Sex differences in the effect of cannabinoid type 1 receptor deletion on locus coeruleus-norepinephrine neurons and corticotropin releasing factor-mediated responses.

Cannabinoids are capable of modulating mood, arousal, cognition and behavior, in part via their effects on the noradrenergic nucleus locus coeruleus (LC). Dysregulation of LC signaling and norepinephrine (NE) efflux in the medial prefrontal cortex (mPFC) can lead to the development of psychiatric disorders, and CB1r deletion results in alterations of α2- and ß1-adrenoceptors in the mPFC, suggestive of increased LC activity. To determine how CB1r deletion alters LC signaling, whole-cell patch-clamp electrophysiology was conducted in LC-NE neurons of male and female wild type (WT) and CB1r-knock out (KO) mice. CB1r deletion caused a significant increase in LC-NE excitability and input resistance in male but not female mice when compared to WT. CB1r deletion also caused adaptations in several indices of noradrenergic function. CB1r/CB2r-KO male mice had a significant increase in cortical NE levels and tyrosine hydroxylase and CRF levels in the LC compared to WT males. CB1r/CB2r-KO female mice showed a significant increase in LC α2-AR levels compared to WT females. To further probe actions of the endocannabinoid system as an anti-stress neuromediator, the effect of CB1r deletion on CRF-induced responses in the LC was investigated. The increase in LC-NE excitability observed in male and female WT mice following CRF (300 nM) bath application was not observed in CB1r-KO mice. These results indicate that cellular adaptations following CB1r deletion cause a disruption in LC-NE signaling in males but not females, suggesting underlying sex differences in compensatory mechanisms in KO mice as well as basal endocannabinoid regulation of LC-NE activity.

Absence of ALOX5 gene prevents stress-induced memory deficits, synaptic dysfunction and tauopathy in a mouse model of Alzheimer's disease.

Although the initial events of Alzheimer's disease (AD) are still not known, it is clear that the disease in its sporadic form results from the combination of genetic and environmental risk factors. Among the latter, behavioral stress has been increasingly recognized as an important factor in the propagation of AD. However, the mechanisms underlying this modulation remain to be fully investigated. Since stress up-regulates the ALOX5 gene product, 5-lipoxygenase (5LO), herein we investigated its role in modulating stress-dependent development of the AD phenotype. To reach this goal, triple transgenic (3xTg) mice and 3xTg genetically deficient for 5LO were investigated after undergoing a restraint/isolation paradigm. In the present paper, we found that 28 days of restraint/isolation stress worsened tau phosphorylation and solubility, increased glycogen synthase kinase 3ß activity, compromised long-term potentiation and impaired fear-conditioned memory recall in 3xTg animals, but not in 3xTg animals lacking 5LO (3xTg/5LO-/-). These results highlight the novel functional role that the ALOX5 gene plays in the development of the biochemical, electrophysiological and behavioral sequelae of stress in the AD context. They provide critical support that this gene and its expressed protein are viable therapeutic targets to prevent the onset or delay the progression of AD in individuals exposed to this risk factor.

The Lysophosphatidylinositol Receptor GPR55 Modulates Pain Perception in the Periaqueductal Gray.

Emerging evidence indicates the involvement of GPR55 and its proposed endogenous ligand, lysophosphatidylinositol (LPI), in nociception, yet their role in central pain processing has not been explored. Using Ca(2+) imaging, we show here that LPI elicits concentration-dependent and GPR55-mediated increases in intracellular Ca(2+) levels in dissociated rat periaqueductal gray (PAG) neurons, which express GPR55 mRNA. This effect is mediated by Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptors and by Ca(2+) entry via P/Q-type of voltage-gated Ca(2+) channels. Moreover, LPI depolarizes PAG neurons and upon intra-PAG microinjection, reduces nociceptive threshold in the hot-plate test. Both these effects are dependent on GPR55 activation, because they are abolished by pretreatment with ML-193 [N-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)-phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide], a selective GPR55 antagonist. Thus, we provide the first pharmacological evidence that GPR55 activation at central levels is pronociceptive, suggesting that interfering with GPR55 signaling in the PAG may promote analgesia.

