Self-Administration of Entactogen Psychostimulants Dysregulates Gamma-Aminobutyric Acid (GABA) and Kappa Opioid Receptor Signaling in the Central Nucleus of the Amygdala of Female Wistar Rats

Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABAA receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-h sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature post-synaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats), however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (post-synaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1 mM U50488) and KOR antagonism (200 nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.

S100A9 is a functional effector of infarct wall thinning after myocardial infarction

Neutrophils infiltrate into the left ventricle (LV) early after myocardial infarction (MI) and launch a pro-inflammatory response. Along with neutrophil infiltration, LV wall thinning due to cardiomyocyte necrosis also peaks at day 1 in the mouse model of MI. To understand the correlation, we examined a previously published dataset that included day 0 (n=10) and MI day 1 (n=10) neutrophil proteome and echocardiography assessments. Out of 123 proteins, 4 proteins positively correlated with the infarct wall thinning index (1/wall thickness): histone 1.2 (r=0.62, p=0.004), S100A9 (r=0.60, p=0.005), histone 3.1 (r=0.55, p=0.01), and fibrinogen (r=0.47, p=0.04). As S100A9 was the highest ranked secreted protein, we hypothesized that S100A9 is a functional effector of infarct wall thinning. We exogenously administered S100A8/A9 at the time of MI to mice (C57BL/6J, male, 3-6 months of age, n=7M (D1), and n=5M (D3)) and compared to saline vehicle control treated mice (n=6M (D1) and n=6M (D3)) at MI days 1 and 3. At MI day 3, the S100A8/A9 group showed a 22% increase in the wall thinning index compared to saline (p=0.02), along with higher dilation and lower ejection fraction. The decline in cardiac physiology occurred subsequent to increased neutrophil and macrophage infiltration at MI day 1 and increased macrophage infiltration at D3. Our results reveal that S100A9 is a functional effector of infarct wall thinning.

Tolerance to the Behavioural and Neurochemical Effects of MDMA Following Repeated Exposure

<p>Rationale: Following repeated +/-3,4- methylenedioxymethamphetamine (MDMA) administration there is tolerance to many behavioural effects and deficits in serotonergic neurotransmission. Objectives: The present studies had three main objectives. 1. To develop a behavioural assay to examine the effects of acute and repeated MDMA exposure. 2. To use this behavioural assay to determine whether functional changes in serotonin (5-HT)2a or 5-HT2c receptors accompany tolerance to the effects of MDMA. 3. To attempt to reverse behavioural tolerance and 5-HT deficits by administering a treatment that has been shown to desensitise the 5-HT1a autoreceptor. Methods: In separate groups of rats the dose effect curves for MDMAproduced hyperactivity were determined (0.0, 1.0, 3.3, 10.0 mg/kg). In additional groups the effect of MDMA pretreatment (4 X 10mg/kg MDMA injections at 2 hour intervals) or saline vehicle on MDMA-produced hyperactivity was assessed. To determine the experimental parameters for MDMA effects in the Emergence Test (ET) separate groups of rats received MDMA (0.0, 3.3 mg/kg) and were either habituated to a hide box for various periods (15, 30, 45mins) or exposed to the test arena 3 times over a period of days (day 1, 5, 9) or injected daily in the home cage with MDMA (0.0, 3.3mg/kg) for 3 days. Emergence latency following injections of MDMA (0.0, 3.3mg/kg), the 5-HT2a /c agonistm-CPP (0.0, 0.3, 0.6 or 1.25 m/kg), the 5-HT releasing stimulant, fenfluramine (0.0, 1.0, 2.0 mg/kg), and the 5-HT2a agonist DOI (0.0, 1.0, 2.0 mg/kg) was measured. The role of 5-HT2c receptors was assessed by determining the effect of the 5-HT2c antagonist, RS102221 (0.0, 0.25, 0.5, 1.0 mg/kg). The effect of MDMA pretreatment on MDMA (0.0, 3.3 mg/kg), m-CPP (1.25 mg/kg), or fenfluramine (2.0mg/kg) induced increases in emergence latency was also assessed. The functional status of the 5-HT1a autoreceptor following MDMA pretreatment was determined by measuring the effect of the 5-HT1a agonist, 8- OHDPAT (0.0, 0.315, 0.0625, 0.125, 0.25, 0.5 mg/kg, SC), on body temperature. The ability of the 5-HT1a antagonist, WAY100635 (0.0, 0.01, 0.1, 1.0mg/kg, SC, 1 X daily for 7 days or by local injection of 0.0 or 500 ng into the dorsal raphe) to reverse the attenuation of MDMA-induced hyperactivity following MDMA pretreatment was examined. Effects of various treatments on tissue levels of 5-HT and 5-HIAA were also measured using HPLC with EC. Results: MDMA produced dose-dependent hyperactivity and tolerance was produced by MDMA pretreatment. MDMA (3.3mg/kg) increased emergence latency following a 30 minute habituation period and this effect was reduced in MDMA-pretreated rats. Fenfluramine and m-CPP but not DOI also increased emergence latency in a dose-dependent manner. RS102221 dose dependently blocked the acute effects of MDMA and m-CPP. Two weeks following MDMA pretreatment rats were tolerant to the effects of MDMA and fenfluramine, but not m-CPP. MDMA pretreatment also produced significant reductions in tissue levels of 5-HT and 5-HIAA. Subcutaneous WAY100635 administration failed to reverse the behavioural and neurochemical deficits produced by MDMA pretreatment but local administration increased MDMA-produced hyperactivity in saline and MDMA pretreated rats and reversed MDMA-produced 5-HT tissue depletions. Conclusion: The Emergence Test is a behavioural assay sensitive to the effects of acute and repeated MDMA exposure. Following MDMA pretreatment behavioural tolerance as measured by the ET is likely to be due to impaired 5-HT release rather than changes in 5-HT2a or 5-HT2c receptor responses. Because partial reversal of tolerance and 5-HT deficits following repeated MDMA administration was achieved through local DRN 5-HT1a antagonist administration the 5-HT1a autoreceptor may prove to be a clinical target for the reversal of MDMA produced deficits.</p>

