The Stressed Gut: Region-specific Immune and Neuroplasticity Changes in Response to Chronic Psychosocial Stress.

Background/Aims: Chronic psychological stress affects gastrointestinal physiology which may underpin alterations in the immune response and epithelial transport, both functions are partly regulated by enteric nervous system. However, its effects on enteric neuroplasticity are still unclear. This study aims to investigate the effects of chronic unpredictable psychological stress on intestinal motility and prominent markers of enteric function. Methods: Adult male C57BL/6J mice were exposed to 19 day of unpredictable stress protocol schedule of social defeat and overcrowding. We investigated the effects on plasma corticosterone, food intake, and body weight. In vivo gastrointestinal motility was assessed by fecal pellet output and by whole-gastrointestinal transit (using the carmine red method). Tissue monoamine level, neural and glial markers, neurotrophic factors, monoamine signaling, and Toll-like receptor expression in the proximal and distal colon, and terminal ileum were also assessed. Results: Following chronic unpredictable psychological stress, stressed mice showed increased food intake and body weight gain (P < 0.001), and reduced corticosterone levels (P < 0.05) compared to control mice. Stressed mice had reduced stool output without differences in water content, and showed a delayed gastrointestinal transit compared to control mice (P < 0.05). Stressed mice exhibited decreased mRNA expression of tyrosine hydroxylase (Th), brain-derived neurotrophic factor (Bdnf) and glial cell-derived neurotrophic factor (Gdnf), as well as Toll-like receptor 2 (Tlr2) compared to control (P < 0.05), only proximal colon. These molecular changes in proximal colon were associated with higher levels of monoamines in tissue. Conclusion: Unpredictable psychological chronic stress induces region-specific impairment in monoamine levels and neuroplasticity markers that may relate to delayed intestinal transit.

Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain.

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-ß1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-ß1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.

Estrous cycle and ovariectomy-induced changes in visceral pain are microbiota-dependent.

Visceral hypersensitivity (VH) is a hallmark of many functional gastrointestinal disorders including irritable bowel syndrome and is categorized by a dull, diffuse sensation of abdominal pain. Recently, the gut microbiota has been implicated in VH in male mice, but the effects in females have yet to be explored fully. To this end, we now show that somewhat surprisingly, female germ-free mice have similar visceral pain responses to colorectal distension (CRD) as their conventional controls. However, we show that although sensitivity to CRD is estrous cycle stage-dependent in conventional mice, it is not in germ-free mice. Further, ovariectomy (OVX) induced VH in conventional but not germ-free mice, and induced weight gain regardless of microbiota status. Finally, we show that estrogen-replacement ameliorated OVX-induced VH. Taken together, this study provides evidence for a major role of female sex hormones and the gut microbiota in sensation of visceral pain in females.

BMP-7 protects male and female rodents against neuropathic pain induced by nerve injury through a mechanism mediated by endogenous opioids.

Neuropathic pain is highly prevalent in pathological conditions such as diabetes, herpes zoster, trauma, etc. The severity and refractoriness to treatments make neuropathic pain a significant health concern. The transforming growth factor (TGF-ß) family of cytokines is involved in pain modulation. Bone morphogenetic proteins (BMPs) constitute the largest subgroup within the TGF-ß family. BMP-7 induces the transcription of genes coding endogenous opioid precursors in vitro. However, a nociception modulatory function for this cytokine remains unexplored in vivo. Herein, we show that BMP-7 and its type I receptors were detected in regions of the nervous system involved in pain transmission, processing, and modulation. BMP-7 haploinsufficiency confers to male and female mice a tactile hyperalgesia phenotype to mechanical stimuli, both at baseline and after sciatic nerve injury (SNI). The administration of recombinant BMP-7 (rBMP-7) reduced the severity of the allodynia after SNI in rodents without sexual dimorphism. Central administration of rBMP-7 delayed allodynia development after SNI and reduced the severity of allodynia. The opioid antagonist naloxone antagonized the antinociceptive effect of rBMP-7 in rats. The analgesic effect of morphine was significantly attenuated in BMP-7+/- mice. The antiallodynic effect of voluntary exercise after SNI, whose mechanism involves the endogenous opioid system, was hampered by BMP-7 deficiency while potentiated by rBMP-7. Our results suggest that BMP-7 may constitute a novel therapeutic target for the treatment of neuropathic pain, which improves the function of the endogenous pain-resolution mechanisms to alleviate chronic pain.