Cannabinoid modulation of alpha2 adrenergic receptor function in rodent medial prefrontal cortex.

Endocannabinoids acting at the cannabinoid type 1 receptor (CB1R) are known to regulate attention, cognition and mood. Previous studies have shown that, in the rat medial prefrontal cortex (mPFC), CB1R agonists increase norepinephrine release, an effect that may be attributed, in part, to CB1Rs localised to noradrenergic axon terminals. The present study was aimed at further characterising functional interactions between CB1R and adrenergic receptor (AR) systems in the mPFC using in vitro intracellular electrophysiology and high-resolution neuroanatomical techniques. Whole-cell patch-clamp recordings of layer V/VI cortical pyramidal neurons in rats revealed that both acute and chronic treatment with the synthetic CB1R agonist WIN 55,212-2 blocked elevations in cortical pyramidal cell excitability and increases in input resistance evoked by the α2-adrenergic receptor (α2-AR) agonist clonidine, suggesting a desensitisation of α2-ARs. These CB1R-α2-AR interactions were further shown to be both action potential- and gamma-aminobutyric acid-independent. To better define sites of cannabinoid-AR interactions, we localised α2A-adrenergic receptors (α2A-ARs) in a genetically modified mouse that expressed a hemoagglutinin (HA) tag downstream of the α2A-AR promoter. Light and electron microscopy indicated that HA-α2A-AR was distributed in axon terminals and somatodendritic processes especially in layer V of the mPFC. Triple-labeling immunocytochemistry revealed that α2A-AR and CB1R were localised to processes that contained dopamine-ß-hydroxylase, a marker of norepinephrine. Furthermore, HA-α2A-AR was localised to processes that were directly apposed to CB1R. These findings suggest multiple sites of interaction between cortical cannabinoid-adrenergic systems that may contribute to understanding the effect of cannabinoids on executive functions and mood.

Stress-dependent opioid and adrenergic modulation of newly retrieved fear memory.

Recent studies on the effect of stress on modulation of fear memory in our laboratory have uncovered endogenous opioid and adrenergic based modulation systems, working in concert, that limit the strengthening or weakening of newly acquired fear memory during consolidation under conditions of mild or intense stress, respectively. The present study sought to determine if similar stress-dependent modulation, mediated by endogenous opioid and adrenergic systems, occurs during reconsolidation of newly retrieved fear memory. Rats underwent contextual fear conditioning followed 24h later by reactivation of fear memory; a retention test was administered the next day. Stress was manipulated by varying duration of recall of fear memory during reactivation. In the first experiment, vehicle or the opioid-receptor blocker naloxone was administered immediately after varied durations (30 or 120 s) of reactivation. The results indicate that (1) reactivation, in the absence of drug, has a marked effect on freezing behavior-as duration of reactivation increases from 30 to 120 s, freezing behavior and presumably fear-induced stress increases and (2) naloxone, administered immediately after 30 s (mild stress) or 120 s (intense stress) of reactivation, enhances or impairs retention, respectively, the next day. In the second experiment, naloxone and the ß-adrenergic blocker propranolol were administered either separately or in combination immediately after 120 s (intense stress) reactivation. The results indicate that separate administration of propranolol and naloxone impairs retention, while the combined administration fails to do so. Taken together the results of the two experiments are consistent with a protective mechanism, mediated by endogenous opioid and adrenergic systems working in concert, that limits enhancement and impairment of newly retrieved fear memory during reactivation in a stress-dependent manner.

Role of serotonin neurons in the dorsal raphe nucleus in heroin self-administration and punishment.

