In vivo photopharmacology with light-activated opioid drugs

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using "caged" opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed photoactivatable oxymorphone (PhOX) and photoactivatable naloxone (PhNX), photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry in response to chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action

S 38093, a histamine H3 antagonist/inverse agonist, promotes hippocampal neurogenesis and improves context discrimination task in aged mice

Strategies designed to increase adult hippocampal neurogenesis (AHN) may have therapeutic potential for reversing memory impairments. H3 receptor antagonists/inverse agonists also may be useful for treating cognitive deficits. However, it remains unclear whether these ligands have effects on AHN. The present study aimed to investigate the effects of a 28-day treatment with S 38093, a novel brain-penetrant antagonist/inverse agonist of H3 receptors, on AHN (proliferation, maturation and survival) in 3-month-old and in aged 16-month-old mice. In addition, the effects of S 38093 treatment on 7-month-old APPSWE Tg2576 transgenic mice, a model of Alzheimer’s disease, were also assessed. In all tested models, chronic treatment with S 38093 stimulated all steps of AHN. In aged animals, S 38093 induced a reversal of age-dependent effects on hippocampal brain-derived neurotrophic factor (BDNF) BDNF-IX, BDNF-IV and BDNF-I transcripts and increased vascular endothelial growth factor (VEGF) expression. Finally, the effects of chronic administration of S 38093 were assessed on a neurogenesis-dependent “context discrimination (CS) test” in aged mice. While ageing altered mouse CS, chronic S 38093 treatment significantly improved CS. Taken together, these results provide evidence that chronic S 38093 treatment increases adult hippocampal neurogenesis and may provide an innovative strategy to improve age-associated cognitive deficits

Diversity of lateral habenula neuron roles in negative valence events

Thesis (Ph.D.)--University of Washington, 2020The lateral habenula (LHb) integrates critical information regarding aversive stimuli that shapes decision making and behavioral responses. Three major LHb outputs innervate dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). LHb neurons that project to these targets are segregated and nonoverlapping, and this led us to consider whether they have distinct molecular phenotypes and adaptations to stress exposure in chapter 2. In order to capture a time-locked profile of gene expression after repeated forced swim stress, we used intersectional expression of RiboTag in rat LHb neurons and next-gen RNA sequencing to interrogate the RNAs actively undergoing translation from each of these pathways. The “translatome” in the neurons comprising these pathways was similar at baseline, but diverged after stress, especially in the neurons projecting to the RMTg. Using weighted gene co-expression network analysis, we found one module, which had an overrepresentation of genes associated with phosphoinositide 3 kinase (PI3K) signaling, comprising genes downregulated after stress in the RMTg-projecting LHb neurons. Reduced PI3K signaling in RMTg-projecting LHb neurons may be a compensatory adaptation that alters the functional balance of LHb outputs to GABAergic vs. monoaminergic neurons following repeated stress exposure.Additionally, while primarily glutamatergic, LHb neurons express numerous neuropeptides, such as pituitary adenylate cyclase-activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. In chapter 3, we explored these neurons. Using Cre-dependent viral vector expression, we characterized these neurons based on their anatomical projections and found that they projected to raphe and rostromedial tegmentum but not ventral tegmental area. Using RiboTag to capture ribosomal-associated RNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP-expressing neurons in LHb. In order to understand the function of these neurons, we chemogenetically excited PACAP-expressing neurons using virally-mediated gene transfer of DREADDs in PACAP-Cre mice and tested the mice using open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that activating these neurons produce behaviors opposite to what is expected from the LHb as a whole – they decreased anxiety-like and fear behavior and produced a conditioned place preference. In conclusion, PACAP-expressing neurons in LHb represents a unique population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events

Sex dependence of opioid-mediated responses to subanesthetic ketamine in rats

Abstract Subanesthetic ketamine is increasingly used for the treatment of varied psychiatric conditions, both on- and off-label. While it is commonly classified as an N-methyl D-aspartate receptor (NMDAR) antagonist, our picture of ketamine’s mechanistic underpinnings is incomplete. Recent clinical evidence has indicated, controversially, that a component of the efficacy of subanesthetic ketamine may be opioid dependent. Using pharmacological functional ultrasound imaging in rats, we found that blocking opioid receptors suppressed neurophysiologic changes evoked by ketamine, but not by a more selective NMDAR antagonist, in limbic regions implicated in the pathophysiology of depression and in reward processing. Importantly, this opioid-dependent response was strongly sex-dependent, as it was not evident in female subjects and was fully reversed by surgical removal of the male gonads. We observed similar sex-dependent effects of opioid blockade affecting ketamine-evoked postsynaptic density and behavioral sensitization, as well as in opioid blockade-induced changes in opioid receptor density. Together, these results underscore the potential for ketamine to induce its affective responses via opioid signaling, and indicate that this opioid dependence may be strongly influenced by subject sex. These factors should be more directly assessed in future clinical trials

Unique pharmacodynamic properties and low abuse liability of the μ-opioid receptor ligand (S)-methadone

(R,S)-methadone ((R,S)-MTD) is a racemic μ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers used for the treatment of opioid use disorder (OUD) and pain. (R)-MTD is used as an OUD treatment, has high MOR potency, and is believed to mediate (R,S)-MTD's therapeutic efficacy. (S)-MTD is in clinical development as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. In opposition to this purported mechanism of action, we found that (S)-MTD does not occupy NMDARs in vivo in rats. Instead, (S)-MTD produced MOR occupancy and induced analgesia with similar efficacy as (R)-MTD. Unlike (R)-MTD, (S)-MTD was not self-administered and failed to increase locomotion or extracellular dopamine levels indicating low abuse liability. Moreover, (S)-MTD antagonized the effects of (R)-MTD in vivo and exhibited unique pharmacodynamic properties, distinct from those of (R)-MTD. Specifically, (S)-MTD acted as a MOR partial agonist with a specific loss of efficacy at the MOR-galanin 1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use, as well as those of (R,S)-MTD

In vivo photopharmacology with light-activated opioid drugs.

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using caged opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed photoactivatable oxymorphone (PhOX) and photoactivatable naloxone (PhNX), photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry in response to chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action