Dorsal hippocampal astrocytes mediate the development of heroin withdrawal-enhanced fear learning.

There is a significant co-occurrence of opioid use disorder (OUD) and post-traumatic stress disorder (PTSD) in clinical populations. However, the neurobiological mechanisms linking chronic opioid use, withdrawal, and the development of PTSD are poorly understood. Our previous research has shown that proinflammatory cytokines, expressed primarily by astrocytes in the dorsal hippocampus (DH), play a role in the development of heroin withdrawal-enhanced fear learning (HW-EFL), an animal model of PTSD-OUD comorbidity. Given the role of astrocytes in memory, fear learning, and opioid use, our experiments aimed to investigate their involvement in HW-EFL. Experiment 1 examined the effect of withdrawal from chronic heroin administration on GFAP surface area and volume, and identified increased surface area and volume of GFAP immunoreactivity in the dentate gyrus (DG) following 24-hour heroin withdrawal. Experiment 2 examined astrocyte morphology and synaptic interactions at the 24-hour withdrawal timepoint using an astroglial membrane-bound GFP (AAV5-GfaABC1D-lck-GFP). Although we did not detect significant changes in surface area and volume of GfaABC1D-Lck-GFP labelled astrocytes, we did observe a significant increase in the colocalization of astrocyte membranes with PSD-95 (postsynaptic density protein 95) in the DG. Experiment 3 tested if stimulating astroglial Gi signaling in the DH alters HW-EFL, and our results demonstrate this manipulation attenuates HW-EFL. Collectively, these findings contribute to our current understanding of the effects of heroin withdrawal on astrocytes and support the involvement of astrocytes in the comorbid relationship between opioid use and anxiety disorders.

Sex-differences in anxiety, neuroinflammatory markers, and enhanced fear learning following chronic heroin withdrawal.

Post-traumatic stress disorder (PTSD) and opioid use disorder (OUD) are comorbid in clinical populations. However, both pre-clinical and clinical studies of these co-occurring disorders have disproportionately represented male subjects, limiting the applicability of these findings. Our previous work has identified chronic escalating heroin administration and withdrawal can produce enhanced fear learning. This behavior is associated with an increase in dorsal hippocampal (DH) interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and glial fibrillary acidic protein (GFAP) immunoreactivity. Further, we have shown that these increases in IL-1ß and TNF-α are mechanistically necessary for the development of enhanced fear learning. Although these are exciting findings, this paradigm has only been studied in males. The current studies aim to examine sex differences in the behavioral and neuroimmune effects of chronic heroin withdrawal and future enhanced fear learning. In turn, we determined that chronic escalating heroin administration can produce withdrawal in female rats comparable to male rats. Subsequently, we examined the consequence of heroin withdrawal on future enhanced fear learning and IL-1ß, TNF-α, and GFAP immunoreactivity. Strikingly, we identified sex differences in these neuroimmune measures, as chronic heroin administration and withdrawal does not produce enhanced fear learning or immunoreactivity changes in females. Moreover, we determined whether heroin withdrawal produces short-term and long-term anxiety behaviors in both female and males. Collectively, these novel experiments are the first to test whether heroin withdrawal can sensitize future fear learning, produce neurobiological changes, and cause short-term and long-term anxiety behaviors in female rats.

Chronic ethanol consumption exacerbates future stress-enhanced fear learning, an effect mediated by dorsal hippocampal astrocytes.

BACKGROUND: Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are highly comorbid, yet there is a lack of preclinical research investigating how prior ethanol (EtOH) dependence influences the development of a PTSD-like phenotype. Furthermore, the neuroimmune system has been implicated in the development of both AUD and PTSD, but the extent of glial involvement in this context remains unclear. A rodent model was developed to address this gap in the literature. METHODS: We used a 15-day exposure to the 5% w/v EtOH low-fat Lieber-DeCarli liquid diet in combination with the stress-enhanced fear learning (SEFL) paradigm to investigate the effects of chronic EtOH consumption on the development of a PTSD-like phenotype. Next, we used a reverse transcription quantitative real-time polymerase chain reaction to quantify mRNA expression of glial cell markers GFAP (astrocytes) and CD68 (microglia) following severe footshock stress in EtOH-withdrawn rats. Finally, we tested the functional contribution of dorsal hippocampal (DH) astrocytes in the development of SEFL in EtOH-dependent rats using astrocyte-specific Gi designer receptors exclusively activated by designer drugs (Gi -DREADD). RESULTS: Results demonstrate that chronic EtOH consumption and withdrawal exacerbate future SEFL. Additionally, we found significantly increased GFAP mRNA expression in the dorsal and ventral hippocampus and amygdalar complex following the severe stressor in EtOH-withdrawn animals. Finally, the stimulation of the astroglial Gi -DREADD during EtOH withdrawal prevented the EtOH-induced enhancement of SEFL. CONCLUSIONS: Collectively, results indicate that prior EtOH dependence and withdrawal combined with a severe stressor potentiate future enhanced fear learning. Furthermore, DH astrocytes significantly contribute to this change in behavior. Overall, these studies provide insight into the comorbidity of AUD and PTSD and the potential neurobiological mechanisms behind increased susceptibility to a PTSD-like phenotype in individuals with AUD.

