Atezolizumab plus Magrolimab, Niraparib, or Tocilizumab versus Atezolizumab Monotherapy in Platinum-Refractory Metastatic Urothelial Carcinoma: A Phase Ib/II Open-Label, Multicenter, Randomized Umbrella Study (MORPHEUS Urothelial Carcinoma).

PURPOSE: The MORPHEUS platform was designed to identify early efficacy signals and evaluate the safety of novel immunotherapy combinations across cancer types. The phase Ib/II MORPHEUS-UC trial (NCT03869190) is evaluating atezolizumab plus magrolimab, niraparib, or tocilizumab in platinum-refractory locally advanced or metastatic urothelial carcinoma (mUC). Additional treatment combinations were evaluated and will be reported separately. PATIENTS AND METHODS: Patients had locally advanced or mUC that progressed during or following treatment with a platinum-containing regimen. The primary efficacy endpoint was investigator-assessed objective response rate (ORR). Key secondary endpoints included investigator-assessed progression-free survival (PFS) and overall survival (OS). Safety and exploratory biomarker analyses were also conducted. RESULTS: Seventy-six patients were randomized to receive either atezolizumab plus magrolimab (n = 16), atezolizumab plus niraparib (n = 15), atezolizumab plus tocilizumab (n = 15), or atezolizumab monotherapy (control; n = 30). No additive benefit in ORR, PFS, or OS was seen in the treatment arms versus the control. The best confirmed ORR was 26.7% with atezolizumab plus magrolimab, 6.7% with atezolizumab plus niraparib, 20.0% with atezolizumab plus tocilizumab, and 27.6% with atezolizumab monotherapy. Overall, the treatment combinations were tolerable, and adverse events were consistent with each agent's known safety profile. Trends were observed for shrinkage of programmed death-ligand 1-positive tumors (atezolizumab, atezolizumab plus magrolimab, atezolizumab plus tocilizumab), inflamed tumors, or tumors with high mutational burden (atezolizumab), and immune excluded tumors (atezolizumab plus magrolimab). CONCLUSIONS: The evaluated regimens in MORPHEUS-UC were tolerable. However, response rates for the combinations did not meet the criteria for further development in platinum-experienced locally advanced or mUC.

Amygdala-Midbrain Connections Modulate Appetitive and Aversive Learning.

The central amygdala (CeA) orchestrates adaptive responses to emotional events. While CeA substrates for defensive behaviors have been studied extensively, CeA circuits for appetitive behaviors and their relationship to threat-responsive circuits remain poorly defined. Here, we demonstrate that the CeA sends robust inhibitory projections to the lateral substantia nigra (SNL) that contribute to appetitive and aversive learning in mice. CeA→SNL neural responses to appetitive and aversive stimuli were modulated by expectation and magnitude consistent with a population-level salience signal, which was required for Pavlovian conditioned reward-seeking and defensive behaviors. CeA→SNL terminal activation elicited reinforcement when linked to voluntary actions but failed to support Pavlovian associations that rely on incentive value signals. Consistent with a disinhibitory mechanism, CeA inputs preferentially target SNL GABA neurons, and CeA→SNL and SNL dopamine neurons respond similarly to salient stimuli. Collectively, our results suggest that amygdala-nigra interactions represent a previously unappreciated mechanism for influencing emotional behaviors.

Distinct neural mechanisms for the prosocial and rewarding properties of MDMA.

The extensively abused recreational drug (±)3,4-methylenedioxymethamphetamine (MDMA) has shown promise as an adjunct to psychotherapy for treatment-resistant psychiatric disease. It is unknown, however, whether the mechanisms underlying its prosocial therapeutic effects and abuse potential are distinct. We modeled both the prosocial and nonsocial drug reward of MDMA in mice and investigated the mechanism of these processes using brain region-specific pharmacology, transgenic manipulations, electrophysiology, and in vivo calcium imaging. We demonstrate in mice that MDMA acting at the serotonin transporter within the nucleus accumbens is necessary and sufficient for MDMA's prosocial effect. MDMA's acute rewarding properties, in contrast, require dopaminergic signaling. MDMA's prosocial effect requires 5-HT1b receptor activation and is mimicked by d-fenfluramine, a selective serotonin-releasing compound. By dissociating the mechanisms of MDMA's prosocial effects from its addictive properties, we provide evidence for a conserved neuronal pathway, which can be leveraged to develop novel therapeutics with limited abuse liability.

Learning what to approach.

Most decisions share a common goal: maximize reward and minimize punishment. Achieving this goal requires learning which choices are likely to lead to favorable outcomes. Dopamine is essential for this process, enabling learning by signaling the difference between what we expect to get and what we actually get. Although all animals appear to use this dopamine prediction error circuit, some do so more than others, and this neural heterogeneity correlates with individual variability in behavior. In this issue of PLOS Biology, Lee and colleagues show that manipulating a simple task parameter can bias the animals' behavioral strategy and modulate dopamine release, implying that how we learn is just as flexible as what we learn.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping.

Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors.

Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.

