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1.
bioRxiv ; 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38746090

ABSTRACT

The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive. Given the complexity of pain as a sensory and emotional experience, and the richness of ethological pain-related behaviors, we developed a standardized, deep-learning platform for deconstructing the behavior dynamics associated with the affective component of pain in mice-LUPE (Light aUtomated Pain Evaluator). LUPE removes human bias in behavior quantification and accelerated analysis from weeks to hours, which we leveraged to discover that morphine altered attentional and motivational pain behaviors akin to affective analgesia in humans. Through activity-dependent genetics and single-nuclei RNA sequencing, we identified specific ensembles of nociceptive cingulate neuron-types expressing mu-opioid receptors. Tuning receptor expression in these cells bidirectionally modulated morphine analgesia. Moreover, we employed a synthetic opioid receptor promoter-driven approach for cell-type specific optical and chemical genetic viral therapies to mimic morphine's pain-relieving effects in the cingulate, without reinforcement. This approach offers a novel strategy for precision pain management by targeting a key nociceptive cortical circuit with on-demand, non-addictive, and effective analgesia.

2.
bioRxiv ; 2024 Apr 21.
Article in English | MEDLINE | ID: mdl-38659775

ABSTRACT

Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), and Crh + neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNST Crh + cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in dBNST or specifically in dBNST Crh + neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

3.
Nat Commun ; 14(1): 5632, 2023 09 13.
Article in English | MEDLINE | ID: mdl-37704594

ABSTRACT

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.


Subject(s)
Analgesia , Chronic Pain , Induced Pluripotent Stem Cells , Animals , Humans , Mice , Rats , Macaca , Receptors, Opioid , Receptors, Opioid, mu/genetics , Transgenes
4.
Nat Commun ; 12(1): 3561, 2021 06 11.
Article in English | MEDLINE | ID: mdl-34117229

ABSTRACT

Active responses to stressors involve motor planning, execution, and feedback. Here we identify an insular cortex to BNST (insula→BNST) circuit recruited during restraint stress-induced active struggling that modulates affective behavior. We demonstrate that activity in this circuit tightly follows struggling behavioral events and that the size of the fluorescent sensor transient reports the duration of the struggle event, an effect that fades with repeated exposure to the homotypic stressor. Struggle events are associated with enhanced glutamatergic- and decreased GABAergic signaling in the insular cortex, indicating the involvement of a larger circuit. We delineate the afferent network for this pathway, identifying substantial input from motor- and premotor cortex, somatosensory cortex, and the amygdala. To begin to dissect these incoming signals, we examine the motor cortex input, and show that the cells projecting from motor regions to insular cortex are engaged shortly before struggle event onset. This study thus demonstrates a role for the insula→BNST pathway in monitoring struggling activity and regulating affective behavior.


Subject(s)
Avoidance Learning , Behavior, Animal , Cerebral Cortex/physiology , Amygdala , Animals , Brain , Cerebral Cortex/diagnostic imaging , Female , Mice , Mice, Inbred C57BL , Neurons , Somatosensory Cortex
5.
J Neurosci ; 40(20): 3949-3968, 2020 05 13.
Article in English | MEDLINE | ID: mdl-32277042

ABSTRACT

Excitatory signaling mediated by NMDARs has been shown to regulate mood disorders. However, current treatments targeting NMDAR subtypes have shown limited success in treating patients, highlighting a need for alternative therapeutic targets. Here, we identify a role for GluN2D-containing NMDARs in modulating emotional behaviors and neural activity in the bed nucleus of the stria terminalis (BNST). Using a GluN2D KO mouse line (GluN2D-/-), we assessed behavioral phenotypes across tasks modeling emotional behavior. We then used a combination of ex vivo electrophysiology and in vivo fiber photometry to assess changes in BNST plasticity, cell-specific physiology, and cellular activity profiles. GluN2D-/- male mice exhibit evidence of exacerbated negative emotional behavior, and a deficit in BNST synaptic potentiation. We also found that GluN2D is functionally expressed on corticotropin-releasing factor (CRF)-positive BNST cells implicated in driving negative emotional states, and recordings in mice of both sexes revealed increased excitatory and reduced inhibitory drive onto GluN2D-/- BNST-CRF cells ex vivo and increased activity in vivo Using a GluN2D conditional KO line (GluN2Dflx/flx) to selectively delete the subunit from the BNST, we find that BNST-GluN2Dflx/flx male mice exhibit increased depressive-like behaviors, as well as altered NMDAR function and increased excitatory drive onto BNST-CRF neurons. Together, this study supports a role for GluN2D-NMDARs in regulating emotional behavior through their influence on excitatory signaling in a region-specific manner, and suggests that these NMDARs may serve as a novel target for selectively modulating glutamate signaling in stress-responsive structures and cell populations.SIGNIFICANCE STATEMENT Excitatory signaling mediated through NMDARs plays an important role in shaping emotional behavior; however, the receptor subtypes/brain regions through which this occurs are poorly understood. Here, we demonstrate that loss of GluN2D-containing NMDARs produces an increase in anxiety- and depressive-like behaviors in mice, deficits in BNST synaptic potentiation, and increased activity in BNST-CRF neurons known to drive negative emotional behavior. Further, we determine that deleting GluN2D in the BNST leads to increased depressive-like behaviors and increased excitatory drive onto BNST-CRF cells. Collectively, these results demonstrate a role for GluN2D-NMDARs in regulating the activity of stress-responsive structures and neuronal populations in the adult brain, suggesting them as a potential target for treating negative emotional states in mood-related disorders.


