Prefrontal cortex output circuits guide reward seeking through divergent cue encoding.

The prefrontal cortex is a critical neuroanatomical hub for controlling motivated behaviours across mammalian species. In addition to intra-cortical connectivity, prefrontal projection neurons innervate subcortical structures that contribute to reward-seeking behaviours, such as the ventral striatum and midline thalamus. While connectivity among these structures contributes to appetitive behaviours, how projection-specific prefrontal neurons encode reward-relevant information to guide reward seeking is unknown. Here we use in vivo two-photon calcium imaging to monitor the activity of dorsomedial prefrontal neurons in mice during an appetitive Pavlovian conditioning task. At the population level, these neurons display diverse activity patterns during the presentation of reward-predictive cues. However, recordings from prefrontal neurons with resolved projection targets reveal that individual corticostriatal neurons show response tuning to reward-predictive cues, such that excitatory cue responses are amplified across learning. By contrast, corticothalamic neurons gradually develop new, primarily inhibitory responses to reward-predictive cues across learning. Furthermore, bidirectional optogenetic manipulation of these neurons reveals that stimulation of corticostriatal neurons promotes conditioned reward-seeking behaviour after learning, while activity in corticothalamic neurons suppresses both the acquisition and expression of conditioned reward seeking. These data show how prefrontal circuitry can dynamically control reward-seeking behaviour through the opposing activities of projection-specific cell populations.

Dorsal Raphe Dopamine Neurons Signal Motivational Salience Dependent on Internal State, Expectation, and Behavioral Context.

The ability to recognize motivationally salient events and adaptively respond to them is critical for survival. Here, we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process in both male and female mice. Population recordings of DRNDA neurons during associative learning tasks showed that their activity dynamically tracks the motivational salience, developing excitation to both reward-paired and shock-paired cues. The DRNDA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRNDA activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRNDA neurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues and reward but not to shock-predicting cues, which was surprising and qualitatively distinct from the population results. Performing the same fear learning procedures in freely-moving and head-fixed groups revealed that head-fixation itself abolished the neural response to aversive cues, indicating its modulation by behavioral context. Overall, these results suggest that DRNDA neurons encode motivational salience, dependent on internal and external factors.SIGNIFICANCE STATEMENT Dopamine (DA) contributes to motivational control, composed of at least two functional cell types, one signaling for motivational value and another for motivational salience. Here, we demonstrate that DA neurons in the dorsal raphe nucleus (DRN) encode the motivational salience in associative learning tasks. Neural responses were dynamic and modulated by the animal's internal state. The majority of single-cells developed responses to reward or paired cues, but not to shock-predicting cues. Additional experiments with freely-moving and head-fixed mice showed that head-fixation abolished the development of cue responses during fear learning. This work provides further characterization on the functional roles of overlooked DRNDA populations and an example that neural responses can be altered by head-fixation, which is commonly used in neuroscience.

Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics.

We engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of fiber-optic cables to produce light-dependent activation of a small volume of tissue. To facilitate expansive optogenetics without the need for invasive implants, our engineering approach leverages the substantial literature of ChR variants to train statistical models for the design of high-performance ChRs. With Gaussian process models trained on a limited experimental set of 102 functionally characterized ChRs, we designed high-photocurrent ChRs with high light sensitivity. Three of these, ChRger1-3, enable optogenetic activation of the nervous system via systemic transgene delivery. ChRger2 enables light-induced neuronal excitation without fiber-optic implantation; that is, this opsin enables transcranial optogenetics.

The G protein-biased κ-opioid receptor agonist RB-64 is analgesic with a unique spectrum of activities in vivo.

The hypothesis that functionally selective G protein-coupled receptor (GPCR) agonists may have enhanced therapeutic benefits has revitalized interest for many GPCR targets. In particular, although κ-opioid receptor (KOR) agonists are analgesic with a low risk of dependence and abuse, their use is limited by a propensity to induce sedation, motor incoordination, hallucinations, and dysphoria-like states. Several laboratories have produced a body of work suggesting that G protein-biased KOR agonists might be analgesic with fewer side effects. Although that has been an intriguing hypothesis, suitable KOR-selective and G protein-biased agonists have not been available to test this idea. Here we provide data using a G protein-biased agonist, RB-64 (22-thiocyanatosalvinorin A), which suggests that KOR-mediated G protein signaling induces analgesia and aversion, whereas ß-arrestin-2 signaling may be associated with motor incoordination. Additionally, unlike unbiased KOR agonists, the G protein-biased ligand RB-64 does not induce sedation and does not have anhedonia-like actions, suggesting that a mechanism other than G protein signaling mediates these effects. Our findings provide the first evidence for a highly selective and G protein-biased tool compound for which many, but not all, of the negative side effects of KOR agonists can be minimized by creating G protein-biased KOR agonists.

