A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Ahnak scaffolds p11/Anxa2 complex and L-type voltage-gated calcium channel and modulates depressive behavior.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the ß subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

Wireless optoelectronic photometers for monitoring neuronal dynamics in the deep brain.

Capabilities for recording neural activity in behaving mammals have greatly expanded our understanding of brain function. Some of the most sophisticated approaches use light delivered by an implanted fiber-optic cable to optically excite genetically encoded calcium indicators and to record the resulting changes in fluorescence. Physical constraints induced by the cables and the bulk, size, and weight of the associated fixtures complicate studies on natural behaviors, including social interactions and movements in environments that include obstacles, housings, and other complex features. Here, we introduce a wireless, injectable fluorescence photometer that integrates a miniaturized light source and a photodetector on a flexible, needle-shaped polymer support, suitable for injection into the deep brain at sites of interest. The ultrathin geometry and compliant mechanics of these probes allow minimally invasive implantation and stable chronic operation. In vivo studies in freely moving animals demonstrate that this technology allows high-fidelity recording of calcium fluorescence in the deep brain, with measurement characteristics that match or exceed those associated with fiber photometry systems. The resulting capabilities in optical recordings of neuronal dynamics in untethered, freely moving animals have potential for widespread applications in neuroscience research.

Stretchable multichannel antennas in soft wireless optoelectronic implants for optogenetics.

Optogenetic methods to modulate cells and signaling pathways via targeted expression and activation of light-sensitive proteins have greatly accelerated the process of mapping complex neural circuits and defining their roles in physiological and pathological contexts. Recently demonstrated technologies based on injectable, microscale inorganic light-emitting diodes (µ-ILEDs) with wireless control and power delivery strategies offer important functionality in such experiments, by eliminating the external tethers associated with traditional fiber optic approaches. Existing wireless µ-ILED embodiments allow, however, illumination only at a single targeted region of the brain with a single optical wavelength and over spatial ranges of operation that are constrained by the radio frequency power transmission hardware. Here we report stretchable, multiresonance antennas and battery-free schemes for multichannel wireless operation of independently addressable, multicolor µ-ILEDs with fully implantable, miniaturized platforms. This advance, as demonstrated through in vitro and in vivo studies using thin, mechanically soft systems that separately control as many as three different µ-ILEDs, relies on specially designed stretchable antennas in which parallel capacitive coupling circuits yield several independent, well-separated operating frequencies, as verified through experimental and modeling results. When used in combination with active motion-tracking antenna arrays, these devices enable multichannel optogenetic research on complex behavioral responses in groups of animals over large areas at low levels of radio frequency power (<1 W). Studies of the regions of the brain that are involved in sleep arousal (locus coeruleus) and preference/aversion (nucleus accumbens) demonstrate the unique capabilities of these technologies.

A GABAergic Projection from the Centromedial Nuclei of the Amygdala to Ventromedial Prefrontal Cortex Modulates Reward Behavior.

The neural circuitry underlying mammalian reward behaviors involves several distinct nuclei throughout the brain. It is widely accepted that the midbrain dopamine (DA) neurons are critical for the reward-related behaviors. Recent studies have shown that the centromedial nucleus of the amygdala (CeMA) has a distinct role in regulating reward-related behaviors. However, the CeMA and ventromedial PFC (vmPFC) interaction in reward regulation remains poorly understood. Here, we identify and dissect a GABAergic projection that originates in the CeMA and terminates in the vmPFC (VGat-CreCeMA-vmPFC) using viral-vector-mediated, cell-type-specific optogenetic techniques in mice. Pathway-specific optogenetic activation of the VGat-CreCeMA-vmPFC circuit in awake, behaving animals produced a positive, reward-like phenotype in real-time place preference and increased locomotor activity in open-field testing. In sucrose operant conditioning, the photoactivation of these terminals increased nose-poking effort with no effect on licking behavior and robustly facilitated the extinction of operant behavior. However, photoactivation of these terminals did not induce self-stimulation in the absence of an external reward. The results described here suggest that the VGat-CreCeMA-vmPFC projection acts to modulate existing reward-related behaviors. SIGNIFICANCE STATEMENT: Many studies have shown that the interactions between the centromedial nucleus of the amygdala (CeMA) and ventromedial PFC (vmPFC) have critical roles for emotional regulation. However, most studies have associated this circuit with fear and anxiety behaviors and emphasized top-down processing from vmPFC to CeMA. Here, we provide new evidence for bottom-up CeMA to vmPFC influence on reward-related behaviors. Although previous work implicated the CeMA in incentive salience, our results isolate the investigation to a specific CeMA GABAergic projection to the vmPFC. This long-range GABAergic interaction between amygdala and frontal cortex adds a new dimension to the complex regulation of reward-related behaviors.

