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.

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.

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.

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.

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.

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.

Ventral tegmental area-basolateral amygdala-nucleus accumbens shell neurocircuitry controls the expression of heroin-conditioned immunomodulation.

The present investigations sought to determine whether the ventral tegmental area (VTA), basolateral amygdala (BLA), and nucleus accumbens shell (NAC) comprise a circuitry that mediates heroin-induced conditioned immunomodulation. Rats were given conditioning trials in which they received an injection of heroin upon placement into a distinctive environment. Prior to testing, rats received unilateral intra-BLA microinfusion of a D(1) antagonist concomitantly with unilateral intra-NAC shell microinfusion of an NMDA antagonist. Disconnection of the VTA-BLA-NAC circuit impaired the ability of the heroin-paired environment to suppress lipopolysaccharide-induced immune responses, defining for the first time a specific neural circuit involved in conditioned neural-immune interactions.

Dopamine D1 receptors within the basolateral amygdala mediate heroin-induced conditioned immunomodulation.

This study investigates the role of basolateral amygdala (BLA) dopamine in heroin-induced conditioned immunomodulation. Animals underwent conditioning in which heroin administration was repeatedly paired with placement into a conditioning chamber. Six days after the final conditioning session animals were returned to the chamber and received intra-BLA microinfusions of dopamine, D(1) or D(2), antagonist. Antagonism of D(1), but not D(2), receptors within the BLA blocked the suppressive effect of heroin-associated environmental stimuli on iNOS, TNF-α and IL-1ß. This study is the first to demonstrate that the expression of heroin's conditioned effects on proinflammatory mediators require dopamine D(1) receptors within the BLA.

Evidence for the nucleus accumbens as a neural substrate of heroin-induced immune alterations.

Administration of opioid drugs such as heroin produces several immunosuppressive effects, including decreases in natural killer (NK) cell activity, lymphocyte proliferative responses, and nitric oxide production. Interestingly, opioids have been shown to alter many immune parameters indirectly by modulating the immunoregulatory actions of the central nervous system. Recently, it has been demonstrated that morphine inhibits NK cell activity through a neural pathway that requires the activation of dopamine D(1) receptors in the nucleus accumbens shell. The present study examined whether the nucleus accumbens also mediates the effects of heroin, a more commonly abused opioid, on several parameters of immune status in Lewis rats. The results showed that bilateral administration of the dopamine D(1) receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390; 0.015 and 0.15 microg/side) into the nucleus accumbens shell blocked decreases in splenic NK activity produced by heroin (3 mg/kg s.c.) but did not attenuate the suppression of splenocyte proliferative responses to concanavalin-A or lipopolysaccharide (LPS). A subsequent experiment was performed to evaluate the effect of D(1) receptor antagonism on LPS-induced expression of inducible nitric-oxide synthase (iNOS) in vivo. These results showed that intra-accumbens SCH-23390 administration prevented heroin-induced reductions of iNOS mRNA expression in spleen, liver, and lung tissues and attenuated the suppression of nitric oxide levels in plasma. Collectively, these findings indicate that nucleus accumbens dopamine D(1) receptors are critically involved in heroin-induced immune alterations.

Conditioned effects of heroin on proinflammatory mediators require the basolateral amygdala.

Heroin administration alters the induction of nitric oxide, a molecule known to play a critical role in immune function. Previous research has shown that these alterations can be conditioned to environmental stimuli that have been associated with drug administration. Little is known about the brain areas that mediate these effects; however, the basolateral amygdala (BLA) has been implicated in the formation of stimulus-reward associations within models of drug abuse. The present study sought to determine whether inactivation of the BLA would alter heroin's conditioned effects on the expression of inducible nitric oxide synthase (iNOS) and the proinflammatory cytokines TNF-alpha and IL-1beta in the rat. The conditioning procedure involved repeated pairing of heroin with placement into a standard conditioning chamber. To test the conditioned response, animals were returned to the previously drug-paired environment 6 days after the final conditioning session. Prior to testing, animals received intra-BLA microinfusions of a mixture of the GABA agonists muscimol and baclofen. Following removal from the chambers on test day, all animals received subcutaneous lipopolysaccharide to induce systemic expression of iNOS, TNF-alpha and IL-1beta. Analyses using real-time RT-PCR indicated that inactivation of the BLA blocked the suppressive effect of heroin-associated environmental stimuli on iNOS induction and on the expression of the proinflammatory cytokines TNF-alpha and IL-1beta in spleen and liver tissue. This study is important because it is the first to demonstrate that heroin's conditioned effects on proinflammatory mediators require the BLA. These findings may have significant implications for the treatment of heroin users.