Different contributions of dopamine D1 and D2 receptor activity to alcohol potentiation of brain stimulation reward in C57BL/6J and DBA/2J mice.

C57BL/6J (C57) and DBA/2J (DBA) mice respond differently to drugs that affect dopamine systems, including alcohol. The current study compared effects of D1 and D2 receptor agonists and antagonists, and the interaction between D1/D2 antagonists and alcohol, on intracranial self-stimulation in male C57 and DBA mice to determine the role of dopamine receptors in the effects of alcohol on brain stimulation reward (BSR). In the initial strain comparison, dose effects on BSR thresholds and maximum operant response rates were determined for the D1 receptor agonist SKF-82958 (±-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 0.1-0.56 mg/kg) and antagonist SCH 23390 (+-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrochloride; 0.003-0.056 mg/kg), and the D2 receptor agonist quinpirole (0.1-3.0 mg/kg) and antagonist raclopride (0.01-0.56 mg/kg). For the alcohol interaction, SCH 23390 (0.003 mg/kg) or raclopride (0.03 mg/kg) was given before alcohol (0.6-2.4 g/kg p.o.). D1 antagonism dose-dependently elevated and SKF-82958 dose-dependently lowered BSR threshold in both strains; DBA mice were more sensitive to SKF-82958 effects. D2 antagonism dose-dependently elevated BSR threshold only in C57 mice. Low doses of quinpirole elevated BSR threshold equally in both strains, whereas higher doses of quinpirole lowered BSR threshold only in C57 mice. SCH 23390, but not raclopride, prevented lowering of BSR threshold by alcohol in DBA mice. Conversely, raclopride, but not SCH 23390, prevented alcohol potentiation of BSR in C57 mice. These results extend C57 and DBA strain differences to D1/D2 sensitivity of BSR, and suggest differential involvement of D1 and D2 receptors in the acute rewarding effects of alcohol in these two mouse strains.

Levetiracetam results in increased and decreased alcohol drinking with different access procedures in C57BL/6J mice.

The antiepileptic levetiracetam (LEV) has been investigated for the treatment of alcohol abuse. However, little is known about how LEV alters the behavioral effects of alcohol in laboratory animals. The acute effects of LEV on alcohol drinking by male C57BL/6J mice were investigated using two different drinking procedures, limited access [drinking-in-the-dark (DID)] and intermittent access (IA) drinking. In the first experiment (DID), mice had access to a single bottle containing alcohol or sucrose for 4 h every other day. In the second experiment (IA), mice had IA to two bottles, one containing alcohol or sucrose and one containing water, for 24 h on Monday, Wednesday, and Friday. In both experiments, mice were administered LEV (0.3-100 mg/kg intraperitoneally) or vehicle 30 min before access to the drinking solutions. In the DID mice, LEV increased alcohol intake from 4.3 to 5.4 g/kg, whereas in the IA mice LEV decreased alcohol intake from 4.8 to 3.0 g/kg in the first 4 h of access and decreased 24 h alcohol intake from 20 to ∼15 g/kg. These effects appear specific to alcohol, as LEV did not affect sucrose intake in either experiment. LEV appears to differentially affect drinking in animal models of moderate and heavier alcohol consumption.

Adult microglial TGFß1 is required for microglia homeostasis via an autocrine mechanism to maintain cognitive function in mice.

While TGF-ß signaling is essential for microglial function, the cellular source of TGF-ß1 ligand and its spatial regulation remains unclear in the adult CNS. Our data supports that microglia but not astrocytes or neurons are the primary producers of TGF-ß1 ligands needed for microglial homeostasis. Microglia-Tgfb1 KO leads to the activation of microglia featuring a dyshomeostatic transcriptome that resembles disease-associated, injury-associated, and aged microglia, suggesting microglial self-produced TGF-ß1 ligands are important in the adult CNS. Astrocytes in MG-Tgfb1 inducible (i)KO mice show a transcriptome profile that is closely aligned with an LPS-associated astrocyte profile. Additionally, using sparse mosaic single-cell microglia KO of TGF-ß1 ligand we established an autocrine mechanism for signaling. Here we show that MG-Tgfb1 iKO mice present cognitive deficits, supporting that precise spatial regulation of TGF-ß1 ligand derived from microglia is required for the maintenance of brain homeostasis and normal cognitive function in the adult brain.

Ventral striatum dopamine release encodes unique properties of visual stimuli in mice.