MicroRNA-30c-5p modulates neuropathic pain in rodents.

Neuropathic pain is a debilitating chronic syndrome that is often refractory to currently available analgesics. Aberrant expression of several microRNAs (miRNAs) in nociception-related neural structures is associated with neuropathic pain in rodent models. We have exploited the antiallodynic phenotype of mice lacking the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transforming growth factor-ß (TGF-ß) pseudoreceptor. We used these mice to identify new miRNAs that might be useful for diagnosing, treating, or predicting neuropathic pain. We show that, after sciatic nerve injury in rats, miR-30c-5p was up-regulated in the spinal cord, dorsal root ganglia, cerebrospinal fluid (CSF) and plasma and that the expression of miR-30c-5p positively correlated with the severity of allodynia. The administration of a miR-30c-5p inhibitor into the cisterna magna of the brain delayed neuropathic pain development and reversed fully established allodynia in rodents. The mechanism was mediated by TGF-ß and involved the endogenous opioid system. In patients with neuropathic pain associated with leg ischemia, the expression of miR-30c-5p was increased in plasma and CSF compared to control patients without pain. Logistic regression analysis in our cohort of patients showed that the expression of miR-30c-5p in plasma and CSF, in combination with other clinical variables, might be useful to help to predict neuropathic pain occurrence in patients with chronic peripheral ischemia.

Microbiota regulates visceral pain in the mouse.

The perception of visceral pain is a complex process involving the spinal cord and higher order brain structures. Increasing evidence implicates the gut microbiota as a key regulator of brain and behavior, yet it remains to be determined if gut bacteria play a role in visceral sensitivity. We used germ-free mice (GF) to assess visceral sensitivity, spinal cord gene expression and pain-related brain structures. GF mice displayed visceral hypersensitivity accompanied by increases in Toll-like receptor and cytokine gene expression in the spinal cord, which were normalized by postnatal colonization with microbiota from conventionally colonized (CC). In GF mice, the volumes of the anterior cingulate cortex (ACC) and periaqueductal grey, areas involved in pain processing, were decreased and enlarged, respectively, and dendritic changes in the ACC were evident. These findings indicate that the gut microbiota is required for the normal visceral pain sensation.

Obesity Takes Its Toll on Visceral Pain: High-Fat Diet Induces Toll-Like Receptor 4-Dependent Visceral Hypersensitivity.

Exposure to high-fat diet induces both, peripheral and central alterations in TLR4 expression. Moreover, functional TLR4 is required for the development of high-fat diet-induced obesity. Recently, central alterations in TLR4 expression have been associated with the modulation of visceral pain. However, it remains unknown whether there is a functional interaction between the role of TLR4 in diet-induced obesity and in visceral pain. In the present study we investigated the impact of long-term exposure to high-fat diet on visceral pain perception and on the levels of TLR4 and Cd11b (a microglial cell marker) protein expression in the prefrontal cortex (PFC) and hippocampus. Peripheral alterations in TLR4 were assessed following the stimulation of spleenocytes with the TLR4-agonist LPS. Finally, we evaluated the effect of blocking TLR4 on visceral nociception, by administering TAK-242, a selective TLR4-antagonist. Our results demonstrated that exposure to high-fat diet induced visceral hypersensitivity. In parallel, enhanced TLR4 expression and microglia activation were found in brain areas related to visceral pain, the PFC and the hippocampus. Likewise, peripheral TLR4 activity was increased following long-term exposure to high-fat diet, resulting in an increased level of pro-inflammatory cytokines. Finally, TLR4 blockage counteracted the hyperalgesic phenotype present in mice fed on high-fat diet. Our data reveal a role for TLR4 in visceral pain modulation in a model of diet-induced obesity, and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity present in pathologies associated to fat diet consumption.

BMP-7 attenuates left ventricular remodelling under pressure overload and facilitates reverse remodelling and functional recovery.

AIMS: TGF-ß regulates tissue fibrosis: TGF-ß promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS: We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-ß and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and ß-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-ß as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-ß-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION: The disequilibrium between BMP-7 and TGF-ß signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.