MDMA administration attenuates hippocampal IL-ß immunoreactivity and subsequent stress-enhanced fear learning: An animal model of PTSD.

Post-traumatic stress disorder (PTSD) is a devastating disorder that involves maladaptive changes in immune status. Using the stress-enhanced fear learning (SEFL) paradigm, an animal model of PTSD, our laboratory has demonstrated increased pro-inflammatory cytokine immunoreactivity in the hippocampus following severe stress. Recent clinical trials have demonstrated 3,4-methylenedioxymethamphetamine (MDMA)-assisted psychotherapy as an effective novel treatment for PTSD. Interestingly, MDMA has been shown to have an immunosuppressive effect in both pre-clinical and clinical studies. Therefore, we predict MDMA administration may attenuate SEFL, in part, due to an immunosuppressive mechanism. The current studies test the hypothesis that MDMA is capable of attenuating SEFL and inducing alterations in expression of TNF-α, IL-1ß, glial fibrillary acidic protein (GFAP), an astrocyte specific marker, and ionized calcium-binding adapter molecule -1 (IBA-1), a microglial specific marker, in the dorsal hippocampus (DH) following a severe stressor in male animals. To this end, experiment 1 determined the effect of MDMA administration 0, 24, and 48 h following a severe foot shock stressor on SEFL. We identified that MDMA administration significantly attenuated SEFL. Subsequently, experiment 2 determined the effect of MDMA administration following a severe stressor on the expression of TNF-α, IL-1ß, GFAP, and IBA-1 in the DH. We found that MDMA administration significantly attenuated stress-induced IL-1ß and stress-reduced IBA-1 but had no effect on TNF-α or GFAP. Overall, these results support the hypothesis that MDMA blocks SEFL through an immunosuppressive mechanism and supports the use of MDMA as a potential therapeutic agent for those experiencing this disorder. Together, these experiments are the first to examine the effect of MDMA in the SEFL model and these data contribute significantly towards the clinical PTSD findings.

GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration.

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

Hippocampal TNF-α Signaling Mediates Heroin Withdrawal-Enhanced Fear Learning and Withdrawal-Induced Weight Loss.

There is significant comorbidity of opioid use disorder (OUD) and post-traumatic stress disorder (PTSD) in clinical populations. However, the neurobiological mechanisms underlying the relationship between chronic opioid use and withdrawal and development of PTSD are poorly understood. Our previous work identified that chronic escalating heroin administration and withdrawal can produce enhanced fear learning, an animal model of hyperarousal, and is associated with an increase in dorsal hippocampal (DH) interleukin-1ß (IL-1ß). However, other cytokines, such as TNF-α, work synergistically with IL-1ß and may have a role in the development of enhanced fear learning. Based on both translational rodent and clinical studies, TNF-α has been implicated in hyperarousal states of PTSD, and has an established role in hippocampal-dependent learning and memory. The first set of experiments tested the hypothesis that chronic heroin administration followed by withdrawal is capable of inducing alterations in DH TNF-α expression. The second set of experiments examined whether DH TNF-α expression is functionally relevant to the development of enhanced fear learning. We identified an increase of TNF-α immunoreactivity and positive cells at 0, 24, and 48 h into withdrawal in the dentate gyrus DH subregion. Interestingly, intra-DH infusions of etanercept (TNF-α inhibitor) 0, 24, and 48 h into heroin withdrawal prevented the development of enhanced fear learning and mitigated withdrawal-induced weight loss. Overall, these findings provide insight into the role of TNF-α in opioid withdrawal and the development of anxiety disorders such as PTSD.

Female rats express heroin-induced and -conditioned suppression of peripheral nitric oxide production in response to endotoxin challenge.

Opioids and opioid-conditioned stimuli (CS) negatively alter host immunity, impairing the response to pathogens during opioid use and following drug cessation. Using male rats, our laboratory has determined that heroin or heroin-CS exposure preceding a lipopolysaccharide (LPS) challenge markedly suppresses normal induction of peripheral pro-inflammatory biomarkers. Presently, it is unknown if these heroin-induced and -conditioned effects extend to the female immune response. To begin this venture, the current study tested the direct effects of heroin and heroin-CS on LPS-induced peripheral nitric oxide (NO) production in female rats. We focused investigations on peripheral NO as it is a critical pro-inflammatory molecule necessary for pathogen resistance. In Experiment 1, male and female Lewis rats were administered 0 (Saline), 1, or 3 mg/kg heroin subcutaneously (s.c). Sixty minutes later, animals were injected with LPS (1 mg/kg, s.c.). Spleen and plasma samples were collected 6 h later to examine NO production through inducible NO synthase (iNOS) expression and nitrate/nitrite concentration, respectively. In Experiment 2, female Lewis rats underwent five, 60-minute context conditioning sessions with heroin (1 mg/kg, s.c.) or saline. On test day, CS-exposed and control (home cage) animals were injected with LPS (1 mg/kg, s.c.). Tissue was collected 6 h later to examine splenic iNOS expression and plasma nitrate/nitrite concentration. Both heroin administration alone and exposure to heroin-CS suppressed LPS-induced indices of NO production in spleen and plasma. Our results are the first to indicate that, similar to males, female rats express heroin-induced and -conditioned immunomodulation to a LPS challenge.

Dorsal hippocampal interleukin-1 signaling mediates heroin withdrawal-enhanced fear learning.

Converging evidence suggests opioid abuse can increase the incidence and severity of post-traumatic stress disorder (PTSD) in clinical populations. Interestingly, opioid withdrawal alone can produce symptoms similar to those of PTSD. Despite this association, the neural mechanisms underlying the relationship of opioid abuse, withdrawal, and PTSD is poorly understood. Our laboratory has investigated the neurobiological underpinnings of stress-enhanced fear learning (SEFL), an animal model of PTSD-like symptoms. We have previously shown that, in SEFL, a severe footshock induces an increase in dorsal hippocampal (DH) interleukin-1ß (IL-1ß), and subsequent fear learning is blocked by DH IL-1 receptor antagonism (IL-1RA). Given that opioids and stress engage similar neuroimmune mechanisms, the present experiments investigate whether the same mechanisms drive heroin withdrawal to induce a PTSD-like phenotype. First, we tested the effect of a chronic escalating heroin dose and withdrawal regimen on fear learning and found it produces enhanced future fear learning. Heroin withdrawal also induces a time-dependent, region-specific increase in IL-1ß and glial fibrillary acidic protein (GFAP) immunoreactivity within the dentate gyrus of the DH. IL-1ß was significantly colocalized with GFAP, indicating astrocytes may be involved in increased IL-1ß. Moreover, intra-DH infusions of IL-1RA 0, 24, and 48 h into heroin withdrawal prevents the development of enhanced fear learning but does not alter withdrawal-induced weight loss. Collectively, our data suggests heroin withdrawal is sufficient to produce enhanced fear learning, astrocytes may play a role in heroin withdrawal-induced IL-1ß, and DH IL-1 signaling during withdrawal mediates the development of heroin withdrawal-enhanced fear learning.

Expression of a heroin contextually conditioned immune effect in male rats requires CaMKIIα-expressing neurons in dorsal, but not ventral, subiculum and hippocampal CA1.

The physiological and motivational effects of heroin and other abused drugs become associated with environmental (contextual) stimuli during repeated drug use. As a result, these contextual stimuli gain the ability to elicit drug-like conditioned effects. For example, after context-heroin pairings, exposure to the heroin-paired context alone produces similar effects on peripheral immune function as heroin itself. Conditioned immune effects can significantly exacerbate the adverse health consequences of heroin use. Our laboratory has shown that exposure to a heroin-paired context suppresses lipopolysaccharide (LPS)-induced splenic nitric oxide (NO) production in male rats, and this effect is mediated in part by the dorsal hippocampus (dHpc). However, specific dHpc output regions, whose efferents might mediate conditioned immune effects, have not been identified, nor has the contribution of ventral hippocampus (vHpc) been investigated. Here, we evaluated the role of CaMKIIα-expressing neurons in the dHpc and vHpc main output regions by expressing Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) under a CaMKIIα promoter in the dorsal subiculum and CA1 (dSub, dCA1) or ventral subiculum and CA1 (vSub, vCA1). After context-heroin conditioning, clozapine-N-oxide (CNO, DREADD agonist) or vehicle was administered systemically prior to heroin-paired context (or home-cage control) exposure and LPS immune challenge. Chemogenetic inhibition of CaMKIIα-expressing neurons in dHpc, but not vHpc, output regions attenuated the expression of conditioned splenic NO suppression. These results establish that the main dHpc output regions, the dSub and dCA1, are critical for this context-heroin conditioned immune effect.

Characterization of the Hippocampal Neuroimmune Response to Binge-Like Ethanol Consumption in the Drinking in the Dark Model.

OBJECTIVES: Alcohol dependence leads to dysregulation of the neuroimmune system, but the effects of excessive alcohol consumption on key players of the neuroimmune response after episodic binge drinking in nondependence has not been readily assessed. These studies seek to determine how the neuroimmune system within the hippocampus responds to binge-like consumption prior to dependence or evidence of brain damage. METHODS: C57BL/6J mice underwent the drinking in the dark (DID) paradigm to recapitulate binge consumption. Immunohistochemical techniques were employed to determine the effects of ethanol on cytokine and astrocyte responses within the hippocampus. Astrocyte activation was also assessed using qRT-PCR. RESULTS: Our results indicated that binge-like ethanol consumption resulted in a 3.6-fold increase in the proinflammatory cytokine interleukin (IL)-1ß immunoreactivity in various regions of the hippocampus. The opposite effect was seen in the anti-inflammatory cytokine IL-10. Binge-like consumption resulted in a 67% decrease in IL-10 immunoreactivity but had no effect on IL-4 or IL-6 compared with the water-drinking control group. Moreover, astrocyte activation occurred following ethanol exposure as GFAP immunoreactivity was increased over 120% in mice that experienced 3 cycles of ethanol binges. PCR analyses indicated that the mRNA increased by almost 4-fold after one cycle of DID, but this effect did not persist in abstinence. CONCLUSIONS: Altogether, these findings suggest that binge-like ethanol drinking prior to dependence causes dysregulation to the neuroimmune system. This altered neuroimmune state may have an impact on behavior but could also result in a heightened neuroimmune response that is exacerbated from further ethanol exposure or other immune-modulating events.

Dorsal hippocampal neural immune signaling regulates heroin-conditioned immunomodulation but not heroin-conditioned place preference.

Repeated pairings of heroin and a context results in Pavlovian associations which manifest as heroin-conditioned appetitive responses and peripheral immunomodulation upon re-exposure to heroin-paired conditioned stimuli (CS). The dorsal hippocampus (DH) plays a key role in the neurocircuitry governing these context-heroin associations. Within the DH, expression of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) is required for heroin-conditioned peripheral immunomodulation to occur. However, the role of signaling via IL-1 receptor type 1 (IL-1R1) has not been examined. Furthermore, it has not been evaluated whether the involvement of IL-1 in associative learning extends to classically conditioned appetitive behaviors, such as conditioned place preference (CPP). The first set of experiments investigated whether DH IL-1R1 signaling during CS re-exposure modulates heroin-conditioned immunomodulation and heroin-CPP. The second set of experiments employed chemogenetic techniques to examine whether DH astroglial signaling during CS re-exposure alters the same Pavlovian responses. This line of investigation is based on previous research indicating that astrocytes support hippocampal-dependent learning and memory through the expression of IL-1ß protein and IL-1R1. Interestingly, IL-1R1 antagonism disrupted heroin-conditioned suppression of peripheral immune parameters but failed to alter heroin-CPP. Similarly, chemogenetic stimulation of Gi-signaling in DH astrocytes attenuated heroin-conditioned peripheral immunomodulation but failed to alter heroin-CPP. Collectively our data show that both IL-1R1 stimulation and astrocyte signaling in the DH are critically involved in the expression of heroin-conditioned immunomodulation but not heroin-CPP. As such these findings strongly suggest hippocampal neuroimmune signaling differentially regulates Pavlovian immunomodulatory and appetitive behaviors.

Chemogenetic Manipulation of Dorsal Hippocampal Astrocytes Protects Against the Development of Stress-enhanced Fear Learning.

Maladaptive behavioral outcomes following stress have been associated with immune dysregulation. For example, we have previously reported that stress-induced dorsal hippocampal interleukin-1ß signaling is critical to the development of stress-enhanced fear learning (SEFL). In parallel, astroglial signaling has been linked to the development of post-traumatic stress disorder (PTSD)-like phenotypes and our most recent studies have revealed astrocytes as the predominant cellular source of stress-induced IL-1ß. Here, we used chemogenetic technology and morphological analyses to further explore dorsal hippocampal astrocyte function in the context of SEFL. Using a glial-expressing DREADD construct (AAV8-GFAP-hM4Di(Gi)-mCherry), we show that dorsal hippocampal astroglial Gi activation is sufficient to attenuate SEFL. Furthermore, our data provide the first initial evidence to support the function of the glial-DREADD construct employed. Specifically, we find that CNO (clozapine-n-oxide) significantly attenuated colocalization of the Gi-coupled DREADD receptor and cyclic adenosine monophosphate (cAMP), indicating functional inhibition of cAMP production. Subsequent experiments examined dorsal hippocampal astrocyte volume, surface area, and synaptic contacts (colocalization with postsynaptic density 95 (PSD95)) following exposure to severe stress (capable of inducing SEFL). While severe stress did not alter dorsal hippocampal astrocyte volume or surface area, the severe stressor exposure reduced dorsal hippocampal PSD95 immunoreactivity and the colocalization analysis showed reduced PSD95 colocalized with astrocytes. Collectively, these data provide evidence to support the functional efficacy of the glial-expressing DREADD employed, and suggest that an astrocyte-specific manipulation, activation of astroglial Gi signaling, is sufficient to protect against the development of SEFL, a PTSD-like behavior.

Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1ß.

Post-traumatic stress disorder (PTSD) is associated with immune dysregulation. We have previously shown that severe stress exposure in a preclinical animal model of the disorder, stress-enhanced fear learning (SEFL), is associated with an increase in hippocampal interleukin-1ß (IL-1ß) and that blocking central IL-1 after the severe stress prevents the development of SEFL. Here, we tested whether blocking hippocampal IL-1 signaling is sufficient to prevent enhanced fear learning and identified the cellular source of stress-induced IL-1ß in this region. Experiment 1 tested whether intra-dorsal hippocampal (DH) infusions of interleukin-1 receptor antagonist (IL-1RA, 1.25µg per hemisphere) 24 and 48h after stress exposure prevents the development of enhanced fear learning. Experiment 2 used triple fluorescence immunohistochemistry to examine hippocampal alterations in IL-1ß, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker, and ionized calcium binding adaptor molecule -1 (Iba-1), a microglial-specific marker, 48h after exposure to the severe stressor of the SEFL paradigm. Intra-DH IL-1RA prevented SEFL and stress-induced IL-1ß was primarily colocalized with astrocytes in the hippocampus. Further, hippocampal GFAP immunoreactivity was not altered, whereas hippocampal Iba-1 immunoreactivity was significantly attenuated following severe stress. These data suggest that hippocampal IL-1 signaling is critical to the development of SEFL and that astrocytes are a predominant source of stress-induced IL-1ß.

Interleukin-1 signaling in the basolateral amygdala is necessary for heroin-conditioned immunosuppression.

Heroin administration suppresses the production of inducible nitric oxide (NO), as indicated by changes in splenic inducible nitric oxide synthase (iNOS) and plasma nitrate/nitrite. Since NO is a measure of host defense against infection and disease, this provides evidence that heroin can increase susceptibility to pathogens by directly interacting with the immune system. Previous research in our laboratory has demonstrated that these immunosuppressive effects of heroin can also be conditioned to environmental stimuli by repeatedly pairing heroin administration with a unique environmental context. Re-exposure to a previously drug-paired context elicits immunosuppressive effects similar to heroin administration alone. In addition, our laboratory has reported that the basolateral amygdala (BLA) and medial nucleus accumbens shell (mNAcS) are critical neural substrates that mediate this conditioned effect. However, our understanding of the contributing mechanisms within these brain regions is limited. It is known that the cytokine interleukin-1 (IL-1) plays an important role in learning and memory. In fact, our laboratory has demonstrated that inhibition of IL-1ß expression in the dorsal hippocampus (DH) prior to re-exposure to a heroin-paired context prevents the suppression of measures of NO production. Therefore, the present studies sought to further investigate the role of IL-1 in heroin-conditioned immunosuppression. Blockade of IL-1 signaling in the BLA, but not in the caudate putamen or mNAcS, using IL-1 receptor antagonist (IL-1Ra) attenuated heroin-conditioned immunosuppression of NO production as measured by plasma nitrate/nitrite and iNOS mRNA expression in spleen tissue. Taken together, these findings suggest that IL-1 signaling in the BLA is necessary for the expression of heroin-conditioned immunosuppression of NO production and may be a target for interventions that normalize immune function in heroin users and patient populations exposed to opiate regimens.