Transcriptomic molecular markers for screening human colon cancer in stool and tissue.

There is a need for sensitive and specific diagnostic molecular markers that can be used to monitor early patterns of gene expression in non-invasive exfoliated colonocytes shed in the stool, and in situ in adenoma-carcinoma epithelium of the colon. RNA-based detection methods are more comprehensive than either DNA-, protein- or methylation-based screening methods. By routinely and systematically being able to perform quantitative gene expression studies on these samples using less than ten colon cancer genes selected by the enormous resources of the National Cancer Institute's Cancer Genome Anatomy Project, we were able to monitor changes at various stages in the neoplastic process, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages. Although the expression of some of the genes tested in tissue showed less variability in normal or cancerous patients than in stool, the stool by itself is suitable for screening. Thus, a transcriptomic approach using stool or tissue samples promises to offer more sensitivity and specificity than currently used molecular screening methods for colon cancer. A larger prospective clinical study utilizing stool and tissue samples derived from many control and colon cancer patients, to allow for a statistically valid analysis, is now urgently required to determine the true sensitivity and specificity of the transcriptomic screening approach for this preventable cancer.

Standardization for transcriptomic molecular markers to screen human colon cancer.

Establishing test performance criteria for a transcriptomic colon cancer marker approach must be carried out in a standardized fashion in order tso ensure that the test will perform the same way in any laboratory, anywhere. Condition of sample preservation and shipping prior to total RNA extraction is critical, and we recommend preserving stool samples in an appropriate preservative and shipping them in cold packs so as to keep stools at 4 degrees C. It is not necessary to isolate colonocytes to obtain adequate RNA for testing. It is, however, important to obtain samples from both mucin-rich and non-mucin rich to have a good representation of both left- and right-side colon cancers. Employing a commercial total RNA extraction kit that contains an RLT buffer from Qiagen Corporation (Valencia, CA, USA) removes bacterial RNA from stool preparations and results in a high yield of undegraded RNA of human origin. Genes selected based on the enormous resources of NCI's Cancer Genome Anatomy project give good results. Primers for PCR should span more than one exon. Use of semiquantitative PCR, preferably with several reference housekeeping genes of various copy numbers, depending on tested genes, should enhance confidence in the quantitative results. Having standardized the testing conditions in our ongoing work, it is now imperative that a larger prospective randomized clinical study utilizing stool and tissue samples derived from several control and colon cancer patients, to allow for statistically valid analyses, be conducted in order to determine the true sensitivity and specificity of the transcriptomic screening approach for this cancer whose incidence is on the rise worldwide.

Morphine-induced alterations of immune status are blocked by the dopamine D2-like receptor agonist 7-OH-DPAT.

Morphine administration produces profound effects on the immune system, including reductions in natural killer cell activity, mitogen-induced lymphocyte proliferation, and cytokine production. Although it has been established that the activation of central nervous system (CNS) micro-opioid receptors by morphine induces immunomodulation, little is known about the neural mechanisms underlying such processes. Interestingly, it has been shown that the dopamine (DA) D2-like receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) blocks the effect of morphine on a number of behaviors that are mediated by central dopamine pathways. The present study examined whether dopamine is involved in the immunomodulatory effects of morphine. In separate experiments, 7-OH-DPAT was administered either systemically (subcutaneous, s.c.) or centrally (intracerebroventricularly, i.c.v.) prior to morphine treatment in male Lewis rats. The results demonstrate that both systemic and central administration of 7-OH-DPAT attenuate the suppressive effect of morphine on several measures of immune status. Overall, these findings provide the first evidence that CNS dopaminergic mechanisms are directly involved in morphine-induced immunomodulation.

Improved methods for extracting RNA from exfoliated human colonocytes in stool and RT-PCR analysis.

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.