TGF-ß and opioid receptor signaling crosstalk results in improvement of endogenous and exogenous opioid analgesia under pathological pain conditions.

Transforming growth factor-ß1 (TGF-ß1) protects against neuroinflammatory events underlying neuropathic pain. TGF-ß signaling enhancement is a phenotypic characteristic of mice lacking the TGF-ß pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-ß1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of µ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of µ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-ß1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-ß1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-ß signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

Toll-like receptor 4 regulates chronic stress-induced visceral pain in mice.

BACKGROUND: Functional gastrointestinal disorders, which have visceral hypersensitivity as a core symptom, are frequently comorbid with stress-related psychiatric disorders. Increasing evidence points to a key role for toll-like receptor 4 (TLR4) in chronic pain states of somatic origin. However, the central contribution of TLR4 in visceral pain sensation remains elusive. METHODS: With pharmacological and genetic approaches, we investigated the involvement of TLR4 in the modulation of visceral pain. The TLR4-deficient and wild-type mice were exposed to chronic stress. Visceral pain was evaluated with colorectal distension. Protein expression levels for TLR4, Cd11b, and glial fibrillary acidic protein (glial cells markers) were quantified in the lumbar region of the spinal cord, prefrontal cortex (PFC), and hippocampus. To evaluate the effect of blocking TLR4 on visceral nociception, TAK-242, a selective TLR4 antagonist, was administered peripherally (intravenous) and centrally (intracerebroventricular and intra-PFC) (n = 10-12/experimental group). RESULTS: The TLR4 deficiency reduced visceral pain and prevented the development of chronic psychosocial stress-induced visceral hypersensitivity. Increased expression of TLR4 coupled with enhanced glia activation in the PFC and increased levels of proinflammatory cytokines were observed after chronic stress in wild-type mice. Administration of a TLR4 specific antagonist, TAK-242, attenuated visceral pain sensation in animals with functional TLR4 when administrated centrally and peripherally. Moreover, intra-PFC TAK-242 administration also counteracted chronic stress-induced visceral hypersensitivity. CONCLUSIONS: Our results reveal a novel role for TLR4 within the PFC in the modulation of visceral nociception and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity.

BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-ß signaling.

Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-ß (TGF-ßs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-ß signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI(-/-)) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-ß. BAMBI deletion led to a gain of myocardial TGF-ß signaling through canonical (Smads) and non-canonical (TAK1-p38 and TAK1-JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI(-/-) mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-ß targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI(-/-) mice involved TGF-ßs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-ß-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-ß remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.

Rodent models of colorectal distension.

Colorectal distension (CRD) is a widely accepted, reproducible method for assessing visceral sensitivity in both clinical and pre-clinical studies. Distension of the colon mirrors the human scenario of visceral pain with regard to intensity and referral of pain in patients. There are several readouts that can be applied to the CRD protocol depending on the species being evaluated, two of which are described in this unit. CRD can be used to measure the impact of novel compounds, strain, or genetic differences as well as the effect of physical and psychological stressors on the sensitivity of the colon. Investigation of the impact of a noxious visceral stimulus (CRD) on other systems within the body can also be carried out. Given that visceral pain is a major clinical problem and one of the most common reasons patients seek out medical advice, the ability to assess this type of pain is essential to the discovery of successful treatments. This unit outlines two protocols that describe CRD of rats and mice.

Chronic psychosocial stress induces visceral hyperalgesia in mice.

Experimental and clinical evidence has shown that chronic stress plays an important role in the onset and/or exacerbation of symptoms of functional gastrointestinal disorders. Here, we aimed to investigate whether exposure to a chronic and temporally unpredictable psychosocial stressor alters visceral and somatic nociception as well as anxiety-related behaviour. In male C57BL/6J mice, chronic stress was induced by repeated exposure to social defeat (SD, 2 h) and overcrowding (OC, 24 h) during 19 consecutive days. Visceral and somatic nociception was evaluated by colorectal distension and a hot plate, respectively. The social interaction test was used to assess social anxiety. Mice exposed to psychosocial stress developed visceral hyperalgesia and somatic hypoalgesia 24 h following the last stress session. SD/OC mice also exhibited social anxiety-like behaviour. All these changes were also associated with physiological alterations, measured as a decreased faecal pellet output and hypothalamic-pituitary-adrenal (HPA) axis disruption. Taken together, these data confirm that this mouse model of chronic psychosocial stress may be useful for studies on the pathophysiological mechanisms underlying such stress-associated disorders and to further test potential therapies.

