Selective Activation of Subthalamic Nucleus Output Quantitatively Scales Movements.

The subthalamic nucleus (STN) is a common target for deep brain stimulation (DBS) treatments of Parkinsonian motor symptoms. According to the dominant model, the STN output can suppress movement by enhancing inhibitory basal ganglia (BG) output via the indirect pathway, and disrupting STN output using DBS can restore movement in Parkinson's patients. But the mechanisms underlying STN DBS remain poorly understood, as previous studies usually relied on electrical stimulation, which cannot selectively target STN output neurons. Here, we selectively stimulated STN projection neurons using optogenetics and quantified behavior in male and female mice using 3D motion capture. STN stimulation resulted in movements with short latencies (10-15 ms). A single pulse of light was sufficient to generate movement, and there was a highly linear relationship between stimulation frequency and kinematic measures. Unilateral stimulation caused movement in the ipsiversive direction (toward the side of stimulation) and quantitatively determined head yaw and head roll, while stimulation of either STN raises the head (pitch). Bilateral stimulation does not cause turning but raised the head twice as high as unilateral stimulation of either STN. Optogenetic stimulation increased the firing rate of STN neurons in a frequency-dependent manner, and the increased firing is responsible for stimulation-induced movements. Finally, stimulation of the STN's projection to the brainstem mesencephalic locomotor region was sufficient to reproduce the behavioral effects of STN stimulation. These results question the common assumption that the STN suppresses movement, and instead suggest that STN output can precisely specify action parameters via direct projections to the brainstem.SIGNIFICANCE STATEMENT Our results question the common assumption that the subthalamic nucleus (STN) suppresses movement, and instead suggest that STN output can precisely specify action parameters via direct projections to the brainstem.

Hypothalamic-Extended Amygdala Circuit Regulates Temporal Discounting.

Choice behavior is characterized by temporal discounting, i.e., preference for immediate rewards given a choice between immediate and delayed rewards. Agouti-related peptide (AgRP)-expressing neurons located in the arcuate nucleus of the hypothalamus (ARC) regulate food intake and energy homeostasis, yet whether AgRP neurons influence choice behavior and temporal discounting is unknown. Here, we demonstrate that motivational state potently modulates temporal discounting. Hungry mice (both male and female) strongly preferred immediate food rewards, yet sated mice were largely indifferent to reward delay. More importantly, selective optogenetic activation of AgRP-expressing neurons or their axon terminals within the posterior bed nucleus of stria terminalis (BNST) produced temporal discounting in sated mice. Furthermore, activation of neuropeptide Y (NPY) type 1 receptors (Y1Rs) within the BNST is sufficient to produce temporal discounting. These results demonstrate a profound influence of hypothalamic signaling on temporal discounting for food rewards and reveal a novel circuit that determine choice behavior.SIGNIFICANCE STATEMENT Temporal discounting is a universal phenomenon found in many species, yet the underlying neurocircuit mechanisms are still poorly understood. Our results revealed a novel neural pathway from agouti-related peptide (AgRP) neurons in the hypothalamus to the bed nucleus of stria terminalis (BNST) that regulates temporal discounting in decision-making.

Striatal mechanisms of turning behaviour following unilateral dopamine depletion in mice.

Unilateral dopamine (DA) depletion produces ipsiversive turning behaviour, and the injection of DA receptor agonists can produce contraversive turning, but the underlying mechanisms remain unclear. We conducted in vivo recording and pharmacological and optogenetic manipulations to study the role of DA and striatal output in turning behaviour. We used a video-based tracking programme while recording single unit activity in both putative medium spiny projection neurons (MSNs) and fast-spiking interneurons (FSIs) in the dorsal striatum bilaterally. Our results suggest that unilateral DA depletion reduced striatal output from the depleted side, resulting in asymmetric striatal output. Depletion systematically altered activity in both MSNs and FSIs, especially in neurons that increased firing during turning movements. Like D1 agonist SKF 38393, optogenetic stimulation in the depleted striatum increased striatal output and reversed biassed turning. These results suggest that relative striatal outputs from the two cerebral hemispheres determine the direction of turning: Mice turn away from the side of higher striatal output and towards the side of the lower striatal output.