One hallmark of substance use disorder is continued drug use despite negative consequences. When drug-taking behavior is punished with aversive stimuli, i.e. footshock, rats can also be categorized into punishment-resistant or compulsive vs. punishment-sensitive or non-compulsive phenotypes. The serotonin (5-hydroxytryptamine, 5-HT) system modulates responses to both reward and punishment. The goal of the current study was to examine punishment phenotypes in heroin self-administration and to determine the role of dorsal raphe nucleus (DRN) 5-HT neurons in both basal and punished heroin self-administration. First, rats were exposed to punished heroin self-administration and neuronal excitability of DRN 5-HT neurons was compared between punishment-resistant and punishment-sensitive phenotypes using ex vivo electrophysiology. Second, DRN 5-HT neuronal activity was manipulated in vivo during basal and punished heroin self-administration using chemogenetic tools in a Tph2-iCre rat line. While rats separated into punishment-resistant and punishment-sensitive phenotypes for punished heroin self-administration, DRN 5-HT neuronal excitability did not differ between the phenotypes. While chemogenetic inhibition of DRN 5-HT neurons was without effect, chemogenetic activation of DRN 5-HT neurons increased both basal and punished heroin self-administration selectively in punishment-resistant animals. Additionally, the responsiveness to chemogenetic activation of DRN 5-HT neurons in basal self-administration and motivation for heroin in progressive ratio each predicted resistance to punishment. Therefore, our data support the role for the DRN 5-HT system in compulsive heroin self-administration.

Role of α1-GABAA receptors in the serotonergic dorsal raphe nucleus in models of opioid reward, anxiety, and depression.

BACKGROUND: The serotonin (5-hydroxytryptamine (5-HT))-mediated system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous studies showed that stress and drug exposure can modulate the dorsal raphe nucleus (DRN)-5-HT system via γ-aminobutyric acid (GABA)A receptors. Moreover, GABAA receptor-mediated inhibition of serotonergic DRN neurons is required for stress-induced reinstatement of opioid seeking. AIM/METHODS: To further test the role of GABAA receptors in the 5-HT system in stress and opioid-sensitive behaviors, our current study generated mice with conditional genetic deletions of the GABAA α1 subunit to manipulate GABAA receptors in either the DRN or the entire population of 5-HT neurons. The GABAA α1 subunit is a constituent of the most abundant GABAA subtype in the brain and the most highly expressed subunit in 5-HT DRN neurons. RESULTS: Our results showed that mice with DRN-specific knockout of α1-GABAA receptors exhibited a normal phenotype in tests of anxiety- and depression-like behaviors as well as swim stress-induced reinstatement of morphine-conditioned place preference. By contrast, mice with 5-HT neuron-specific knockout of α1-GABAA receptors exhibited an anxiolytic phenotype at baseline and increased sensitivity to post-morphine withdrawal-induced anxiety. CONCLUSIONS: Our data suggest that GABAA receptors on 5-HT neurons contribute to anxiety-like behaviors and sensitivity of those behaviors to opioid withdrawal.

Sex differences in ethanol consumption and drinking despite negative consequences following adolescent social isolation stress in male and female rats.

Alcohol use disorder (AUD) is a debilitating psychiatric disorder characterized by drinking despite negative social and biological consequences. AUDs make up 71% of substance use disorders, with relapse rates as high as 80%. Current treatments stem from data conducted largely in males and fail to target the psychological distress motivating drinking in stress-vulnerable and at-risk populations. Here we employed a rat model and hypothesized that early life stress would reveal sex differences in ethanol intake and drinking despite negative consequences in adulthood. Rats were group housed or isolated postweaning to evaluate sex and stress effects on ethanol consumption in homecage drinking, self-administration (SA), and punished SA (drinking despite negative consequences) in adulthood. Stressed rats showed elevated homecage ethanol intake, an effect more pronounced in females. During SA, males were more sensitive to stress-induced elevations of drinking over time, but females drank more overall. Stressed rats, regardless of sex, responded more for ethanol than their non-stressed counterparts. Stressed females showed greater resistance to punishment-suppressed SA than stressed males, indicating a more stress-resistant drinking phenotype. Results support our hypothesis that adolescent social isolation stress enhances adult ethanol intake in a sex- and model-dependent manner with females being especially sensitive to early life stress-induced elevations in ethanol intake and punished SA in adulthood. Our findings echo the clinical literature which indicates that stress-vulnerable populations are more likely to 'self-medicate' with substances. Elucidating a potential mechanism that underlies why vulnerable populations 'self-medicate' with alcohol can lead towards developing catered pharmacotherapeutics that could reduce punishment-resistant drinking and relapse.