Modulation of Binge-like Ethanol Consumption by IL-10 Signaling in the Basolateral Amygdala.

Excessive ethanol consumption alters the neuroimmune system and particularly impacts the cytokine milieu of the CNS. Cytokine dysregulation has been shown to underlie addictive-like behaviors including alcohol abuse; however, many studies focus primarily on the proinflammatory cytokine profile during alcohol dependence. The current study furthers this research by determining the impact of excessive ethanol consumption on interleukin-10 (IL-10) and interleukin-4 (IL-4) activity in a model of non-dependent binge consumption called the "drinking in the dark" (DID) paradigm. Furthermore, the ability of IL-10 to modulate ethanol consumption was tested using site-directed pharmacology. Immunohistochemistry analyses determined that ethanol decreased IL-10 by 50 % in the basolateral amygdala (BLA) but had no effect on IL-4. Neither IL-10 nor IL-4, however, were altered in the central amygdala (CEA). Enzyme linked immunosorbent assays confirmed that IL-10 was decreased in the amygdala but not in the serum, suggesting changes of this cytokine with the DID paradigm are restricted to the central nervous system. Finally, bilateral infusions of IL-10 into the BLA, but not CeA, reduced binge-like drinking and corresponding blood ethanol concentrations without impacting either locomotor activity or anxiety-like behavioral correlates. Together, these data support the idea that alcohol abuse dysregulates specific anti-inflammatory cytokines; however, ameliorating alcohol-induced effects on cytokines, like IL-10, may prove to be an effective therapy in curbing excessive consumption.

Acquisition of heroin conditioned immunosuppression requires IL-1 signaling in the dorsal hippocampus.

Opioid users experience increased incidence of infection, which may be partially attributable to both direct opiate-immune interactions and conditioned immune responses. Previous studies have investigated the neural circuitry governing opioid conditioned immune responses, but work remains to elucidate the mechanisms mediating this effect. Our laboratory has previously shown that hippocampal IL-1 signaling, specifically, is required for the expression of heroin conditioned immunosuppression following learning. The current studies were designed to further characterize the role of hippocampal IL-1 in this phenomenon by manipulating IL-1 during learning. Experiment 1 tested whether hippocampal IL-1 is also required for the acquisition of heroin conditioned immunosuppression, while Experiment 2 tested whether hippocampal IL-1 is required for the expression of unconditioned heroin immunosuppression. We found that blocking IL-1 signaling in the dorsal hippocampus with IL-1RA during each conditioning session, but not on interspersed non-conditioning days, significantly attenuated the acquisition of heroin conditioned immunosuppression. Strikingly, we found that the same IL-1RA treatment did not alter unconditioned immunosuppression to a single dose of heroin. Thus, IL-1 signaling is not a critical component of the response to heroin but rather may play a role in the formation of the association between heroin and the context. Collectively, these studies suggest that IL-1 signaling, in addition to being involved in the expression of a heroin conditioned immune response, is also involved in the acquisition of this effect. Importantly, this effect is likely not due to blocking the response to the unconditioned stimulus since IL-1RA did not affect heroin's immunosuppressive effects.

IL-1 receptor signaling in the basolateral amygdala modulates binge-like ethanol consumption in male C57BL/6J mice.