Transforming growth factor-ß in normal nociceptive processing and pathological pain models.

The transforming growth factor-ß (TGF-ß) superfamily is a multifunctional, contextually acting family of cytokines that participate in the regulation of development, disease and tissue repair in the nervous system. The TGF-ß family is composed of several members, including TGF-ßs, bone morphogenetic proteins (BMPs) and activins. In this review, we discuss recent findings that suggest TGF-ß function as important pleiotropic modulators of nociceptive processing both physiologically and under pathological painful conditions. The strategy of increasing TGF-ß signaling by deleting "BMP and activin membrane-bound inhibitor" (BAMBI), a TGF-ß pseudoreceptor, has demonstrated the inhibitory role of TGF-ß signaling pathways in normal nociception and in inflammatory and neuropathic pain models. In particular, strong evidence suggests that TGF-ß1 is a relevant mediator of nociception and has protective effects against the development of chronic neuropathic pain by inhibiting the neuroimmune responses of neurons and glia and promoting the expression of endogenous opioids within the spinal cord. In the peripheral nervous system, activins and BMPs function as target-derived differentiation factors that determine and maintain the phenotypic identity and circuit assembly of peptidergic nociceptors. In this context, activin is involved in the complex events of neuroinflammation that modulate the expression of pain during wound healing. These findings have provided new insights into the physiopathology of nociception. Moreover, specific members of the TGF-ß family and their signaling effectors and modulator molecules may be promising molecular targets for novel therapeutic agents for pain management.

Riluzole normalizes early-life stress-induced visceral hypersensitivity in rats: role of spinal glutamate reuptake mechanisms.

BACKGROUND & AIMS: The molecular basis underlying visceral hypersensitivity in functional irritable bowel syndrome remains elusive, resulting in poor treatment effectiveness. Because alterations in spinal non-neuronal (astrocytic) glutamate reuptake are suspected to participate in chronic pain, we asked whether such processes occur in visceral hypersensitivity. METHODS: Visceral hypersensitivity was induced in Sprague-Dawley rats by maternal separation. Separated adults were given a systemic administration of riluzole (5 mg/kg), an approved neuroprotective agent activating glutamate reuptake. Visceral hypersensitivity was assessed using colorectal distension (40 mm Hg). Somatic nociception was quantified using Hot Plate, Randall-Sellito, and Hargreaves tests. Spinal proteins were quantified using immunofluorescence and Western blot. The dependence of visceral sensory function upon spinal glutamate transport was evaluated by intrathecal injection of glutamate transport antagonist DL-threo-beta-benzyloxyaspartate (TBOA). For in vitro testing of riluzole and TBOA, primary cultures of astrocytes were used. RESULTS: We show that riluzole counteracts stress-induced visceral hypersensitivity without affecting visceral response in nonseparated rats or altering nociceptive responses to somatic pain stimulation. In addition, maternal separation produces a reduction in glial excitatory amino acid transporter (EAAT)-1 with no change in EAAT-2 or gamma-amino butyric acid transporters. Stress was not associated with changes in glial fibrillary acidic protein or astrocytic morphology per se. Furthermore, visceral normosensitivity relies on spinal EAAT, as intrathecal TBOA is sufficient to induce hypersensitivity in normal rats. CONCLUSIONS: We identify spinal EAAT as a therapeutic target, and establish riluzole as a candidate to counteract gastrointestinal hypersensitivity in disorders such as irritable bowel syndrome.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-beta family in pain modulation.

Transforming growth factors-beta (TGF-betas) signal through type I and type II serine-threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-beta family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-beta signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI(-/-) mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI(-/-) mice also appeared attenuated through a mechanism involving delta-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI(-/-). Transcript levels of TGF-betas and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-beta family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.

Facilitation of avoidance behaviour in mice chronically treated with heroin or methadone.