ANK2 autism mutation targeting giant ankyrin-B promotes axon branching and ectopic connectivity.

Giant ankyrin-B (ankB) is a neurospecific alternatively spliced variant of ANK2, a high-confidence autism spectrum disorder (ASD) gene. We report that a mouse model for human ASD mutation of giant ankB exhibits increased axonal branching in cultured neurons with ectopic CNS axon connectivity, as well as with a transient increase in excitatory synapses during postnatal development. We elucidate a mechanism normally limiting axon branching, whereby giant ankB localizes to periodic axonal plasma membrane domains through L1 cell-adhesion molecule protein, where it couples microtubules to the plasma membrane and prevents microtubule entry into nascent axon branches. Giant ankB mutation or deficiency results in a dominantly inherited impairment in selected communicative and social behaviors combined with superior executive function. Thus, gain of axon branching due to giant ankB-deficiency/mutation is a candidate cellular mechanism to explain aberrant structural connectivity and penetrant behavioral consequences in mice as well as humans bearing ASD-related ANK2 mutations.

Mediodorsal Thalamus Contributes to the Timing of Instrumental Actions.

The perception of time is critical to adaptive behavior. While prefrontal cortex and basal ganglia have been implicated in interval timing in the seconds to minutes range, little is known about the role of the mediodorsal thalamus (MD), which is a key component of the limbic cortico-basal ganglia-thalamocortical loop. In this study, we tested the role of the MD in timing, using an operant temporal production task in male mice. In this task, that the expected timing of available rewards is indicated by lever pressing. Inactivation of the MD with muscimol produced rightward shifts in peak pressing on probe trials as well as increases in peak spread, thus significantly altering both temporal accuracy and precision. Optogenetic inhibition of glutamatergic projection neurons in the MD also resulted in similar changes in timing. The observed effects were found to be independent of significant changes in movement. Our findings suggest that the MD is a critical component of the neural circuit for interval timing, without playing a direct role in regulating ongoing performance.SIGNIFICANCE STATEMENT The mediodorsal nucleus (MD) of the thalamus is strongly connected with the prefrontal cortex and basal ganglia, areas which have been implicated in interval timing. Previous work has shown that the MD contributes to working memory and learning of action-outcome contingencies, but its role in behavioral timing is poorly understood. Using an operant temporal production task, we showed that inactivation of the MD significantly impaired timing behavior.

Postnatal TrkB ablation in corticolimbic interneurons induces social dominance in male mice.

The tight balance between synaptic excitation and inhibition (E/I) within neocortical circuits in the mammalian brain is important for complex behavior. Many loss-of-function studies have demonstrated that brain-derived neurotrophic factor (BDNF) and its cognate receptor tropomyosin receptor kinase B (TrkB) are essential for the development of inhibitory GABAergic neurons. However, behavioral consequences of impaired BDNF/TrkB signaling in GABAergic neurons remain unclear, largely due to confounding motor function deficits observed in previous animal models. In this study, we generated conditional knockout mice (TrkB cKO) in which TrkB was ablated from a majority of corticolimbic GABAergic interneurons postnatally. These mice showed intact motor coordination and movement, but exhibited enhanced dominance over other mice in a group-housed setting. In addition, immature fast-spiking GABAergic neurons of TrkB cKO mice resulted in an E/I imbalance in layer 5 microcircuits within the medial prefrontal cortex (mPFC), a key region regulating social dominance. Restoring the E/I imbalance via optogenetic modulation in the mPFC of TrkB cKO mice normalized their social dominance behavior. Taken together, our results provide strong evidence for a role of BDNF/TrkB signaling in inhibitory synaptic modulation and social dominance behavior in mice.

Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation.

Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.

Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling.

Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and ß-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of ß4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.

Human Umbilical Tissue-Derived Cells Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins.