The mesolimbic dopamine system is an evolutionarily conserved set of brain circuits that play a role in attention, appetitive behavior, and reward processing. In this circuitry, ascending dopaminergic projections from the ventral midbrain innervate targets throughout the limbic forebrain, such as the ventral striatum/nucleus accumbens (NAc). Dopaminergic signaling in the NAc has been widely studied for its role in behavioral reinforcement, reward prediction error encoding, and motivational salience. Less well characterized is the role of dopaminergic neurotransmission in the response to surprising or alerting sensory events. To address this, we used the genetically encoded dopamine sensor dLight1 and fiber photometry to explore the ability of striatal dopamine release to encode the properties of salient sensory stimuli in mice, such as threatening looming discs. Here, we report that lateral NAc (LNAc) dopamine release encodes the rate and magnitude of environmental luminance changes rather than the visual stimulus threat level. This encoding is highly sensitive, as LNAc dopamine could be evoked by light intensities that were imperceptible to human experimenters. We also found that light-evoked dopamine responses are wavelength-dependent at low irradiances, independent of the circadian cycle, robust to previous exposure history, and involve multiple phototransduction pathways. Thus, we have further elaborated the mesolimbic dopamine system's ability to encode visual information in mice, which is likely relevant to a wide body of scientists employing light sources or optical methods in behavioral research involving rodents.

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.

Prepronociceptin-Expressing Neurons in the Extended Amygdala Encode and Promote Rapid Arousal Responses to Motivationally Salient Stimuli.

Motivational states consist of cognitive, emotional, and physiological components controlled by multiple brain regions. An integral component of this neural circuitry is the bed nucleus of the stria terminalis (BNST). Here, we identify that neurons within BNST that express the gene prepronociceptin (PnocBNST) modulate rapid changes in physiological arousal that occur upon exposure to motivationally salient stimuli. Using in vivo two-photon calcium imaging, we find that PnocBNST neuronal responses directly correspond with rapid increases in pupillary size when mice are exposed to aversive and rewarding odors. Furthermore, optogenetic activation of these neurons increases pupillary size and anxiety-like behaviors but does not induce approach, avoidance, or locomotion. These findings suggest that excitatory responses in PnocBNST neurons encode rapid arousal responses that modulate anxiety states. Further histological, electrophysiological, and single-cell RNA sequencing data reveal that PnocBNST neurons are composed of genetically and anatomically identifiable subpopulations that may differentially tune rapid arousal responses to motivational stimuli.

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 and 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 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.

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

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.

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.

Deep tissue optical focusing and optogenetic modulation with time-reversed ultrasonically encoded light.

Noninvasive light focusing deep inside living biological tissue has long been a goal in biomedical optics. However, the optical scattering of biological tissue prevents conventional optical systems from tightly focusing visible light beyond several hundred micrometers. The recently developed wavefront shaping technique time-reversed ultrasonically encoded (TRUE) focusing enables noninvasive light delivery to targeted locations beyond the optical diffusion limit. However, until now, TRUE focusing has only been demonstrated inside nonliving tissue samples. We present the first example of TRUE focusing in 2-mm-thick living brain tissue and demonstrate its application for optogenetic modulation of neural activity in 800-µm-thick acute mouse brain slices at a wavelength of 532 nm. We found that TRUE focusing enabled precise control of neuron firing and increased the spatial resolution of neuronal excitation fourfold when compared to conventional lens focusing. This work is an important step in the application of TRUE focusing for practical biomedical uses.

Hormonal gain control of a medial preoptic area social reward circuit.

Neural networks that control reproduction must integrate social and hormonal signals, tune motivation, and coordinate social interactions. However, the neural circuit mechanisms for these processes remain unresolved. The medial preoptic area (mPOA), an essential node for social behaviors, comprises molecularly diverse neurons with widespread projections. Here we identify a steroid-responsive subset of neurotensin (Nts)-expressing mPOA neurons that interface with the ventral tegmental area (VTA) to form a socially engaged reward circuit. Using in vivo two-photon imaging in female mice, we show that mPOANts neurons preferentially encode attractive male cues compared to nonsocial appetitive stimuli. Ovarian hormone signals regulate both the physiological and cue-encoding properties of these cells. Furthermore, optogenetic stimulation of mPOANts-VTA circuitry promotes rewarding phenotypes, social approach and striatal dopamine release. Collectively, these data demonstrate that steroid-sensitive mPOA neurons encode ethologically relevant stimuli and co-opt midbrain reward circuits to promote prosocial behaviors critical for species survival.

Stroke-Like Presentation Following Febrile Seizure in a Patient with 1q43q44 Deletion Syndrome.

Hemiconvulsion-hemiplegia-epilepsy syndrome (HHE) is a rare outcome of prolonged hemiconvulsion that is followed by diffuse unilateral hemispheric edema, hemiplegia, and ultimately hemiatrophy of the affected hemisphere and epilepsy. Here, we describe the case of a 3-year-old male with a 1;3 translocation leading to a terminal 1q43q44 deletion and a terminal 3p26.1p26.3 duplication that developed HHE after a prolonged febrile seizure and discuss the pathogenesis of HHE in the context of the patient's complex genetic background.