The ventral hippocampus and nucleus accumbens as neural substrates for cocaine contextual memory reconsolidation.

Drug craving triggered by cues that were once associated with drug intoxication is a major contributor to continued drug-seeking behaviors. Addictive drugs engage molecular pathways of associative learning and memory. Reactivated memories are vulnerable to disruption by interference with the process of reconsolidation, hence targeting reconsolidation could be a strategy to reduce cue-induced drug craving and relapse. Here we examined the circuitry of cocaine contextual memory reconsolidation and explored neuroplasticity following memory reactivation. Mice underwent chemogenetic inhibition of either nucleus accumbens (NA) neurons or the glutamatergic projection neurons from the ventral hippocampus (vHPC) to NA using inhibitory designer receptors exclusively activated by designer drugs (iDREADD). Mice underwent cocaine conditioned place preference followed by reactivation of the cocaine contextual memory. Clozapine-N-oxide (CNO) was administered after memory reactivation to inhibit either NA neurons or the accumbens-projecting vHPC neurons during the reconsolidation period. When retested 3 days later, a significant reduction in the previously established preference for the cocaine context was found in both conditions. FosTRAP2-Ai14 mice were used to identify neurons activated by cocaine memory recall and to evaluate plasticity in NA medium spiny neurons (MSNs) and vHPC pyramidal neurons upon recall of cocaine memories. Results indicate a significant increase in dendritic spine density in NA MSNs activated by cocaine memory recall, particularly of the thin spine type. Sholl analysis indicated longer dendritic length and more branching of NA MSNs after cocaine memory recall than without memory reactivation. vHPC neurons showed increased spine density, with the most robust change in stubby spines. These results implicate a circuit involving glutamatergic projections from the vHPC onto NA neurons which is necessary for the reconsolidation of cocaine memories. Interruption of cocaine memory reconsolidation reduced drug-seeking behavior.

Interactions between chemokine and mu-opioid receptors: anatomical findings and electrophysiological studies in the rat periaqueductal grey.

Opioids have immunomodulatory functions and may alter susceptibility to immune disorders. Behavioral studies also indicate that chemokines, molecules expressed by immune cells, block opioid-induced analgesia in the periaqueductal grey (PAG). Bi-directional heterologous desensitization of opioid and chemokine receptors has been described in cell systems. We report the anatomical and functional interactions of chemokine receptors with the mu-opioid receptor (MOR) in the rat brain. The chemokine receptors, CXCR4 and CX3CR1, as well as their chemokine substrates, CXCL12 and CX3CL1, are widely expressed in the central nervous system (CNS). Immunohistochemical techniques were utilized to investigate MOR-CXCR4 and MOR-CX3CR1 receptor colocalization in multiple brain areas. Our results demonstrate co-expression of these receptors on individual neurons in several regions including cingulate cortex, hippocampus, and PAG, suggesting functional receptor interactions. Whole-cell patch-clamp recordings of PAG neurons in a rat brain slice preparation were used to examine morphine or chemokine (CXCL12, CX3CL1) effects alone, or in combination on neuronal membrane properties. Morphine (10 µM) hyperpolarized and reduced input resistance of PAG neurons. CXCL12 and CX3CL1 (10 nM) had no impact on either parameter. In the presence of CXCL12, morphine's electrophysiological effects were blocked in all neurons examined, whereas with CX3CL1, morphine's effects were blocked in 57% of neurons studied. The data provide electrophysiological evidence for MOR-CXCR4 and MOR-CX3CR1 heterologous desensitization in the PAG at the single-cell level. These interactions may contribute to the limited utility of opioid analgesics for inflammatory pain treatment and supports chemokines as neuromodulators.

The effect of gp120 on morphine's antinociceptive and neurophysiological actions.