Cell therapy demonstrates great potential for the treatment of neurological disorders. Human umbilical tissue-derived cells (hUTCs) were previously shown to have protective and regenerative effects in animal models of stroke and retinal degeneration, but the underlying therapeutic mechanisms are unknown. Because synaptic dysfunction, synapse loss, degeneration of neuronal processes, and neuronal death are hallmarks of neurological diseases and retinal degenerations, we tested whether hUTCs contribute to tissue repair and regeneration by stimulating synapse formation, neurite outgrowth, and neuronal survival. To do so, we used a purified rat retinal ganglion cell culture system and found that hUTCs secrete factors that strongly promote excitatory synaptic connectivity and enhance neuronal survival. Additionally, we demonstrated that hUTCs support neurite outgrowth under normal culture conditions and in the presence of the growth-inhibitory proteins chondroitin sulfate proteoglycan, myelin basic protein, or Nogo-A (reticulon 4). Furthermore, through biochemical fractionation and pharmacology, we identified the major hUTC-secreted synaptogenic factors as the thrombospondin family proteins (TSPs), TSP1, TSP2, and TSP4. Silencing TSP expression in hUTCs, using small RNA interference, eliminated both the synaptogenic function of these cells and their ability to promote neurite outgrowth. However, the majority of the prosurvival functions of hUTC-conditioned media was spared after TSP knockdown, indicating that hUTCs secrete additional neurotrophic factors. Together, our findings demonstrate that hUTCs affect multiple aspects of neuronal health and connectivity through secreted factors, and each of these paracrine effects may individually contribute to the therapeutic function of these cells. SIGNIFICANCE STATEMENT: Human umbilical tissue-derived cells (hUTC) are currently under clinical investigation for the treatment of geographic atrophy secondary to age-related macular degeneration. These cells show great promise for the treatment of neurological disorders; however, the therapeutic effects of these cells on CNS neurons are not fully understood. Here we provide compelling evidence that hUTCs secrete multiple factors that work synergistically to enhance synapse formation and function, and support neuronal growth and survival. Moreover, we identified thrombospondins (TSPs) as the hUTC-secreted factors that mediate the synaptogenic and growth-promoting functions of these cells. Our findings highlight novel paracrine effects of hUTC on CNS neuron health and connectivity and begin to unravel potential therapeutic mechanisms by which these cells elicit their effects.

Basal ganglia outputs map instantaneous position coordinates during behavior.

The basal ganglia (BG) are implicated in many movement disorders, yet how they contribute to movement remains unclear. Using wireless in vivo recording, we measured BG output from the substantia nigra pars reticulata (SNr) in mice while monitoring their movements with video tracking. The firing rate of most nigral neurons reflected Cartesian coordinates (either x- or y-coordinates) of the animal's head position during movement. The firing rates of SNr neurons are either positively or negatively correlated with the coordinates. Using an egocentric reference frame, four types of neurons can be classified: each type increases firing during movement in a particular direction (left, right, up, down), and decreases firing during movement in the opposite direction. Given the high correlation between the firing rate and the x and y components of the position vector, the movement trajectory can be reconstructed from neural activity. Our results therefore demonstrate a quantitative and continuous relationship between BG output and behavior. Thus, a steady BG output signal from the SNr (i.e., constant firing rate) is associated with the lack of overt movement, when a stable posture is maintained by structures downstream of the BG. Any change in SNr firing rate is associated with a change in position (i.e., movement). We hypothesize that the SNr output quantitatively determines the direction, velocity, and amplitude of voluntary movements. By changing the reference signals to downstream position control systems, the BG can produce transitions in body configurations and initiate actions.

Striatonigral control of movement velocity in mice.

The basal ganglia have long been implicated in action initiation. Using three-dimensional motion capture, we quantified the effects of optogenetic stimulation of the striatonigral (direct) pathway on movement kinematics. We generated transgenic mice with channelrhodopsin-2 expression in striatal neurons that express the D1-like dopamine receptor. With optic fibres placed in the sensorimotor striatum, an area known to contain movement velocity-related single units, photo-stimulation reliably produced movements that could be precisely quantified with our motion capture programme. A single light pulse was sufficient to elicit movements with short latencies (< 30 ms). Increasing stimulation frequency increased movement speed, with a highly linear relationship. These findings support the hypothesis that the sensorimotor striatum is part of a velocity controller that controls rate of change in body configurations.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Spotlight on movement disorders: What optogenetics has to offer.