Although the repercussion of chronic treatment with large amounts of opioids on cognitive performance is a matter of concern, the effects of opioid drugs on passive avoidance learning have been scarcely studied. Here, we analyzed the effects of prolonged administration of heroin and methadone, as well as the impact of suffering repeated episodes of withdrawal on fear-motivated learning using the passive avoidance test. Mice received chronic treatment (39 days) with methadone (10 mg/kg/24 h), associated or not with repeated withdrawal episodes, or with heroin (5 mg/kg/12 h). Our results show that, regardless of the type of treatment received, all mice displayed similar basal thermal nociceptive thresholds during 25 days of treatment. In the hot plate test, both methadone and heroin induced antinociception 30 min after drug administration. The analgesic effect was absent when measured 4 h after heroin and 12 h after methadone. Pain behavioural responses elicited by growing intensities of electric shock, applied on day 28th of treatment, were similar in all groups of mice. Our results indicate that chronic opioid treatment had promnesic effects on passive avoidance behaviour in mice, unrelated to changes in the nociceptive state.

Chronic administration of heroin to mice produces up-regulation of brain apoptosis-related proteins and impairs spatial learning and memory.

Several studies open up the possibility that chronic exposure to opioid drugs in the CNS would interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Here, we have analyzed the effects of prolonged heroin administration on sensorimotor and cognitive performance in mice, as well as the associated changes in brain expression of proteins regulating the extrinsic (FasL and Fas) and the mitochondrial (Bcl-2, Bcl-X(L), Bad and Bax) apoptotic pathways. Our findings indicate that chronic heroin did not interfere with mice performance in a battery of sensorimotor tests. On the other hand, cognitive ability in the Morris water maze and cognitive flexibility-related performance were strongly impaired by chronic heroin. These effects were associated with up-regulation of pro-apoptotic proteins such as Fas, FasL and Bad, in the cortex and hippocampus, indicating the activation of both the death receptor and the mitochondrial apoptotic pathways. Another indicator of apoptosis was the presence of TUNEL (TdT-mediated dUTP nick-end labeling) positive cells scattered throughout the brain.

Chronic methadone treatment and repeated withdrawal impair cognition and increase the expression of apoptosis-related proteins in mouse brain.

OBJECTIVES: This study analyzes the effects of prolonged administration of methadone and withdrawal on sensorimotor and cognitive performance in mice and explores the associated changes in brain expression of proteins regulating the extrinsic (FasL, Fas, and caspase-8) and the mitochondrial (Bcl-2, Bcl-x(L), Bad, and Bax) apoptotic pathways. RESULTS: Our findings indicate that, although acute methadone administration impairs some sensorimotor abilities, tolerance to most of the deleterious effects develops after chronic administration. Cognitive abilities in the Morris water maze were impaired by chronic methadone and, to a greater extent, by exposure to precipitated withdrawal every week in the course of methadone treatment. Both the chronic methadone and repeated withdrawal groups showed up-regulation of several pro-apoptotic proteins (FasL, the active fragment of caspase-8, and Bad) in the cortex and hippocampus, indicating activation of both the death-receptor and mitochondrial apoptotic pathways. In contrast, reduced expression of the apoptosis regulatory proteins FasL and Bad was found after a single administration of methadone. CONCLUSIONS: Our data suggest that neural apoptotic damage could contribute to impairment of the cognitive abilities of mice observed after chronic methadone administration and withdrawal.

Modulation of apoptosis in the mouse brain after morphine treatments and morphine withdrawal.

We have examined the effects of acute or chronic morphine and naltrexone-precipitated withdrawal on mouse brain apoptotic cell death. The associated changes in the expression of apoptosis regulatory proteins were also analyzed. After a single dose of morphine, no apoptotic cells were detected by TUNEL or active caspase-3 immunocytochemistry. Concurrently, a down-regulation of the proapoptotic proteins FasL and Bad was detected in cortical lysates. On the other hand, the brains of chronic-morphine-treated mice and abstinent mice exhibited scattered apoptotic neurons and astrocytes throughout the brain. This neurotoxic effect was accompanied by up-regulation of the proapoptotic proteins FasL, Fas, and Bad and the active fragments of caspases-8 and -3 in cortical and hippocampal lysates. Abstinent mice also displayed a reduced expression of the antiapoptotic protein Bcl-2. No changes on t-Bid expression were detected under any experimental condition. These results suggest a neurotoxic effect exerted by chronic, but not acute, morphine and its withdrawal by activating both the intrinsic and the extrinsic apoptotic pathways. The possible clinical implications of our findings are discussed.