A dorsal, but not ventral, hippocampal circuit is required for expression of heroin's contextually conditioned immune effects

Drugs of abuse, like opioids, cause a diverse array of physiological effects. These effects can become conditioned to occur to any stimulus, for example an environmental context, that becomes associated with drug use. In terms of drug reward conditioning, exposure to drug-paired contexts can elicit craving and re-engagement in drug seeking behaviors, promoting relapse to drug use. Similarly, the immune modulating effects of opioids can be conditioned to occur with exposure to an opioid-paired context. Therefore, exposure to drug-paired contexts can significantly exacerbate both the health consequences and risk of relapse with drugs of abuse. Understanding the neurological mechanisms that allow for the expression of contextually conditioned effects will allow us to better combat the problem of drug abuse. Brain regions governing opioid conditioned reward and immune responses have previously been investigated, but how these regions interact with each other is not fully understood. Previous studies show that the nucleus accumbens, basolateral amygdala, and hippocampus are required for expression of heroin contextually conditioned immune modulation, and this overlaps with what brain regions are required for contextually conditioned reward. The hippocampus is vital for encoding context, and we hypothesize that it instigates the motivational and immunological changes with exposure to a drug-paired context by engaging the other brain regions involved. The present studies are designed to further characterize the hippocampus’ role in contextually conditioned drug behaviors by manipulating this region’s predominant outgoing projections prior to expression of heroin contextually conditioned immune modulation. The hippocampus is not a homogeneous structure, and the dorsal and ventral aspects of the hippocampus connect anatomically to distinct groups of brain regions. Therefore, understanding the relative importance of the dorsal and ventral outputs from the hippocampus will give a clearer understanding of how the hippocampus relays information about context to other brain regions. The chemogenetic technique, designer receptors exclusively activated by designer drugs (DREADDs), lends itself well to inhibiting particular neurons in hippocampal output regions and building toward an understanding of hippocampal circuits. An experiment in Chapter 2 tested whether output from the dorsal hippocampus is required for the expression of heroin contextually conditioned immune modulation. Chapter 3’s experiment tested whether output from the ventral hippocampus is required for the expression of heroin contextually conditioned immune modulation. Chemogenetic inhibition of the dorsal hippocampus output, but not inhibition of the ventral hippocampus output, attenuated the expression of heroin conditioned immune modulation. Thus, Chapter 4’s experiment tested whether a specific anatomical connection between the dorsal hippocampus and retrosplenial cortex is required for expression of conditioned immune modulation. Chemogenetic inhibition of the specific projection from the dorsal hippocampus to the retrosplenial cortex did not attenuate the expression of heroin conditioned immune modulation, leading to the conclusion that this specific projection is not required. The results from these experiments begin to build a picture of how the hippocampus, and its representation of context, can influence immune function and perhaps also the behaviors that lead to cycles of drug abuse.Doctor of Philosoph

Acquisition of Heroin Conditioned Immunosuppression Requires IL-1 Signaling in the Dorsal Hippocampus

Opioid users experience increased infection rates. While partially attributable to direct opiate-immune interactions, conditioned immune responses contribute as well. Neural circuitry governing opioid conditioned immune responses has been investigated, but mediating mechanisms are unknown. Previous studies showed that hippocampal IL-1 signaling is required for expression of heroin conditioned immunosuppression following learning. Current studies were designed to further characterize hippocampal IL-1’s role in this phenomenon by manipulating IL-1 during learning. Experiment 1 tested whether hippocampal IL-1 is required for the acquisition of heroin conditioned immunosuppression. Experiment 2 tested whether hippocampal IL-1 is required for unconditioned heroin immunosuppression. Blocking IL-1 signaling in the dorsal hippocampus with IL-1RA during each conditioning session, but not on interspersed non-conditioning days, attenuated the acquisition of heroin conditioned immunosuppression. IL-1RA treatment did not alter unconditioned immunosuppression to a single dose of heroin. Thus, IL-1 signaling may play a role in learning the association between heroin and context.Master of Art

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-1β expression in the dorsal hippocampus (DH) prior to reexposure 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

Morphine prevents the development of stress-enhanced fear learning

The current study investigates the pharmacotherapeutic use of morphine as a preventative treatment for stress-enhanced fear learning, an animal model that closely mimics symptoms of post-traumatic stress disorder (PTSD). PTSD is a chronic and debilitating anxiety disorder characterized by exaggerated fear and/or anxiety that may develop as a result of exposure to a traumatic event. In this model, rats are exposed to a severe stressor (15 foot shocks) in one environment (Context A) and then subsequently exposed to a milder form of the same stressor (single foot shock) in a different environment (Context B). Animals that did not receive prior shock treatment exhibit fear responsiveness to Context B in line with the severity of the single shock given in this context. Animals that had received prior shock treatment in Context A exhibit an exaggerated learned fear response to Context B. Furthermore, animals receiving a single dose of morphine immediately following the severe stressor in Context A continue to show an enhanced fear response in Context B. However, animals receiving repeated morphine administration (three injections) after exposure to the severe stressor in Context A or a single dose of morphine at 48 h after the severe stressor no longer exhibit an enhancement in fear learning to Context B. These results are consistent with clinical studies suggesting that morphine treatment following a severe stressor may be useful in preventing or reducing the severity of PTSD in at-risk populations

