Probes for INS

Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information.

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.

Background The central amygdala (CeA) is a bilateral hub of pain and emotional processing with well-established functional lateralization. We reported that optogenetic manipulation of neural activity in the left and right CeA has opposing effects on bladder pain. Methods To determine the influence of calcitonin gene-related peptide (CGRP) signaling from the parabrachial nucleus (PBN) on this diametrically opposed lateralization, we administered CGRP and evaluated the activity of CeA neurons in acute brain slices as well as the behavioral signs of bladder pain in the mouse. Results We found that CGRP increased firing in both the right and left CeA neurons. Furthermore, we found that CGRP administration in the right CeA increased behavioral signs of bladder pain and decreased bladder pain-like behavior when administered in the left CeA. Conclusions These studies reveal a parabrachial-to-amygdala circuit driven by opposing actions of CGRP that determines hemispheric lateralization of visceral pain.

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.

Exposure to irregular lighting schedules leads to deficits in affective behaviors. The retino-recipient perihabenular nucleus (PHb) of the dorsal thalamus has been shown to mediate these effects in mice. However, the mechanisms of how light information is processed within the PHb remains unknown. Here, we show that the PHb contains a distinct cluster of GABAergic neurons that receive direct retinal input. These neurons are part of a larger inhibitory network composed of the thalamic reticular nucleus and zona incerta, known to modulate thalamocortical communication. In addition, PHbGABA neurons locally modulate excitatory-relay neurons, which project to limbic centers. Chronic exposure to irregular light-dark cycles alters photo-responsiveness and synaptic output of PHbGABA neurons, disrupting daily oscillations of genes associated with inhibitory and excitatory PHb signaling. Consequently, selective and chronic PHbGABA manipulation results in mood alterations that mimic those caused by irregular light exposure. Together, light-mediated disruption of PHb inhibitory networks underlies mood deficits.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

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.