Subject(s)
Anxiety/psychology , Behavior, Animal/physiology , Depression/psychology , Receptors, N-Methyl-D-Aspartate/physiology , Septal Nuclei/physiology , Synapses/physiology , Animals , Corticotropin-Releasing Hormone/physiology , Electrophysiological Phenomena/physiology , Feeding Behavior/physiology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/physiology
6.
Nat Neurosci ; 22(12): 2000-2012, 2019 12.
Article in English | MEDLINE | ID: mdl-31712775

ABSTRACT

Acquisition and extinction of learned fear responses utilize conserved but flexible neural circuits. Here we show that acquisition of conditioned freezing behavior is associated with dynamic remodeling of relative excitatory drive from the basolateral amygdala (BLA) away from corticotropin releasing factor-expressing (CRF+) centrolateral amygdala neurons, and toward non-CRF+ (CRF-) and somatostatin-expressing (SOM+) neurons, while fear extinction training remodels this circuit back toward favoring CRF+ neurons. Importantly, BLA activity is required for this experience-dependent remodeling, while directed inhibition of the BLA-centrolateral amygdala circuit impairs both fear memory acquisition and extinction memory retrieval. Additionally, ectopic excitation of CRF+ neurons impairs fear memory acquisition and facilities extinction, whereas CRF+ neuron inhibition impairs extinction memory retrieval, supporting the notion that CRF+ neurons serve to inhibit learned freezing behavior. These data suggest that afferent-specific dynamic remodeling of relative excitatory drive to functionally distinct subcortical neuronal output populations represents an important mechanism underlying experience-dependent modification of behavioral selection.


Subject(s)
Basolateral Nuclear Complex/physiology , Central Amygdaloid Nucleus/physiology , Fear/physiology , Glutamic Acid/physiology , Animals , Conditioning, Classical/physiology , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism , Excitatory Postsynaptic Potentials/physiology , Extinction, Psychological/physiology , Freezing Reaction, Cataleptic/physiology , Mice, Transgenic , Neural Pathways/physiology , Somatostatin/genetics , Somatostatin/metabolism
7.
Alcohol Clin Exp Res ; 43(8): 1695-1701, 2019 08.
Article in English | MEDLINE | ID: mdl-31141179

ABSTRACT

BACKGROUND: Relapse is a critical barrier to effective long-term treatment of alcoholism, and stress is often cited as a key trigger to relapse. Numerous studies suggest that stress-induced reinstatement to drug-seeking behaviors is mediated by norepinephrine (NE) and corticotropin-releasing factor (CRF) signaling interactions in the bed nucleus of the stria terminalis (BNST), a brain region critical to many behavioral and physiologic responses to stressors. Here, we sought to directly examine the effects of NE on BNST CRF neuron activity and determine whether these effects may be modulated by chronic intermittent EtOH (CIE) exposure or a single restraint stress. METHODS: Adult male CRF-tomato reporter mice were treatment-naïve, or either exposed to CIE for 2 weeks or to a single 1-hour restraint stress. Effects of application of exogenous NE on BNST CRF neuron activity were assessed via whole-cell patch-clamp electrophysiological techniques. RESULTS: We found that NE depolarized BNST CRF neurons in naïve mice in a ß-adrenergic receptor (AR)-dependent mechanism. CRF neurons from CIE- or stress-exposed mice had significantly elevated basal resting membrane potential compared to naïve mice. Furthermore, CIE and stress individually disrupted the ability of NE to depolarize CRF neurons, suggesting that both stress and CIE utilize ß-AR signaling to modulate BNST CRF neurons. Neither stress nor CIE altered the ability of exogenous NE to inhibit evoked glutamatergic transmission onto BNST CRF neurons as shown in naïve mice, a mechanism previously shown to be α-AR-dependent. CONCLUSIONS: Altogether, these findings suggest that stress and CIE interact with ß-AR signaling to modulate BNST CRF neuron activity, potentially disrupting the α/ß-AR balance of BNST CRF neuronal excitability. Restoration of α/ß-AR balance may lead to novel therapies for the alleviation of many stress-related disorders.


Subject(s)
Adrenergic Neurons/physiology , Corticotropin-Releasing Hormone/physiology , Ethanol/adverse effects , Norepinephrine/pharmacology , Restraint, Physical/physiology , Septal Nuclei/physiology , Adrenergic Neurons/drug effects , Adrenergic Neurons/metabolism , Animals , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Gene Knock-In Techniques , Glutamic Acid/physiology , Kynurenic Acid/pharmacology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Norepinephrine/antagonists & inhibitors , Picrotoxin/pharmacology , Propranolol/pharmacology , Septal Nuclei/drug effects , Substance Withdrawal Syndrome/physiopathology
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