA indicator, to enable optical recording of DA dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging DA dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond DA transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous DA transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators. Together, this tool provides a unique opportunity to optically monitor DA release dynamics in vivo with long-term stability and unprecedented spatiotemporal resolution. In Chapter III, I have characterized the functional roles of sparse DA populations in the dorsal raphe nucleus (DRN) and discovered that these neurons play key roles in promoting behavioral arousal. I first demonstrated that DRNDA neurons are activated by diverse forms of motivationally salient stimuli, irrespective of valence. Simultaneous fiber photometry and polysomnographic recordings showed that DRNDA neuronal activity is correlated with distinct sleep-wake states, showing highest activities during wakefulness over sleep states. Optogenetic activation of DRNDA neurons was sufficient to cause immediate sleep-to-wake transitions and promote longer wakefulness upon sustained stimulation. On contrary, DRNDA inhibition via chemogenetics reduced wakefulness and promoted non-rapid eye movement sleep, even in the presence of ethologically relevant salient stimuli. Taken together, this pinpoints DRNDA neurons as the critical contributor of arousal-promoting DA system in the brain. In Chapter IV, I further characterized the encoding dynamics of DRNDA neurons during classical conditioning tasks where mice learned the association between neutral cues and outcomes with positive or negative outcomes. DRNDA neurons developed phasic, positive responses to cues predicting both positive and negative unconditioned stimuli across learning, suggesting that these populations track motivational salience rather than valence. In addition, DRNDA neurons encoded unsigned prediction error, demonstrating higher neuronal activity to unexpected reward or punishment over fully expected outcomes. Collectively with Chapter III, these results expand on the existing literature on functionally heterogeneous roles of DA in the brain and propose that DRNDA neurons play critical roles in signaling arousal and motivational salience to the forebrain regions to coordinate appropriate behavior, depending on the nature of environmental stimuli.</p

Dorsal Raphe Dopamine Neurons Modulate Arousal and Promote Wakefulness by Salient Stimuli

Ventral midbrain dopamine (DA) is unambiguously involved in motivation and behavioral arousal, yet the contributions of other DA populations to these processes are poorly understood. Here, we demonstrate that the dorsal raphe nucleus DA neurons are critical modulators of behavioral arousal and sleep-wake patterning. Using simultaneous fiber photometry and polysomnography, we observed time-delineated dorsal raphe nucleus dopaminergic (DRNDA) activity upon exposure to arousal-evoking salient cues, irrespective of their hedonic valence. We also observed broader fluctuations of DRNDA activity across sleep-wake cycles with highest activity during wakefulness. Both endogenous DRNDA activity and optogenetically driven DRNDA activity were associated with waking from sleep, with DA signal strength predictive of wake duration. Conversely, chemogenetic inhibition opposed wakefulness and promoted NREM sleep, even in the face of salient stimuli. Therefore, the DRNDA population is a critical contributor to wake-promoting pathways and is capable of modulating sleep-wake states according to the outside environment, wherein the perception of salient stimuli prompts vigilance and arousal

Cholinergic Mesopontine Signals Govern Locomotion and Reward through Dissociable Midbrain Pathways

The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms, we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons; however, although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders

Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose neurodevelopmental symptoms include impaired executive function, attention, and spatial learning that 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 stimulus-dependent, 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

Resection of individually identified high-rate high-frequency oscillations region is associated with favorable outcome in neocortical epilepsy

Objectives: High-frequency oscillations (HFOs) represent a novel electrophysiologic marker of endogenous epileptogenicity. Clinically, this propensity can be utilized to more accurately delineate the resection margin before epilepsy surgery. Currently, prospective application of HFOs is limited because of a lack of an exact quantitative measure to reliably identify HFO-generating areas necessary to include in the resection. Here, we evaluated the potential of a patient-individualized approach of identifying high-rate HFO regions to plan the neocortical resection. Methods: Fifteen patients with neocortical seizure-onset zones (SOZs) underwent intracranial electroencephalographic monitoring. To identify interictal HFOs, we applied an automated, hypersensitive HFO-detection algorithm followed by post hoc processing steps to reject false detections. The spatial relationship between HFO distribution and the SOZ was evaluated. To address high interpatient variability in HFO properties, we evaluated the high-rate HFO region, an unbiased statistical parameter, in each patient. The relationship between resection of the high-rate HFO region and postoperative outcome was examined. Results: Grouped data demonstrated that the rate of ripple (60–200 Hz) and fast ripple (200–500 Hz) was increased in the SOZ (both p < 0.01). Intrapatient analysis of the HFO distribution localized the SOZ in 11 patients. High-rate HFO regions were determined in all patients by an individually adjusted threshold. Resection of high-rate HFO regions was significantly associated with a seizure-free outcome (p < 0.01). The extent/ratio of SOZ or spiking region resection did not differ between seizure-free and seizure-persistent groups. Significance: Intrapatient analysis of high-rate HFOs provides more detailed description of HFO-generating areas and can mark the areas of clinically significant epileptogenicity—a crucial component of the neocortical epileptic network that should be removed to achieve a good outcome. Validating and adopting an unbiased quantitative HFO parameter has the potential to propel wider and prospective utilization of HFOs in the surgical treatment of neocortical epilepsy and to improve its outcome

Light-guided sectioning for precise in situ localization and tissue interface analysis for brain-implanted optical fibers and GRIN lenses

Optical implants to control and monitor neuronal activity in vivo have become foundational tools of neuroscience. Standard two-dimensional histology of the implant location, however, often suffers from distortion and loss during tissue processing. To address that, we developed a three-dimensional post hoc histology method called “light-guided sectioning” (LiGS), which preserves the tissue with its optical implant in place and allows staining and clearing of a volume up to 500 μm in depth. We demonstrate the use of LiGS to determine the precise location of an optical fiber relative to a deep brain target and to investigate the implant-tissue interface. We show accurate cell registration of ex vivo histology with single-cell, two-photon calcium imaging, obtained through gradient refractive index (GRIN) lenses, and identify subpopulations based on immunohistochemistry. LiGS provides spatial information in experimental paradigms that use optical fibers and GRIN lenses and could help increase reproducibility through identification of fiber-to-target localization and molecular profiling

Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose neurodevelopmental symptoms include impaired executive function, attention, and spatial learning that 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 stimulus-dependent, 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

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators