Probes for INS

Background Selective serotonin reuptake inhibitors such as fluoxetine have a limited treatment efficacy. The mechanism by which some patients respond to fluoxetine while others do not remains poorly understood, limiting treatment effectiveness. We have found the opioid system to be involved in the responsiveness to fluoxetine treatment in a mouse model for anxiety- and depressive-like behavior. Methods We analyzed gene expression changes in the dentate gyrus of mice chronically treated with corticosterone and fluoxetine. After identifying a subset of genes of interest, we studied their expression patterns in relation to treatment responsiveness. We further characterized their expression through in situ hybridization and the analysis of a single-cell RNA-Seq data set. Finally, we behaviorally tested mu and delta opioid receptor knockout mice in the Novelty Suppressed Feeding test and the Forced Swim Test after chronic corticosterone and fluoxetine treatment. Results Chronic fluoxetine treatment upregulates proenkephalin expression in the dentate gyrus, and this upregulation is associated with treatment responsiveness. The expression of several of the most significantly upregulated genes, including proenkephalin, is localized to an anatomically and transcriptionally specialized subgroup of mature granule cells in the dentate gyrus. We have also found that the delta opioid receptor contributes to some, but not all, of the behavioral effects of fluoxetine. Conclusions These data indicate that the opioid system is involved in the antidepressant effects of fluoxetine, and this effect may be mediated through the upregulation of proenkephalin in a subpopulation of mature granule cells.

The parabrachial nucleus (PBN) integrates interoceptive and exteroceptive information to control various behavioral and physiological processes including breathing, emotion, and sleep/wake regulation through the neural circuits that connect to the forebrain and the brainstem. However, the precise identity and function of distinct PBN subpopulations are still largely unknown. Here, we leveraged molecular characterization, retrograde tracing, optogenetics, chemogenetics, and electrocortical recording approaches to identify a small subpopulation of neurotensin-expressing neurons in the PBN that largely project to the emotional control regions in the forebrain, rather than the medulla. Their activation induces freezing and anxiety-like behaviors, which in turn result in tachypnea. In addition, optogenetic and chemogenetic manipulations of these neurons revealed their function in promoting wakefulness and maintaining sleep architecture. We propose that these neurons comprise a PBN subpopulation with specific gene expression, connectivity, and function, which play essential roles in behavioral and physiological regulation.

Green light exposure has been shown to reduce pain in animal models. Here, we report a vision-associated enkephalinergic neural circuit responsible for green light-mediated analgesia. Full-field green light exposure at an intensity of 10 lux produced analgesic effects in healthy mice and in a model of arthrosis. Ablation of cone photoreceptors completely inhibited the analgesic effect, whereas rod ablation only partially reduced pain relief. The analgesic effect was not modulated by the ablation of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are atypical photoreceptors that control various nonvisual effects of light. Inhibition of the retino-ventrolateral geniculate nucleus (vLGN) pathway completely abolished the analgesic effects. Activation of this pathway reduced nociceptive behavioral responses; such activation was blocked by the inhibition of proenkephalin (Penk)-positive neurons in the vLGN (vLGNPenk). Moreover, green light analgesia was prevented by knockdown of Penk in the vLGN or by ablation of vLGNPenk neurons. In addition, activation of the projections from vLGNPenk neurons to the dorsal raphe nucleus (DRN) was sufficient to suppress nociceptive behaviors, whereas its inhibition abolished the green light analgesia. Our findings indicate that cone-dominated retinal inputs mediated green light analgesia through the vLGNPenk-DRN pathway and suggest that this signaling pathway could be exploited for reducing pain.