Recently, we have shown that morphine's analgesic activity can be attenuated by chemokines, specifically CCL5 and CXCL12. Because the HIV-1 coat protein, glycoprotein 120 (gp120), binds to the same receptors as do CCL5 and CXCL12, experiments were designed to investigate the effect of gp120 in the brain on antinociception induced by morphine in the cold-water (-3°C) tail-flick (CWT) and hot-plate (+54°C) tests. In addition, mu-opioid-receptor-mediated effects in brain periaqueductal grey (PAG) slices were examined with whole-cell patch-clamp recordings. The results showed that (1) pretreatment with gp120 itself (10, 25, 50, 100 or 133 ng, PAG) had no nociceptive effect in the CWT; (2) pretreatment with gp120 (25 or 100 ng) dose-dependently reduced antinociception induced by subcutaneous (sc) injection of morphine (3 or 6 mg/kg) or PAG injection of morphine (100 ng) in the CWT; (3) a PAG injection of gp120 (133 ng), given 30 min before sc injection of morphine (6 mg/kg), similarly reduced morphine antinociception in the hot-plate test; (4) the inhibitory effect of gp120 on morphine-induced antinociception in the CWT was reversed by AMD3100, an antagonist of CXCR4; (5) pretreatment of slices with gp120 (200 pM) prevented morphine (10 µM)-induced hyperpolarization and reduction of input resistance in PAG neurons. Electrophysiology studies paralleled gp120-induced desensitization of a mu-opioid-receptor-mediated response in PAG neurons at the single-cell level. These studies are the first to demonstrate that the analgesic activity of morphine can be reduced by the presence of gp120 in the PAG and that pretreatment with AMD3100 is able to restore the analgesic effects of morphine.

CRF-5-HT interactions in the dorsal raphe nucleus and motivation for stress-induced opioid reinstatement.

RATIONALE: The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous data show that stressors can inhibit 5-HT neuronal activity and release by stimulating the release of the stress neurohormone corticotropin-releasing factor (CRF) within the serotonergic dorsal raphe nucleus (DRN). The inhibitory effects of CRF on 5-HT DRN neurons are indirect, mediated by CRF-R1 receptors located on GABAergic afferents. OBJECTIVES: We tested the hypothesis that DRN CRF-R1 receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). We also examined the role of this circuitry in stress-induced negative affective state with 22-kHz distress ultrasonic vocalizations (USVs), which are naturally emitted by rats in response to environmental challenges such as pain, stress, and drug withdrawal. METHODS: First, we tested if activation of CRF-R1 receptors in the DRN with the CRF-R1-preferring agonist ovine CRF (oCRF) would reinstate morphine CPP and then if blockade of CRF-R1 receptors in the DRN with the CRF-R1 antagonist NBI 35965 would attenuate swim stress-induced reinstatement of morphine CPP. Second, we tested if intra-DRN pretreatment with NBI 35965 would attenuate foot shock stress-induced 22-kHz USVs. RESULTS: Intra-DRN injection of oCRF reinstated morphine CPP, while intra-DRN injection of NBI 35965 attenuated swim stress-induced reinstatement. Moreover, intra-DRN pretreatment with NBI 35965 significantly reduced 22-kHz distress calls induced by foot shock. CONCLUSIONS: These data provide evidence that stress-induced negative affective state is mediated by DRN CRF-R1 receptors and may contribute to reinstatement of morphine CPP.

Corticotropin-releasing factor increases GABA synaptic activity and induces inward current in 5-hydroxytryptamine dorsal raphe neurons.

Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.

Endocannabinoids, stress signaling, and the locus coeruleus-norepinephrine system.

