MUPET-Mouse Ultrasonic Profile ExTraction: A Signal Processing Tool for Rapid and Unsupervised Analysis of Ultrasonic Vocalizations.

Vocalizations play a significant role in social communication across species. Analyses in rodents have used a limited number of spectro-temporal measures to compare ultrasonic vocalizations (USVs), which limits the ability to address repertoire complexity in the context of behavioral states. Using an automated and unsupervised signal processing approach, we report the development of MUPET (Mouse Ultrasonic Profile ExTraction) software, an open-access MATLAB tool that provides data-driven, high-throughput analyses of USVs. MUPET measures, learns, and compares syllable types and provides an automated time stamp of syllable events. Using USV data from a large mouse genetic reference panel and open-source datasets produced in different social contexts, MUPET analyzes the fine details of syllable production and repertoire use. MUPET thus serves as a new tool for USV repertoire analyses, with the capability to be adapted for use with other species.

Quantitative Trait Loci and a Novel Genetic Candidate for Fear Learning.

UNLABELLED: Trauma- and stress-related disorders are clinically heterogeneous and associated with substantial genetic risk. Understanding the biological origins of heterogeneity of key intermediate phenotypes such as cognition and emotion can provide novel mechanistic insights into disorder pathogenesis. Performing quantitative genetics in animal models is a tractable strategy for examining both the genetic basis of intermediate phenotypes and functional testing of candidate quantitative traits genes (QTGs). Here, existing and newly collected data were used for collaborative genome-wide mapping of cued fear acquisition and expression in 65 mouse strains from the BXD genetic reference panel. For fear acquisition, we identified a significant locus on chromosome (Chr) 10 and eight suggestive loci on Chr 2, 4, 5, 11, 13, and 15. For fear expression, we identified one significant and another highly suggestive locus on Chr 13, as well as four suggestive loci on Chr 10, 11, and X. Across these loci, 60 putative QTGs were identified. The quantitative trait locus on distal Chr 13 contained a single, highly promising gene at the location of the peak likelihood ratio statistic score. The gene, hyperpolarization-activated cyclic nucleotide-gated channel 1 (Hcn1), regulates neuronal excitability. Validation experiments using behavioral pharmacology revealed that functional Hcn channels in the basolateral amygdala are necessary for conditioned fear acquisition and expression. Hcn1, together with the other candidate QTGs, thus provide new targets for neurobiological and treatment studies of fear learning and trauma- and stress-related disorders. SIGNIFICANCE STATEMENT: There is a knowledge gap in understanding the genetic contributions to behavioral heterogeneity in typical and atypical populations. Mouse genetic reference panels (GRPs) provide one approach for identifying genetic sources of variation. Here, we identified three loci for conditioned fear acquisition and expression in a mouse GRP. Each locus contained candidate quantitative trait genes (QTGs). One locus had a single QTG, Hcn1 (hyperpolarization-activated cyclic nucleotide-gated channel 1), which has been implicated in neuronal excitability and learning. This discovery was validated using behavioral pharmacology, revealing that Hcn channels in the basolateral amygdala are required for fear acquisition and expression. The study thus identifies novel candidate QTGs that may contribute to variation in emotional learning and highlight the utility of mouse GRPs for the identification of genes underlying complex traits.

Social housing conditions and oxytocin and vasopressin receptors contribute to ethanol conditioned social preference in female mice.

Social behavior modulates response to alcohol. Because oxytocin (OXT) and vasopressin (AVP) contribute to rewarding social behavior, the present study utilized a genetic strategy to determine whether OXT and AVP receptors (OXTR, AVPR1a) are essential for female mice to demonstrate a conditioned social preference for ethanol. The study compared wild-type (WT) and knock-out (KO) females lacking either Oxtr or Avpr1a in a conditioned social preference (CSP) test. KO females and WT females from Het-Het crosses were pair-housed: KO and WT(ko). WT females from Het-WT crosses were pair-housed: WT(wt). Test mice received 2g/kg ethanol or saline ip, and were paired four times each with one stimulus female (CS-) after saline, and with another female (CS+) following ethanol. After pairing, the time spent with CS+ and CS- females was measured. WT(wt) females showed conditioned preference for the CS+ female paired with ethanol, demonstrated by greater interaction time (p<0.05). In both KO lines, ethanol significantly reduced interaction with the CS+ female (p<0.05), and there was no change in interaction for WT(ko) females. Response to odors by habituation-dishabituation was unaffected in both KO lines, and the response to a hypnotic dose of ethanol also was the same as in WT mice. However, anxiety, measured as time on the open arms of the elevated plus maze, was reduced in KO(Oxtr) females compared with WT(wt). The results suggest that Oxtr and Avpr1a are required for conditioned effects of an ethanol-associated social stimulus. The lack of CSP in WT(ko) females suggests that the quality of social interactions during postnatal and postweaning life may modulate development and expression of normal social responses.

Sprouty2 in the dorsal hippocampus regulates neurogenesis and stress responsiveness in rats.

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders.

Kappa opioid receptor signaling in the basolateral amygdala regulates conditioned fear and anxiety in rats.

BACKGROUND: The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats. METHODS: We assessed whether fear conditioning induces plasticity in KOR or dynorphin (the endogenous KOR ligand) messenger RNA (mRNA) expression in the basolateral (BLA) and central (CeA) nuclei of the amygdala, hippocampus, or striatum. We then assessed whether microinfusions of the KOR antagonist JDTic (0-10 µg/side) into the BLA or CeA affect the expression of conditioned fear or anxiety. Finally, we examined whether fear extinction induces plasticity in KOR mRNA expression that relates to the quality of fear extinction. RESULTS: Fear conditioning upregulated KOR mRNA in the BLA by 65% and downregulated it in the striatum by 22%, without affecting KOR levels in the CeA or hippocampus, or dynorphin levels in any region. KOR antagonism in either the BLA or CeA decreased conditioned fear in the fear-potentiated startle paradigm, whereas KOR antagonism in the BLA, but not the CeA, produced anxiolytic-like effects in the elevated plus maze. Effective fear extinction was associated with a 67% reduction in KOR mRNA in the BLA. CONCLUSIONS: These findings suggest that fear conditioning and extinction dynamically regulate KOR expression in the BLA and provide evidence that the BLA and CeA are important neural substrates mediating the anxiolytic-like effects of KOR antagonists in models of fear and anxiety.

Dynorphin, stress, and depression.

Stress is most often associated with aversive states. It rapidly induces the release of hormones and neuropeptides including dynorphin, which activates kappa opioid receptors (KORs) in the central and peripheral nervous systems. In animal models, many aversive effects of stress are mimicked or exacerbated by stimulation of KORs in limbic brain regions. Although KOR signaling during acute stress may increase physical ability (by producing analgesia) and motivation to escape a threat (by producing aversion), prolonged KOR signaling in response to chronic or uncontrollable stress can lead to persistent expression of behavioral signs that are characteristic of human depressive disorders (i.e., "prodepressive-like" signs). Accumulating evidence suggests that KORs contribute to the progressive amplification (sensitization) of stress-induced behaviors that occurs with repeated exposure to stress. Many of the aversive effects of stress are blocked by KOR antagonists, suggesting that these agents may have potential as therapeutics for stress-related conditions such as depression and anxiety disorders. This review summarizes current data on how KOR systems contribute to the acute (rapid), delayed, and cumulative molecular and behavioral effects of stress. We focus on behavioral paradigms that provide insight on interactions between stress and KOR function within each of these temporal categories. Using a simplified model, we consider the time course and mechanism of KOR-mediated effects in stress and suggest future directions that may be useful in determining whether KOR antagonists exert their therapeutic effects by preventing the development of stress-induced behaviors, the expression of stress-induced behaviors, or both.

Kappa-opioid ligands in the study and treatment of mood disorders.

The biological basis of mood is not understood. Most research on mood and affective states has focused on the roles of brain systems containing monoamines (e.g., dopamine, norepinephrine, serotonin). However, it is becoming clear that endogenous opioid systems in the brain may also be involved in the regulation of mood. In this review, we focus on the potential utility of kappa-opioid receptor (KOR) ligands in the study and treatment of psychiatric disorders. Research from our group and others suggests that KOR antagonists might be useful for depression, KOR agonists might be useful for mania, and KOR partial agonists might be useful for mood stabilization. Currently available KOR agents have some unfavorable properties that might be addressed through medicinal chemistry. The development of KOR-selective agents with improved drug-like characteristics would facilitate preclinical and clinical studies designed to evaluate the possibility that KORs are a feasible target for new medications.

Anxiolytic-like effects of kappa-opioid receptor antagonists in models of unlearned and learned fear in rats.

Endogenous opioid systems regulate neurobiological responses to threatening stimuli. Stimulation of kappa-opioid receptors (KORs) produces analgesia but induces prodepressive-like effects in a variety of animal models. In contrast, KOR antagonists have antidepressant-like effects. KORs and their endogenous ligand dynorphin are expressed throughout brain areas involved in fear and anxiety, including the extended amygdala. Here, we examined whether KOR antagonists would affect unlearned fear (anxiety) in the elevated plus maze (EPM) and open field (OF) paradigms and learned fear in the fear-potentiated startle (FPS) paradigm. These studies were designed to accommodate the slow onset (approximately 24 h) and extended time course (>3 weeks) of the prototypical KOR antagonists nor-binaltorphimine hydrochloride (norBNI) and JDTic [(3R)-7-hydroxy-N-[(1S)-1-[[(3R, 4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide hydrochloride]. Rats received an i.p. injection of norBNI (3.0-30 mg/kg) or JDTic (1.0-10 mg/kg) 48 h before EPM testing. One day later, they were tested in the OF, and 5 and 7 days later, they were trained and tested in the FPS paradigm. Both KOR antagonists dose-dependently increased open arm exploration in the EPM without affecting OF behavior. They also decreased conditioned fear in the FPS paradigm. The anxiolytic-like effects of KOR antagonists were qualitatively similar to those of the benzodiazepine chlordiazepoxide in the EPM. The selective serotonin reuptake inhibitor fluoxetine had no effect in the EPM and anxiogenic-like effects in the OF. Our results indicate that KOR antagonists produce a unique combination of antidepressant- and anxiolytic-like effects and suggest that this class of drugs may be particularly effective for the treatment of comorbid depressive and anxiety disorders.

Behavioral and anatomical interactions between dopamine and corticotropin-releasing factor in the rat.

The neuropeptide corticotropin-releasing factor (CRF) is believed to play a role in a number of psychiatric conditions, including anxiety disorders and depression. In the present study, male Sprague Dawley rats were used to examine the behavioral effects of altering dopamine transmission on CRF-enhanced startle, a behavioral assay believed to reflect stress- or anxiety-like states. Systemic administration of the selective dopamine D1 receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] (0, 0.01, 0.05, 0.1, 0.5 mg/kg) dose dependently blocked the effect of CRF (1 microg, i.c.v.) on startle at doses that had no effect on baseline startle response. Immunohistochemical studies showed that most CRF-containing cells in the dorsolateral division of the bed nucleus of the stria terminalis (BSTld), part of the critical brain area mediating CRF-enhanced startle, are surrounded by a dense plexus of tyrosine hydroxylase (TH)-positive fibers. Intra-BSTld injections of the retrograde tracer Fluorogold (FG) into the TH field identified neurons in the major dopaminergic areas (A8-A10), but not the major noradrenergic areas [A5, A6 (locus ceruleus), A7], as a significant source of TH-positive innervation. The majority of FG-filled cells double-labeled for TH were found in the dorsocaudal A10 cell group (A10dc) located in the periaqueductal gray area. Together, these data suggest that neuronal regulation of the BSTld by specific dopaminergic pathways and receptors may be an important mechanism for controlling CRF-dependent moods and affective states. These data also suggest that compounds with D1 receptor antagonist properties might have anxiolytic-like effects that could be useful for treating conditions associated with hyperactive CRF systems.