Elucidating the neuronal mechanisms underlying movement disorders is a major challenge because of the intricacy of the relevant neural circuits, which are characterized by diverse cell types and complex connectivity. A major limitation of traditional techniques, such as electrical stimulation or lesions, is that individual elements of a neural circuit cannot be selectively manipulated. Moreover, available treatments are largely based on trial and error rather than a detailed understanding of the circuit mechanisms. Gaps in our knowledge of the circuit mechanisms for movement disorders, as well as mechanisms underlying known treatments such as deep brain stimulation, make it difficult to design new and improved treatment options. In this perspective, we discuss how optogenetics, which allows researchers to use light to manipulate neuronal activity, can contribute to the understanding and treatment of movement disorders. We outline the advantages and limitations of optogenetics and discuss examples of studies that have used this tool to clarify the role of the basal ganglia circuitry in movement.

Striatal firing rate reflects head movement velocity.

Although the basal ganglia have long been implicated in the initiation of actions, their contribution to movement remains a matter of dispute. Using wireless multi-electrode recording and motion tracking, we examined the relationship between single-unit activity in the sensorimotor striatum and movement kinematics. We recorded single-unit activity from medium spiny projection neurons and fast-spiking interneurons while monitoring the movements of mice using motion tracking. In Experiment 1, we trained mice to generate movements reliably by water-depriving them and giving them periodic cued sucrose rewards. We found high correlations between single-unit activity and movement velocity in particular directions. This correlation was found in both putative medium spiny projection neurons and fast-spiking interneurons. In Experiment 2, to rule out the possibility that the observed correlations were due to reward expectancy, we repeated the same procedure but added trials in which sucrose delivery was replaced by an aversive air puff stimulus. The air puff generated avoidance movements that were clearly different from movements on rewarded trials, but the same neurons that showed velocity correlation on reward trials exhibited a similar correlation on air puff trials. These experiments show for the first time that the firing rate of striatal neurons reflects movement velocity for different types of movements, whether to seek rewards or to avoid harm.

The role of pedunculopontine nucleus in choice behavior under risk.

The dopaminergic projections to the basal ganglia have long been implicated in reward-guided behavior and decision-making, yet little is known about the role of the posterior pedunculopontine nucleus (pPPN), a major source of excitatory input to the mesolimbic dopamine system. Here we studied the contributions of the pPPN to decision-making under risk, using excitoxic lesions and reversible inactivation in rats. Rats could choose between two options - a small but certain reward on one lever; or a large but uncertain reward on the other lever. The overall payoff associated with each choice is the same, but the reward variance (risk) associated with the risky choice is much higher. In Experiment 1, we showed that excitotoxic lesions of the pPPN before training did not affect acquisition of lever pressing. But whereas the controls strongly preferred the safe choice, the lesioned rats did not. In Experiment 2, we found that muscimol inactivation of the pPPN also produced similar effects, but reversibly. These results show that permanent lesions or reversible inactivation of the pPPN both abolish risk aversion in decision-making.

The role of the substantia nigra in posture control.

Disorders implicating the basal ganglia are often characterized by postural deficits, but little is known about the role of the basal ganglia in posture control. Using wireless multi-electrode recording, we measured single unit activity from GABAergic and dopaminergic neurons in the substantia nigra as unrestrained mice stood on an elevated platform while introducing continuous postural disturbances in the roll plane. We found two major types of neurons - those activated by tilt to the left side of the body and suppressed by tilt to the right side, and others activated by tilt to the right side and suppressed by tilt to the left side. Contrary to the prevailing view that the basal ganglia output from the substantia nigra pars reticulata either inhibits or disinhibits downstream structures in an all or none fashion, we showed that it continuously sends anti-phase signals to their downstream targets. We also demonstrated for the first time that nigrostriatal dopaminergic transmission is modulated by postural disturbances.