IntroductionExact measurement of renal function is essential for the treatment of patients. Elevated serum-creatinine levels, while established are influenced by other parameters and show a significant time-lag. This drives the search for novel biomarkers of renal function and injury. Beside Lipocalin-2 and kidney-injury-molecule-1(KIM-1), the endogenous opioid precursor proenkephalin-A(Penk) has recently emerged as a promising marker for renal function. But the cellular origin and regulation of Penk outside the brain has not yet been investigated in depth.Materials and MethodsThis study characterizes the cellular origin of Penk expression with high resolution in-situ hybridization in two models of renal fibrosis in mice and human tissue.ResultsInterstitial cells are the main expression site for renal Penk. This classifies Penk as biomarker for interstitial damage as opposed to tubular damage markers like Lipocalin-2 and KIM-1. Furthermore, our data indicate that renal Penk expression is not regulated by classical profibrotic pathways.DiscussionThis study characterizes changing Penk expression in the kidneys. The similarity of Penk expression across species gives rise to further investigations into the function of Penk in healthy and injured kidneys.ConclusionPenk is a promising biomarker for interstitial renal damage that warrants further studies to utilize its predictive potential.

Opioid medications are the mainstay of pain management but present substantial side-effects such as respiratory depression which can be lethal with overdose. Most opioid drugs, such as fentanyl, act on opioid receptors such as the G-protein-coupled µ-opioid receptors (MOR). G-protein-coupled receptors activate pertussis toxin-sensitive G-proteins to inhibit neuronal activity. Binding of opioid ligands to MOR and subsequent activation G proteins βγ is modulated by regulator of G-protein signaling (RGS). The roles of G-proteins βγ and RGS in MOR-mediated inhibition of the respiratory network are not known. Using rodent models to pharmacologically modulate G-protein signaling, we aim to determine the roles of βγ G-proteins and RGS4. We showed that inhibition of βγ G-proteins using gallein perfused in the brainstem circuits regulating respiratory depression by opioid drugs results in complete reversal of respiratory depression. Blocking of RGS4 using CCG55014 did not change the respiratory depression induced by MOR activation despite co-expression of RGS4 and MORs in the brainstem. Our results suggest that neuronal inhibition by opioid drugs is mediated by G-proteins, but not by RGS4, which supports the concept that βγ G-proteins could be molecular targets to develop opioid overdose antidotes without the risks of re-narcotization often found with highly potent opioid drugs. On the other hand, RGS4 mediates opioid analgesia, but not respiratory depression, and RGS4 may be molecular targets to develop pain therapies without respiratory liability.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6% of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7% of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

Mechanical allodynia (MA) represents one prevalent symptom of chronic pain. Previously we and others have identified spinal and brain circuits that transmit or modulate the initial establishment of MA. However, brain-derived descending pathways that control the laterality and duration of MA are still poorly understood. Here we report that the contralateral brain-to-spinal circuits, from Oprm1 neurons in the lateral parabrachial nucleus (lPBNOprm1), via Pdyn neurons in the dorsal medial regions of hypothalamus (dmHPdyn), to the spinal dorsal horn (SDH), act to prevent nerve injury from inducing contralateral MA and reduce the duration of bilateral MA induced by capsaicin. Ablating/silencing dmH-projecting lPBNOprm1 neurons or SDH-projecting dmHPdyn neurons, deleting Dyn peptide from dmH, or blocking spinal κ-opioid receptors all led to long-lasting bilateral MA. Conversely, activation of dmHPdyn neurons or their axonal terminals in SDH can suppress sustained bilateral MA induced by lPBN lesion.

Opioids are potent analgesics broadly used for pain management; however, they can produce dangerous side effects including addiction and respiratory depression. These harmful effects have led to an epidemic of opioid abuse and overdose deaths, creating an urgent need for the development of both safer pain medications and treatments for opioid use disorders. Both the analgesic and addictive properties of opioids are mediated by the mu opioid receptor (MOR), making resolution of the cell types and neural circuits responsible for each of the effects of opioids a critical research goal. Single-cell RNA sequencing (scRNA-seq) technology is enabling the identification of MOR-expressing cell types throughout the nervous system, creating new opportunities for mapping distinct opioid effects onto newly discovered cell types. Here, we describe molecularly defined MOR-expressing neuronal cell types throughout the peripheral and central nervous systems and their potential contributions to opioid analgesia and addiction.