Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

Divergent profiles of fentanyl withdrawal and associated pain in mice and rats.

Opioid abuse has devastating effects on patients, their families, and society. Withdrawal symptoms are severely unpleasant, prolonged, and frequently hinder recovery or lead to relapse. The sharp increase in abuse and overdoses arising from the illicit use of potent and rapidly-acting synthetic opioids, such as fentanyl, highlights the urgency of understanding the withdrawal mechanisms related to these drugs. Progress is impeded by inconsistent reports on opioid withdrawal in different preclinical models. Here, using rats and mice of both sexes, we quantified withdrawal behaviors during spontaneous and naloxone-precipitated withdrawal, following two weeks of intermittent fentanyl exposure. We found that both mice and rats lost weight during exposure and showed increased signs of distress during spontaneous and naloxone precipitated withdrawal. However, these species differed in their expression of withdrawal associated pain, a key contributor to relapse in humans. Spontaneous or ongoing pain was preferentially expressed in rats in both withdrawal conditions, while no change was observed in mice. In contrast, withdrawal associated thermal hyperalgesia was found only in mice. These data suggest that rats and mice diverge in how they experience withdrawal and which aspects of the human condition they most accurately model. These differences highlight each species' strengths as model systems and can inform experimental design in studies of opioid withdrawal.

Contemporary resuscitation of hemorrhagic shock: What will the future hold?

Resuscitation of the critically ill patient with fluid and blood products is one of the most widespread interventions in medicine. This is especially relevant for trauma patients, as hemorrhagic shock remains the most common cause of preventable death after injury. Consequently, the study of the ideal resuscitative product for patients in shock has become an area of great scientific interest and investigation. Recently, the pendulum has swung towards increased utilization of blood products for resuscitation. However, pathogens, immune reactions and the limited availability of this resource remain a challenge for clinicians. Technologic advances in pathogen reduction and innovations in blood product processing will allow us to increase the safety profile and efficacy of blood products, ultimately to the benefit of patients. The purpose of this article is to review the current state of blood product based resuscitative strategies as well as technologic advancements that may lead to safer resuscitation.

An Amygdalo-Parabrachial Pathway Regulates Pain Perception and Chronic Pain.

The parabrachial (PB) complex mediates both ascending nociceptive signaling and descending pain modulatory information in the affective/emotional pain pathway. We have recently reported that chronic pain is associated with amplified activity of PB neurons in a rat model of neuropathic pain. Here we demonstrate that similar activity amplification occurs in mice, and that this is related to suppressed inhibition to lateral parabrachial (LPB) neurons from the CeA in animals of either sex. Animals with pain after chronic constriction injury of the infraorbital nerve (CCI-Pain) displayed higher spontaneous and evoked activity in PB neurons, and a dramatic increase in after-discharges, responses that far outlast the stimulus, compared with controls. LPB neurons in CCI-Pain animals showed a reduction in inhibitory, GABAergic inputs. We show that, in both rats and mice, LPB contains few GABAergic neurons, and that most of its GABAergic inputs arise from CeA. These CeA GABA neurons express dynorphin, somatostatin, and/or corticotropin releasing hormone. We find that the efficacy of this CeA-LPB pathway is suppressed in chronic pain. Further, optogenetically stimulating this pathway suppresses acute pain, and inhibiting it, in naive animals, evokes pain behaviors. These findings demonstrate that the CeA-LPB pathway is critically involved in pain regulation, and in the pathogenesis of chronic pain.SIGNIFICANCE STATEMENT We describe a novel pathway, consisting of inhibition by dynorphin, somatostatin, and corticotropin-releasing hormone-expressing neurons in the CeA that project to the parabrachial nucleus. We show that this pathway regulates the activity of pain-related neurons in parabrachial nucleus, and that, in chronic pain, this inhibitory pathway is suppressed, and that this suppression is causally related to pain perception. We propose that this amygdalo-parabrachial pathway is a key regulator of both chronic and acute pain, and a novel target for pain relief.

(2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions.

Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3-/-, gene. Combined subeffective doses of the mGlu2/3 receptor antagonist LY341495 and (2R,6R)-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu2 receptor function in clinical trials for treatment-resistant depression either alone or in combination.

Sex-Specific Involvement of Estrogen Receptors in Behavioral Responses to Stress and Psychomotor Activation.

Fluctuating hormone levels, such as estradiol might underlie the difference in the prevalence of psychiatric disorders observed in women vs. men. Estradiol exert its effects primarily through binding on the two classical estrogen receptor subtypes, alpha (ERα) and beta (ERß). Both receptors have been suggested to a have role in the development of psychiatric disorders, however, most of the current literature is limited to their role in females. We investigated the role of estrogen receptors on cognition (novel-object recognition), anxiety (open-field test, elevated-plus maze, and light/dark box), stress-responsive behaviors (forced-swim test, learned helplessness following inescapable shock, and sucrose preference), pre-pulse inhibition (PPI) and amphetamine-induced hyperlocomotion in both male and female mice either lacking the ERα or ERß receptor. We found that female Esr1 -/- mice have attenuated pre-pulse inhibition, whereas female Esr2 -/- mice manifested enhanced pre-pulse inhibition. No pre-pulse inhibition difference was observed in male Esr1 -/- and Esr2 -/- mice. Moreover, amphetamine-induced hyperlocomotion was decreased in male Esr1 -/-, but not Esr2 -/- mice, while female Esr1 -/- and Esr2 -/- mice showed an enhanced response. Genetic absence of ERα did not alter the escape capability or sucrose preference following inescapable shock in both male and female mice. In contrast, female, but not male Esr2 -/- mice, manifested decreased escape failures compared with controls. Lack of Esr2 gene in male mice was associated with decreased sucrose preference following inescapable shock, suggesting susceptibility for development of anhedonia following stress. No sucrose preference differences were found in female Esr2 -/- mice following inescapable shock stress. Lastly, we demonstrated that lack of Esr1 or Esr2 genes had no effect on memory and anxiety-like behaviors in both male and female mice. Our findings indicate a differential sex-specific involvement of estrogen receptors in the development of stress-mediated maladaptive behaviors as well as psychomotor activation responses suggesting that these receptors might act as potential treatment targets in a sex-specific manner.

Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.