Proinflammatory cytokines have been implicated in alcohol-induced neurodegeneration, but the role of the neuroimmune system in alcohol related behaviors has only recently come to the forefront. Herein, the effects of binge-like drinking on IL-1ß mRNA and immunoreactivity within the amygdala were measured following the "drinking in the dark" (DID) paradigm, a model of binge-like ethanol drinking in C57BL/6J mice. Moreover, the role of IL-1 receptor signaling in the amygdala on ethanol consumption was assessed. Results indicated that a history of binge-like ethanol drinking promoted a significant increase of IL-1ß mRNA expression within the amygdala, and immunohistochemistry analyses revealed that the basolateral amygdala (BLA), but not central amygdala (CeA), exhibited significantly increased IL-1ß immunoreactivity. However, Fluoro-Jade® C labeling indicated that multiple cycles of the DID paradigm were not sufficient to elicit neuronal death. Bilateral infusions of IL-1 receptor antagonist (IL-1Ra) reduced ethanol consumption when infused into the BLA but not the CeA. These observations were specific to ethanol drinking as the IL-1Ra did not alter either sucrose drinking or open-field locomotor activity. The current findings highlight a specific role for IL-1 receptor signaling in modulating binge-like ethanol consumption and indicate that proinflammatory cytokines can be induced prior to dependence or any evidence of neuronal cell death. These findings provide a framework in which to understand how neuroimmune adaptations may alter ethanol consumption and therein contribute to alcohol abuse.

The role of brain interleukin-1 in stress-enhanced fear learning.

Posttraumatic stress disorder (PTSD) has been shown to be associated with pro-inflammatory markers, including elevated plasma levels of interleukin-1ß (IL-1ß). However, the precise role of neuroinflammation and central immune signaling on the development of this debilitating psychological disorder is not known. Here, we used stress-enhanced fear learning (SEFL), an animal model of the disorder, to examine the role of central IL-1ß in PTSD. The results show that the severe stressor in SEFL induces a time-dependent increase in IL-1ß immunoreactivity and mRNA expression within the dentate gyrus of the dorsal hippocampus (DH). There was no increase in IL-1ß in the basolateral amygdala or the perirhinal cortex. Moreover, blocking the action of IL-1ß following the severe stressor with IL-1 receptor antagonist (10 µg, intracerebroventricular (i.c.v.), 24 and 48 h after the stressor) prevented the development of SEFL. To provide further support for the role of IL-1ß in the development of SEFL, we show that systemic morphine, a treatment which is known to reduce both PTSD and SEFL, also reduces IL-1ß expression in the DH induced by the severe stressor. These studies provide the first evidence that IL-1 is involved SEFL and suggest that IL-1 signaling in the brain may have a critical role in the development of PTSD.

Region-specific contribution of the ventral tegmental area to heroin-induced conditioned immunomodulation.

Dopamine receptor stimulation is critical for heroin-conditioned immunomodulation; however, it is unclear whether the ventral tegmental area (VTA) contributes to this phenomenon. Hence, rats received repeated pairings of heroin with placement into a distinct environmental context. At test, they were re-exposed to the previously heroin-paired environment followed by systemic lipopolysaccharide treatment to induce an immune response. Bilateral GABA agonist-induced neural inactivation of the anterior, but not the posterior VTA, prior to context re-exposure inhibited the ability of the heroin-paired environment to suppress peripheral nitric oxide and tumor necrosis factor-α expression, suggesting a role for the anterior VTA in heroin-conditioned immunomodulation.

Heroin-induced conditioned immunomodulation requires expression of IL-1ß in the dorsal hippocampus.

Opioid-associated environmental stimuli elicit robust immune-altering effects via stimulation of a neural circuitry that includes the basolateral amygdala and nucleus accumbens. These brain regions are known to have both direct and indirect connections with the hippocampus. Thus, the present study evaluated whether the dorsal hippocampus (DH), and more specifically interleukin-1 beta (IL-1ß) within the DH, is necessary for the expression of heroin-induced conditioned immunomodulation. Rats received five Pavlovian pairings of systemic heroin administration (1.0mg/kg, SC) with placement into a distinct environment (conditioned stimulus, CS). Six days after conditioning, a GABAA/B agonist cocktail or IL-1ß small interfering RNA (siRNA) was microinfused into the DH to inhibit neuronal activity or IL-1ß gene expression prior to CS or home cage exposure. Control animals received saline or negative control siRNA microinfusions. Furthermore, all rats received systemic administration of lipopolysaccharide (LPS) to stimulate proinflammatory nitric oxide production. CS exposure suppressed LPS-induced nitric oxide production relative to home cage exposure. Inactivation of, or IL-1ß silencing in, the DH disrupted the CS-induced suppression of nitric oxide production relative to vehicle or negative control siRNA treatment. These results are the first to show a role for DH IL-1ß expression in heroin-conditioned suppression of a proinflammatory immune response.