Tolerance to the Behavioural and Neurochemical Effects of MDMA Following Repeated Exposure

<p>Rationale: Following repeated +/-3,4- methylenedioxymethamphetamine (MDMA) administration there is tolerance to many behavioural effects and deficits in serotonergic neurotransmission. Objectives: The present studies had three main objectives. 1. To develop a behavioural assay to examine the effects of acute and repeated MDMA exposure. 2. To use this behavioural assay to determine whether functional changes in serotonin (5-HT)2a or 5-HT2c receptors accompany tolerance to the effects of MDMA. 3. To attempt to reverse behavioural tolerance and 5-HT deficits by administering a treatment that has been shown to desensitise the 5-HT1a autoreceptor. Methods: In separate groups of rats the dose effect curves for MDMAproduced hyperactivity were determined (0.0, 1.0, 3.3, 10.0 mg/kg). In additional groups the effect of MDMA pretreatment (4 X 10mg/kg MDMA injections at 2 hour intervals) or saline vehicle on MDMA-produced hyperactivity was assessed. To determine the experimental parameters for MDMA effects in the Emergence Test (ET) separate groups of rats received MDMA (0.0, 3.3 mg/kg) and were either habituated to a hide box for various periods (15, 30, 45mins) or exposed to the test arena 3 times over a period of days (day 1, 5, 9) or injected daily in the home cage with MDMA (0.0, 3.3mg/kg) for 3 days. Emergence latency following injections of MDMA (0.0, 3.3mg/kg), the 5-HT2a /c agonistm-CPP (0.0, 0.3, 0.6 or 1.25 m/kg), the 5-HT releasing stimulant, fenfluramine (0.0, 1.0, 2.0 mg/kg), and the 5-HT2a agonist DOI (0.0, 1.0, 2.0 mg/kg) was measured. The role of 5-HT2c receptors was assessed by determining the effect of the 5-HT2c antagonist, RS102221 (0.0, 0.25, 0.5, 1.0 mg/kg). The effect of MDMA pretreatment on MDMA (0.0, 3.3 mg/kg), m-CPP (1.25 mg/kg), or fenfluramine (2.0mg/kg) induced increases in emergence latency was also assessed. The functional status of the 5-HT1a autoreceptor following MDMA pretreatment was determined by measuring the effect of the 5-HT1a agonist, 8- OHDPAT (0.0, 0.315, 0.0625, 0.125, 0.25, 0.5 mg/kg, SC), on body temperature. The ability of the 5-HT1a antagonist, WAY100635 (0.0, 0.01, 0.1, 1.0mg/kg, SC, 1 X daily for 7 days or by local injection of 0.0 or 500 ng into the dorsal raphe) to reverse the attenuation of MDMA-induced hyperactivity following MDMA pretreatment was examined. Effects of various treatments on tissue levels of 5-HT and 5-HIAA were also measured using HPLC with EC. Results: MDMA produced dose-dependent hyperactivity and tolerance was produced by MDMA pretreatment. MDMA (3.3mg/kg) increased emergence latency following a 30 minute habituation period and this effect was reduced in MDMA-pretreated rats. Fenfluramine and m-CPP but not DOI also increased emergence latency in a dose-dependent manner. RS102221 dose dependently blocked the acute effects of MDMA and m-CPP. Two weeks following MDMA pretreatment rats were tolerant