Region-specific impairments in striatal synaptic transmission and impaired instrumental learning in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation and impaired speech. Because patients with this disorder often exhibit motor tremor and stereotypical behaviors, which are associated with basal ganglia pathology, we hypothesized that AS is accompanied by abnormal functioning of the striatum, the input nucleus of the basal ganglia. Using mutant mice with maternal deficiency of AS E6-AP ubiquitin protein ligase Ube3a (Ube3a(m-/p+) ), we assessed the effects of Ube3a deficiency on instrumental conditioning, a striatum-dependent task. We used whole-cell patch-clamp recording to measure glutamatergic transmission in the dorsomedial striatum (DMS) and dorsolateral striatum (DLS). Ube3a(m-/p+) mice were severely impaired in initial acquisition of lever pressing. Whereas the lever pressing of wild-type controls was reduced by outcome devaluation and instrumental contingency reversal, the performance of Ube3a(m-/p+) mice were more habitual, impervious to changes in outcome value and action-outcome contingency. In the DMS, but not the DLS, Ube3a(m-/p+) mice showed reduced amplitude and frequency of miniature excitatory postsynaptic currents. These results show for the first time a selective deficit in instrumental conditioning in the Ube3a deficient mouse model, and suggest a specific impairment in glutmatergic transmission in the associative corticostriatal circuit in AS.

Mechanisms of action selection and timing in substantia nigra neurons.

The timing of actions is critical for adaptive behavior. In this study we measured neural activity in the substantia nigra as mice learned to change their action duration to earn food rewards. We observed dramatic changes in single unit activity during learning: both dopaminergic and GABAergic neurons changed their activity in relation to behavior to reflect the learned instrumental contingency and the action duration. We found the emergence of "action-on" neurons that increased firing for the duration of the lever press and mirror-image "action-off" neurons that paused at the same time. This pattern is especially common among GABAergic neurons. The activity of many neurons also reflected confidence about the just completed action and the prospect of reward. Being correlated with the relative duration of the completed action, their activity could predict the likelihood of reward collection. Compared with the GABAergic neurons, the activity of dopaminergic neurons was more commonly modulated by the discriminative stimulus signaling the start of each trial, suggesting that their phasic activity reflected sensory salience rather than any reward prediction error found in previous work. In short, these results suggest that (1) nigral activity is highly plastic and modified by the learning of the instrumental contingency; (2) GABAergic output from the substantia nigra can simultaneously inhibit and disinhibit downstream structures, while the dopaminergic output also provide bidirectional modulation of the corticostriatal circuits; (3) dopaminergic and GABAergic neurons show similar task-related activity, although DA neurons are more responsive to the trial start signal.

Cortical regulation of neurogenesis and cell proliferation in the ventral subventricular zone.

Neurogenesis and differentiation of neural stem cells (NSCs) are controlled by cell-intrinsic molecular pathways that interact with extrinsic signaling cues. In this study, we identify a circuit that regulates neurogenesis and cell proliferation in the lateral ventricle-subventricular zone (LV-SVZ). Our results demonstrate that direct glutamatergic projections from the anterior cingulate cortex (ACC), as well as inhibitory projections from calretinin+ local interneurons, modulate the activity of cholinergic neurons in the subependymal zone (subep-ChAT+). Furthermore, in vivo optogenetic stimulation and inhibition of the ACC-subep-ChAT+ circuit are sufficient to control neurogenesis in the ventral SVZ. Both subep-ChAT+ and local calretinin+ neurons play critical roles in regulating ventral SVZ neurogenesis and LV-SVZ cell proliferation.

A one-photon endoscope for simultaneous patterned optogenetic stimulation and calcium imaging in freely behaving mice.

Optogenetics and calcium imaging can be combined to simultaneously stimulate and record neural activity in vivo. However, this usually requires two-photon microscopes, which are not portable nor affordable. Here we report the design and implementation of a miniaturized one-photon endoscope for performing simultaneous optogenetic stimulation and calcium imaging. By integrating digital micromirrors, the endoscope makes it possible to activate any neuron of choice within the field of view, and to apply arbitrary spatiotemporal patterns of photostimulation while imaging calcium activity. We used the endoscope to image striatal neurons from either the direct pathway or the indirect pathway in freely moving mice while activating any chosen neuron in the field of view. The endoscope also allows for the selection of neurons based on their relationship with specific animal behaviour, and to recreate the behaviour by mimicking the natural neural activity with photostimulation. The miniaturized endoscope may facilitate the study of how neural activity gives rise to behaviour in freely moving animals.