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-1β (IL-1β). 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-1β in PTSD. The results show that the severe stressor in SEFL induces a time-dependent increase in IL-1β immunoreactivity and mRNA expression within the dentate gyrus of the dorsal hippocampus (DH). There was no increase in IL-1β in the basolateral amygdala or the perirhinal cortex. Moreover, blocking the action of IL-1β 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-1β in the development of SEFL, we show that systemic morphine, a treatment which is known to reduce both PTSD and SEFL, also reduces IL-1β 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

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

Heroin-induced conditioned immunomodulation requires expression of IL-1β 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-1β) within the DH, is necessary for the expression of heroin-induced conditioned immunomodulation. Rats received five Pavlovian pairings of systemic heroin administration (1.0 mg/kg, SC) with placement into a distinct environment (conditioned stimulus, CS). Six days after conditioning, a GABAA/B agonist cocktail or IL-1β small interfering RNA (siRNA) was microinfused into the DH to inhibit neuronal activity or IL-1β 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-1β 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-1β expression in heroin-conditioned suppression of a proinflammatory immune response

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

Morphine prevents the development of stress-enhanced fear learning

The current study investigates the pharmacotherapeutic use of morphine as a preventative treatment for stress-enhanced fear learning, an animal model that closely mimics symptoms of post-traumatic stress disorder (PTSD). PTSD is a chronic and debilitating anxiety disorder characterized by exaggerated fear and/or anxiety that may develop as a result of exposure to a traumatic event. In this model, rats are exposed to a severe stressor (15 foot shocks) in one environment (Context A) and then subsequently exposed to a milder form of the same stressor (single foot shock) in a different environment (Context B). Animals that did not receive prior shock treatment exhibit fear responsiveness to Context B in line with the severity of the single shock given in this context. Animals that had received prior shock treatment in Context A exhibit an exaggerated learned fear response to Context B. Furthermore, animals receiving a single dose of morphine immediately following the severe stressor in Context A continue to show an enhanced fear response in Context B. However, animals receiving repeated morphine administration (three injections) after exposure to the severe stressor in Context A or a single dose of morphine at 48 h after the severe stressor no longer exhibit an enhancement in fear learning to Context B. These results are consistent with clinical studies suggesting that morphine treatment following a severe stressor may be useful in preventing or reducing the severity of PTSD in at-risk populations. Highlights We utilize the stress-enhanced fear learning (SEFL) model of PTSD. -Repeated morphine administration following trauma blocks SEFL -Single morphine injection given immediately following trauma has no effect on SEFL. -A single morphine injection given 48-hours after the trauma block SEFL -Morphine may be an effective preventative therapy for populations at-risk for PTSD

Morphine prevents the development of stress-enhanced fear learning

The current study investigates the pharmacotherapeutic use of morphine as a preventative treatment for stress-enhanced fear learning, an animal model that closely mimics symptoms of post-traumatic stress disorder (PTSD). PTSD is a chronic and debilitating anxiety disorder characterized by exaggerated fear and/or anxiety that may develop as a result of exposure to a traumatic event. In this model, rats are exposed to a severe stressor (15 foot shocks) in one environment (Context A) and then subsequently exposed to a milder form of the same stressor (single foot shock) in a different environment (Context B). Animals that did not receive prior shock treatment exhibit fear responsiveness to Context B in line with the severity of the single shock given in this context. Animals that had received prior shock treatment in Context A exhibit an exaggerated learned fear response to Context B. Furthermore, animals receiving a single dose of morphine immediately following the severe stressor in Context A continue to show an enhanced fear response in Context B. However, animals receiving repeated morphine administration (three injections) after exposure to the severe stressor in Context A or a single dose of morphine at 48 h after the severe stressor no longer exhibit an enhancement in fear learning to Context B. These results are consistent with clinical studies suggesting that morphine treatment following a severe stressor may be useful in preventing or reducing the severity of PTSD in at-risk populations. Highlights We utilize the stress-enhanced fear learning (SEFL) model of PTSD. -Repeated morphine administration following trauma blocks SEFL -Single morphine injection given immediately following trauma has no effect on SEFL. -A single morphine injection given 48-hours after the trauma block SEFL -Morphine may be an effective preventative therapy for populations at-risk for PTSD