Ahnak in the prefrontal cortex mediates behavioral correlates of stress resilience and rapid antidepressant action in mice.

The prefrontal cortex (PFC) is a key neural node mediating behavioral responses to stress and the actions of ketamine, a fast-acting antidepressant. The molecular mechanisms underlying these processes, however, are not fully understood. Our recent study revealed a pivotal role of hippocampal Ahnak as a regulator of cellular and behavioral adaptations to chronic stress. However, despite its significant expression in the PFC, the contribution of cortical Ahnak to behavioral responses to stress and antidepressants remains unknown. Here, using a mouse model for chronic social stress, we find that Ahnak expression in the PFC is significantly increased in stress-resilient mice and positively correlated with social interaction after stress exposure. Conditional deletion of Ahnak in the PFC or forebrain glutamatergic neurons facilitates stress susceptibility, suggesting that Ahnak is required for behavioral resilience. Further supporting this notion, Ahnak expression in the PFC is increased after the administration of ketamine or its metabolite (2R, 6R)-hydroxynorketamine (HNK). Moreover, Ahnak deletion in forebrain glutamatergic neurons blocks the restorative behavioral effects of ketamine or HNK in stress-susceptible mice. This forebrain excitatory neuron-specific Ahnak deletion reduces the frequency of mini excitatory postsynaptic currents in layer II/III pyramidal neurons, suggesting that Ahnak may induce its behavioral effects via modulation of glutamatergic transmission in the PFC. Altogether, these data suggest that Ahnak in glutamatergic PFC neurons may be critical for behavioral resilience and antidepressant actions of ketamine or HNK in chronic social stress-exposed mice.

Molecular and Cellular Adaptations in Hippocampal Parvalbumin Neurons Mediate Behavioral Responses to Chronic Social Stress.

Parvalbumin-expressing interneurons (PV neurons) maintain inhibitory control of local circuits implicated in behavioral responses to environmental stressors. However, the roles of molecular and cellular adaptations in PV neurons in stress susceptibility or resilience have not been clearly established. Here, we show behavioral outcomes of chronic social defeat stress (CSDS) are mediated by differential neuronal activity and gene expression in hippocampal PV neurons in mice. Using in vivo electrophysiology and chemogenetics, we find increased PV neuronal activity in the ventral dentate gyrus is required and sufficient for behavioral susceptibility to CSDS. PV neuron-selective translational profiling indicates mitochondrial oxidative phosphorylation is the most significantly altered pathway in stress-susceptible versus resilient mice. Among differentially expressed genes associated with stress-susceptibility and resilience, we find Ahnak, an endogenous regulator of L-type calcium channels which are implicated in the regulation of mitochondrial function and gene expression. Notably, Ahnak deletion in PV neurons impedes behavioral susceptibility to CSDS. Altogether, these findings indicate behavioral effects of chronic stress can be controlled by selective modulation of PV neuronal activity or a regulator of L-type calcium signaling in PV neurons.

Extended amygdala-parabrachial circuits alter threat assessment and regulate feeding.