Ventral tegmental area (VTA) dopamine (DA) neurons have been implicated in reward, aversion, salience, cognition, and several neuropsychiatric disorders. Optogenetic approaches involving transgenic Cre-driver mouse lines provide powerful tools for dissecting DA-specific functions. However, the emerging complexity of VTA circuits requires Cre-driver mouse lines that restrict transgene expression to a precisely defined cell population. Because of recent work reporting that VTA DA neurons projecting to the lateral habenula release GABA, but not DA, we performed an extensive anatomical, molecular, and functional characterization of prominent DA transgenic mouse driver lines. We find that transgenes under control of the tyrosine hydroxylase, but not the dopamine transporter, promoter exhibit dramatic non-DA cell-specific expression patterns within and around VTA nuclei. Our results demonstrate how Cre expression in unintentionally targeted cells in transgenic mouse lines can confound the interpretation of supposedly cell-type-specific experiments. This Matters Arising paper is in response to Stamatakis et al. (2013), published in Neuron. See also the Matters Arising Response paper by Stuber et al. (2015), published concurrently with this Matters Arising in Neuron.

Illuminating circuitry relevant to psychiatric disorders with optogenetics.

The brain's remarkable capacity to generate cognition and behavior is mediated by an extraordinarily complex set of neural interactions that remain largely mysterious. This complexity poses a significant challenge in developing therapeutic interventions to ameliorate psychiatric disease. Accordingly, few new classes of drugs have been made available for patients with mental illness since the 1950s. Optogenetics offers the ability to selectively manipulate individual neural circuit elements that underlie disease-relevant behaviors and is currently accelerating the pace of preclinical research into neurobiological mechanisms of disease. In this review, we highlight recent findings from studies that employ optogenetic approaches to gain insight into normal and aberrant brain function relevant to mental illness. Emerging data from these efforts offers an exquisitely detailed picture of disease-relevant neural circuits in action, and hints at the potential of optogenetics to open up entirely new avenues in the treatment of psychiatric disorders.

Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens.

The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.

A causal link between prediction errors, dopamine neurons and learning.

Situations in which rewards are unexpectedly obtained or withheld represent opportunities for new learning. Often, this learning includes identifying cues that predict reward availability. Unexpected rewards strongly activate midbrain dopamine neurons. This phasic signal is proposed to support learning about antecedent cues by signaling discrepancies between actual and expected outcomes, termed a reward prediction error. However, it is unknown whether dopamine neuron prediction error signaling and cue-reward learning are causally linked. To test this hypothesis, we manipulated dopamine neuron activity in rats in two behavioral procedures, associative blocking and extinction, that illustrate the essential function of prediction errors in learning. We observed that optogenetic activation of dopamine neurons concurrent with reward delivery, mimicking a prediction error, was sufficient to cause long-lasting increases in cue-elicited reward-seeking behavior. Our findings establish a causal role for temporally precise dopamine neuron signaling in cue-reward learning, bridging a critical gap between experimental evidence and influential theoretical frameworks.

Establishing causality for dopamine in neural function and behavior with optogenetics.

Dopamine (DA) is known to play essential roles in neural function and behavior. Accordingly, DA neurons have been the focus of intense experimental investigation that has led to many important advances in our understanding of how DA influences these processes. However, it is becoming increasingly appreciated that delineating the precise contributions of DA neurons to cellular, circuit, and systems-level phenomena will require more sophisticated control over their patterns of activity than conventional techniques can provide. Specifically, the roles played by DA neurons are likely to depend on their afferent and efferent connectivity, the timing and length of their neural activation, and the nature of the behavior under investigation. Recently developed optogenetic tools hold great promise for disentangling these complex issues. Here we discuss the use of light-sensitive microbial opsins in the context of outstanding questions in DA research. A major technical advance offered by these proteins is the ability to bidirectionally modulate DA neuron activity in in vitro and in vivo preparations on a time scale that more closely approximates those of neural, perceptual and behavioral events. In addition, continued advances in rodent genetics and viral-mediated gene delivery have contributed to the ability to selectively target DA neurons or their individual afferent and efferent connections. Further, these tools are suitable for use in experimental subjects engaged in complex behaviors. After reviewing the strengths and limitations of optogenetic methodologies, we conclude by describing early efforts in the application of this valuable new approach that demonstrate its potential to improve our understanding of the neural and behavioral functions of DA. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement.

Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.

Perceptual learning directs auditory cortical map reorganization through top-down influences.

The primary sensory cortex is positioned at a confluence of bottom-up dedicated sensory inputs and top-down inputs related to higher-order sensory features, attentional state, and behavioral reinforcement. We tested whether topographic map plasticity in the adult primary auditory cortex and a secondary auditory area, the suprarhinal auditory field, was controlled by the statistics of bottom-up sensory inputs or by top-down task-dependent influences. Rats were trained to attend to independent parameters, either frequency or intensity, within an identical set of auditory stimuli, allowing us to vary task demands while holding the bottom-up sensory inputs constant. We observed a clear double-dissociation in map plasticity in both cortical fields. Rats trained to attend to frequency cues exhibited an expanded representation of the target frequency range within the tonotopic map but no change in sound intensity encoding compared with controls. Rats trained to attend to intensity cues expressed an increased proportion of nonmonotonic intensity response profiles preferentially tuned to the target intensity range but no change in tonotopic map organization relative to controls. The degree of topographic map plasticity within the task-relevant stimulus dimension was correlated with the degree of perceptual learning for rats in both tasks. These data suggest that enduring receptive field plasticity in the adult auditory cortex may be shaped by task-specific top-down inputs that interact with bottom-up sensory inputs and reinforcement-based neuromodulator release. Top-down inputs might confer the selectivity necessary to modify a single feature representation without affecting other spatially organized feature representations embedded within the same neural circuitry.