A pharmacogenetic determinant of mu-opioid receptor antagonist effects on alcohol reward and consumption: evidence from humanized mice.

BACKGROUND: It has been proposed that therapeutic responses to naltrexone in alcoholism are moderated by variation at the mu-opioid receptor gene locus (OPRM1). This remains controversial because human results vary and no prospectively genotyped studies have been reported. We generated humanized mice carrying the respective human OPRM1 A118G alleles. Here, we used this model system to examine the role of OPRM1 A118G variation for opioid antagonist effects on alcohol responses. METHODS: Effects of naltrexone on alcohol reward were examined using intracranial self-stimulation. Effects of naltrexone or nalmefene on alcohol intake were examined in continuous access home cage two-bottle free-choice drinking and operant alcohol self-administration paradigms. RESULTS: Alcohol lowered brain stimulation reward thresholds in 118GG mice in a manner characteristic of rewarding drugs, and this effect was blocked by naltrexone. Brain stimulation reward thresholds were unchanged by alcohol or naltrexone in 118AA mice. In the home cage, increased alcohol intake emerged in 118GG mice with increasing alcohol concentrations and was 33% higher at 17% alcohol. At this concentration, naltrexone selectively suppressed alcohol intake in 118GG animals to a level virtually identical to that of 118AA mice. No effect of naltrexone was found in the latter group. Similarly, both naltrexone and nalmefene were more effective in suppressing operant alcohol self-administration in 118GG mice. CONCLUSIONS: In a model that allows close experimental control, OPRM1 A118G variation robustly moderates effects of opioid antagonism on alcohol reward and consumption. These findings strongly support a personalized medicine approach to alcoholism treatment that takes into account OPRM1 genotype.

Receptor Reserve Moderates Mesolimbic Responses to Opioids in a Humanized Mouse Model of the OPRM1 A118G Polymorphism.

The OPRM1 A118G polymorphism is the most widely studied µ-opioid receptor (MOR) variant. Although its involvement in acute alcohol effects is well characterized, less is known about the extent to which it alters responses to opioids. Prior work has shown that both electrophysiological and analgesic responses to morphine but not to fentanyl are moderated by OPRM1 A118G variation, but the mechanism behind this dissociation is not known. Here we found that humanized mice carrying the 118GG allele (h/mOPRM1-118GG) were less sensitive than h/mOPRM1-118AA littermates to the rewarding effects of morphine and hydrocodone but not those of other opioids measured with intracranial self-stimulation. Reduced morphine reward in 118GG mice was associated with decreased dopamine release in the nucleus accumbens and reduced effects on GABA release in the ventral tegmental area that were not due to changes in drug potency or efficacy in vitro or receptor-binding affinity. Fewer MOR-binding sites were observed in h/mOPRM1-118GG mice, and pharmacological reduction of MOR availability unmasked genotypic differences in fentanyl sensitivity. These findings suggest that the OPRM1 A118G polymorphism decreases sensitivity to low-potency agonists by decreasing receptor reserve without significantly altering receptor function.

A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior.

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq-coupled M3-DREADD within the same neuronal population facilitated the sequential and bidirectional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators.

Effects of the neuroactive steroid allopregnanolone on intracranial self-stimulation in C57BL/6J mice.

RATIONALE: The neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP, allopregnanolone) has effects on reward-related behaviors in mice and rats that suggest that it may activate brain reward circuits. Intracranial self-stimulation (ICSS) is an operant behavioral technique that detects changes in the sensitivity of brain reward circuitry following drug administration. OBJECTIVE: To examine the effects of the neuroactive steroid allopregnanolone on ICSS and to compare these effects to those of cocaine. METHODS: Male C57BL/6J mice implanted with stimulating electrodes implanted into the medial forebrain bundle responded for reinforcement by electrical stimulation (brain stimulation reward (BSR)). Mice received cocaine (n = 11, 3.0-30.0 mg/kg, intraperitoneal (i.p.)) or the neuroactive steroid allopregnanolone (n = 11, 3.0-17.0 mg/kg, i.p.). BSR thresholds (θ 0) and maximum (MAX) operant response rates after drug treatments were compared to those after vehicle injections. RESULTS: Cocaine and allopregnanolone dose dependently lowered BSR thresholds relative to vehicle injections. Cocaine was maximally effective (80 % reduction) in the second 15 min following the 30 mg/kg dose, while allopregnanolone was maximally effective (30 % reduction) 15-45 min after the 17 mg/kg dose. Neither drug had significant effects on MAX response rates. CONCLUSIONS: The effects of allopregnanolone on BSR thresholds are consistent with the previously reported effects of benzodiazepines and alcohol, suggesting that positive modulation of GABAA receptors can facilitate reward-related behaviors in C57BL/6J mice.