An animal's evolutionary success depends on the ability to seek and consume foods while avoiding environmental threats. However, how evolutionarily conserved threat detection circuits modulate feeding is unknown. In mammals, feeding and threat assessment are strongly influenced by the parabrachial nucleus (PBN), a structure that responds to threats and inhibits feeding. Here, we report that the PBN receives dense inputs from two discrete neuronal populations in the bed nucleus of the stria terminalis (BNST), an extended amygdala structure that encodes affective information. Using a series of complementary approaches, we identify opposing BNST-PBN circuits that modulate neuropeptide-expressing PBN neurons to control feeding and affective states. These previously unrecognized neural circuits thus serve as potential nodes of neural circuitry critical for the integration of threat information with the intrinsic drive to feed.

Galanin administration into the prelimbic cortex impairs consolidation and expression of contextual fear conditioning.

The neuropeptide galanin is a potential therapeutic target for treating stress-related disorders, such as post-traumatic stress disorder (PTSD); however, its effects on contextual fear conditioning (CFC), an accepted animal model of PTSD, are not well understood. Dysregulation of the medial prefrontal cortex (mPFC) is implicated in PTSD. We investigated the effects of galanin (1 ug) administrated bilaterally into the prelimbic cortex, a division of the mPFC, on the consolidation, expression, and extinction of CFC of male Sprague-Dawley rats. Galanin administration significantly reduced consolidation and expression of CFC, but had no effect on retention or retrieval of extinction learning. These data further implicate galanin as a potential therapeutic target for treating stress-related disorders, particularly those characterized by aberrant emotional memory.

Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid System.

Negative affective states affect quality of life for patients suffering from pain. These maladaptive emotional states can lead to involuntary opioid overdose and many neuropsychiatric comorbidities. Uncovering the mechanisms responsible for pain-induced negative affect is critical in addressing these comorbid outcomes. The nucleus accumbens (NAc) shell, which integrates the aversive and rewarding valence of stimuli, exhibits plastic adaptations in the presence of pain. In discrete regions of the NAc, activation of the kappa opioid receptor (KOR) decreases the reinforcing properties of rewards and induces aversive behaviors. Using complementary techniques, we report that in vivo recruitment of NAc shell dynorphin neurons, acting through KOR, is necessary and sufficient to drive pain-induced negative affect. Taken together, our results provide evidence that pain-induced adaptations in the kappa opioid system within the NAc shell represent a functional target for therapeutic intervention that could circumvent pain-induced affective disorders. VIDEO ABSTRACT.

Molecular and Functional Sex Differences of Noradrenergic Neurons in the Mouse Locus Coeruleus.

Preclinical work has long focused on male animals, though biological sex clearly influences risk for certain diseases, including many psychiatric disorders. Such disorders are often treated by drugs targeting the CNS norepinephrine system. Despite roles for noradrenergic neurons in behavior and neuropsychiatric disease models, their molecular characterization has lagged. We profiled mouse noradrenergic neurons in vivo, defining over 3,000 high-confidence transcripts expressed therein, including druggable receptors. We uncovered remarkable sex differences in gene expression, including elevated expression of the EP3 receptor in females-which we leverage to illustrate the behavioral and pharmacologic relevance of these findings-and of Slc6a15 and Lin28b, both major depressive disorder (MDD)-associated genes. Broadly, we present a means of transcriptionally profiling locus coeruleus under baseline and experimental conditions. Our findings underscore the need for preclinical work to include both sexes and suggest that sex differences in noradrenergic neurons may underlie behavioral differences relevant to disease.

Locus coeruleus to basolateral amygdala noradrenergic projections promote anxiety-like behavior.