The endocannabinoid (eCB) system has been implicated in a variety of physiological functions due to abundant expression of its receptors and endogenous ligands in the central nervous system. Substantial progress has been made in understanding how the eCB system influences the brain norepinephrine (NE) system, an important neurochemical target in the continued development of new therapies for stress-induced psychiatric disorders. We, and others, have characterized the neuroanatomical, biochemical and pharmacological effects of cannabinoid receptor modulation on brain noradrenergic circuitry and defined how molecular elements of the eCB system are positioned to directly impact the locus coeruleus (LC)-prefrontal cortex pathway, a neural circuit well recognized for contributing to symptoms of hyperarousal, a key pathophysiological feature of stress-related disorders. We also described molecular and electrophysiological properties of LC noradrenergic neurons and NE release in the medial prefrontal cortex under conditions of cannabinoid type 1 receptor deletion. Finally, we identified how stress influences cannabinoid modulation of the coeruleo-cortical pathway. A number of significant findings emerged from these studies that will be summarized in the present review and have important implications for clinical studies targeting the eCB system in the treatment of stress-induced psychiatric disorders.

PLDT (planarian light/dark test): an invertebrate assay to quantify defensive responding and study anxiety-like effects.

BACKGROUND: Planarians, like rodents, instinctively spend more time in dark versus light environments when given a choice. This behavioral phenomenon is called negative phototaxis, which may reflect defensive responding related to an anxiety-like phenotype. NEW METHOD: We propose a planarian light/dark test, designated PLDT, to predict anxiogenic- or anxiolytic-like effects. Experimentally, we placed a planarian at the midline of a Petri dish, containing test compound or water, that was split evenly into light and dark compartments and determined time spent in the light over 10min. RESULTS: A clinically-approved benzodiazepine agonist (clorazepate; 10µM) increased time spent in the light whereas an inverse benzodiazepine agonist (FG-7142; 1, 10µM) produced the opposite response. Fluoxetine (1µM) or ethanol (1%), as well as the 'bath salt' cathinone S-mephedrone (300µM), enhanced time spent in the light. Planarians exposed to predator (frog) odor spent more time in the dark. COMPARISON WITH EXISTING METHODS: The light/dark box (LDB) test in rodents is used to screen putative medications for possible anxiolytic and anxiogenic effects. Our results showing that time spent in the light by planarians is enhanced by common anxiety-relieving drugs (e.g. benzodiazepine agonist, ethanol, fluoxetine) and decreased by anxiogenic substances (e.g. predator odor, benzodiazepine inverse agonist) reveal directionally similar effects in the established (LDB) and new (PLDT) assays. CONCLUSION: Our data identify the PLDT as a cost-effective, invertebrate assay for quantifying the effects of practically any water-soluble substance on defensive responding and for studying and teaching anxiety-like responses in a living organism.

Cellular effects of swim stress in the dorsal raphe nucleus.

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.

Effects of cocaine history on postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats.

The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Stressors and stress hormones can inhibit the dorsal raphe nucleus (DRN)-5-HT system, which composes the majority of forebrain-projecting 5-HT. This inhibition is mediated via stimulation of GABA synaptic activity at DRN-5-HT neurons. Using swim stress-induced reinstatement of morphine conditioned place-preference, recent data from our laboratory indicate that morphine history sensitizes DRN-5-HT neurons to GABAergic inhibitory effects of stress. Moreover, GABAA receptor-mediated inhibition of the serotonergic DRN is required for this reinstatement. In our current experiment, we tested the hypothesis that GABAergic sensitization of DRN-5-HT neurons is a neuroadaptation elicited by multiple classes of abused drugs across multiple models of stress-induced relapse by applying a chemical stressor (yohimbine) to induce reinstatement of previously extinguished cocaine self-administration in Sprague-Dawley rats. Whole-cell patch-clamp recordings of GABA synaptic activity in DRN-5-HT neurons were conducted after the reinstatement. Behavioral data indicate that yohimbine triggered reinstatement of cocaine self-administration. Electrophysiology data indicate that 5-HT neurons in the cocaine group exposed to yohimbine had increased amplitude of inhibitory postsynaptic currents compared to yoked-saline controls exposed to yohimbine or unstressed animals in both drug groups. These data, together with previous findings, indicate that interaction between psychostimulant or opioid history and chemical or physical stressors may increase postsynaptic GABA receptor density and/or sensitivity in DRN-5-HT neurons. Such mechanisms may result in serotonergic hypofunction and consequent dysphoric mood states which confer vulnerability to stress-induced drug reinstatement.

Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant.

Ethanol consumption in the Sprague-Dawley rat increases sensitivity of the dorsal raphe nucleus to 5,7-dihydroxytryptamine.

Alcoholism afflicts 1 in 13 US adults, and comorbidity with depression is common. Levels of serotonin (5-HT) metabolites in alcoholic or depressed humans and rat strains are lower compared to healthy counterparts. Rats bred for ethanol (EtOH) preference are common in EtOH studies, however out-bred strains better model the range of EtOH consumption in humans. We examined voluntary EtOH consumption in out-bred Sprague-Dawley (SD) rats placed in the 20% EtOH intermittent access drinking paradigm (IA). Acquisition of 20% EtOH consumption (g EtOH/kg/24h) was assessed during the first 6-8 weeks of IA. Rats naturally separated into two groups (Drinkers or Non-drinkers) based on EtOH intake above or below 0.5 g/kg/24h prior to treatment intervention. We examined the effect of central 5-HT depletion on EtOH consumption by infusing 5,7-dihyroxytryptamine (5,7-DHT; i.c.v., 200-300 µg) or vehicle and measured EtOH consumption for 4 weeks post-operatively in IA. Compared to baseline, there was no effect of vehicle or 5,7-DHT on EtOH consumption during the post-operative period. Quantification of 5-HT depletion in the dorsal raphe nucleus (DRN) using tryptophan hydroxylase-2 (TPH2) immunohistochemistry resulted in a 76% decrease in staining with 5,7-DHT treatment. Interestingly, preservation of the ventromedial (VM) sub-regions was evident in all animals treated with 5,7-DHT, regardless of drinking behavior. In addition, Drinkers treated with 5,7-DHT had significantly more TPH2 depletion in the DRN compared to Non-drinkers. Our findings indicate that out-bred SD rats exhibit a natural EtOH consumption behavior (Drinker or Non-drinker) that is stable across time and independent of 5-HT depletion in the CNS. In addition, rats that regularly consumed >0.5 g EtOH/kg had greater sensitivity to 5,7-DHT in the DRN, indicating an interaction between EtOH and sensitivity of DRN 5-HT cells to neurotoxic substances. This may contribute to the dysfunctionality of the 5-HT system in alcoholic humans and lead to a better understanding of current pharmacological treatments for this addiction.

Dorsal raphe 5-HT(2C) receptor and GABA networks regulate anxiety produced by cocaine withdrawal.

The serotonin system is intimately linked to both the mediation of anxiety and long-term effects of cocaine, potentially through interaction of inhibitory 5-HT2C receptor and gamma-aminobutyric acid (GABA) networks. This study characterized the function of the dorsal raphe (DR) 5-HT2C receptor and GABA network in anxiety produced by chronic cocaine withdrawal. C57BL/6 mice were injected with saline or cocaine (15 mg/kg) 3 times daily for 10 days, and tested on the elevated plus maze 30 min, 25 h, or 7 days after the last injection. Cocaine-withdrawn mice showed heightened anxiety-like behavior at 25 h of withdrawal, as compared to saline controls. Anxiety-like behavior was not different when mice were tested 30 min or 7 days after the last cocaine injection. Electrophysiology data revealed that serotonin cells from cocaine-withdrawn mice exhibited increased GABA inhibitory postsynaptic currents (IPSCs) in specific DR subregions dependent on withdrawal time (25 h or 7 d), an effect that was absent in cells from non-withdrawn mice (30 min after the last cocaine injection). Increased IPSC activity was restored to baseline levels following bath application of the 5-HT2C receptor antagonist, SB 242084. In a separate cohort of cocaine-injected mice at 25 h of withdrawal, both global and intra-DR blockade of 5-HT2C receptors prior to elevated plus maze testing attenuated anxiety-like behavior. This study demonstrates that DR 5-HT2C receptor blockade prevents anxiety-like behavior produced by cocaine withdrawal, potentially through attenuation of heightened GABA activity, supporting a role for the 5-HT2C receptor in mediating anxiety produced by cocaine withdrawal.