Increased tonic activity of locus coeruleus noradrenergic (LC-NE) neurons induces anxiety-like and aversive behavior. While some information is known about the afferent circuitry that endogenously drives this neural activity and behavior, the downstream receptors and anatomical projections that mediate these acute risk aversive behavioral states via the LC-NE system remain unresolved. Here we use a combination of retrograde tracing, fast-scan cyclic voltammetry, electrophysiology, and in vivo optogenetics with localized pharmacology to identify neural substrates downstream of increased tonic LC-NE activity in mice. We demonstrate that photostimulation of LC-NE fibers in the BLA evokes norepinephrine release in the basolateral amygdala (BLA), alters BLA neuronal activity, conditions aversion, and increases anxiety-like behavior. Additionally, we report that ß-adrenergic receptors mediate the anxiety-like phenotype of increased NE release in the BLA. These studies begin to illustrate how the complex efferent system of the LC-NE system selectively mediates behavior through distinct receptor and projection-selective mechanisms.

Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States.

Anxiety disorders are debilitating psychiatric illnesses with detrimental effects on human health. These heightened states of arousal are often in the absence of obvious threatening cues and are difficult to treat owing to a lack of understanding of the neural circuitry and cellular machinery mediating these conditions. Activation of noradrenergic circuitry in the basolateral amygdala is thought to have a role in stress, fear, and anxiety, and the specific cell and receptor types responsible is an active area of investigation. Here we take advantage of two novel cellular approaches to dissect the contributions of G-protein signaling in acute and social anxiety-like states. We used a chemogenetic approach utilizing the Gαs DREADD (rM3Ds) receptor and show that selective activation of generic Gαs signaling is sufficient to induce acute and social anxiety-like behavioral states in mice. Second, we use a recently characterized chimeric receptor composed of rhodopsin and the ß2-adrenergic receptor (Opto-ß2AR) with in vivo optogenetic techniques to selectively activate Gαs ß-adrenergic signaling exclusively within excitatory neurons of the basolateral amygdala. We found that optogenetic induction of ß-adrenergic signaling in the basolateral amygdala is sufficient to induce acute and social anxiety-like behavior. These findings support the conclusion that activation of Gαs signaling in the basolateral amygdala has a role in anxiety. These data also suggest that acute and social anxiety-like states may be mediated through signaling pathways identical to ß-adrenergic receptors, thus providing support that inhibition of this system may be an effective anxiolytic therapy.

Ethinyl estradiol and levonorgestrel alter cognition and anxiety in rats concurrent with a decrease in tyrosine hydroxylase expression in the locus coeruleus and brain-derived neurotrophic factor expression in the hippocampus.

In the United States, more than ten million women use contraceptive hormones. Ethinyl estradiol and levonorgestrel have been mainstay contraceptive hormones for the last four decades. Surprisingly, there is scant information regarding their action on the central nervous system and behavior. Intact female rats received three weeks of subcutaneous ethinyl estradiol (10 or 30µg/rat/day), levonorgestrel (20 or 60µg/rat/day), a combination of both (10/20µg/rat/day and 30/60µg/rat/day), or vehicle. Subsequently, the rats were tested in three versions of the novel object recognition test to assess learning and memory, and a battery of tests for anxiety-like behavior. Serum estradiol and ovarian weights were measured. All treatment groups exhibited low endogenous 17ß-estradiol levels at the time of testing. Dose-dependent effects of drug treatment manifested in both cognitive and anxiety tests. All low dose drugs decreased anxiety-like behavior and impaired performance on novel object recognition. In contrast, the high dose ethinyl estradiol increased anxiety-like behavior and improved performance in cognitive testing. In the cell molecular analyses, low doses of all drugs induced a decrease in tyrosine hydroxylase mRNA and protein in the locus coeruleus. At the same time, low doses of ethinyl estradiol and ethinyl estradiol/levonorgestrel increased galanin protein in this structure. Consistent with the findings above, the low dose treatments of ethinyl estradiol and combination ethinyl estradiol/levonorgestrel reduced brain-derived neurotrophic factor mRNA in the hippocampus. These effects of ethinyl estradiol 10µg alone and in combination with levonorgestrel 20µg suggest a diminution of norepinephrine input into the hippocampus resulting in a decline in learning and memory.