to the effects of MDMA and fenfluramine, but not m-CPP. MDMA pretreatment also produced significant reductions in tissue levels of 5-HT and 5-HIAA. Subcutaneous WAY100635 administration failed to reverse the behavioural and neurochemical deficits produced by MDMA pretreatment but local administration increased MDMA-produced hyperactivity in saline and MDMA pretreated rats and reversed MDMA-produced 5-HT tissue depletions. Conclusion: The Emergence Test is a behavioural assay sensitive to the effects of acute and repeated MDMA exposure. Following MDMA pretreatment behavioural tolerance as measured by the ET is likely to be due to impaired 5-HT release rather than changes in 5-HT2a or 5-HT2c receptor responses. Because partial reversal of tolerance and 5-HT deficits following repeated MDMA administration was achieved through local DRN 5-HT1a antagonist administration the 5-HT1a autoreceptor may prove to be a clinical target for the reversal of MDMA produced deficits.</p>

A Behavioural Analysis of Serotonergic Functional Status Following MDMA Exposure

<p>Rationale +/- 3,4-Methylenedioxymethamphetamine (MDMA) produces effects on a number of neurochemical systems. Many studies have shown that repeated MDMA administration produces deficits in central serotonergic neurotransmission, which have been suggested to underlie some of the behavioural changes associated with use. Objectives The present studies sought to evaluate the functional statuses of the serotonin transporter (SERT) and the serotonin2c (5-HT2c) and serotonin2a (5-HT2a) receptors following treatment with MDMA to determine whether behavioural deficits could be attributed to alterations in these proteins. Methods Rats received a pretreatment regimen of MDMA (4 x 10mg/kg MDMA injections administered at 2h intervals) or the saline vehicle and, 2 weeks later, [3H] paroxetine binding was undertaken to assess densities of SERT. In other groups, dose-effect curves for MDMA-produced hyperactivity were determined. Additional groups were tested following a 12-week withdrawal period from MDMA in order to assess whether there was recovery of function. The functional status of the SERT was further examined by determining the effect of MDMA pretreatment on the reduction in MDMA-produced hyperactivity (0.0 - 10.0mg/kg) produced by the selective serotonin reuptake inhibitor, clomipramine (0.0 - 5.0mg/kg). The ability for the 5-HT2c receptor agonist, m-CPP (0.0 - 2.5mg/kg) to produce hypolocomotion or increased emergence latency or for the 5-HT2a receptor agonist, DOI (0.0 - 2.0mg/kg) to produce wetdog shakes (WDS) were examined in MDMA pretreated rats. The ability for the 5-HT2c receptors to modulate MDMA-produced hyperactivity was assessed by examining the effect of MDMA pretreatment on the potentiation of MDMA-produced hyperactivity produced by the selective antagonist, RS102221 (0.0 - 1.0mg/kg). Conversely, the modulatory abilities of the 5-HT2a receptors were assessed by examining the effect of MDMA pretreatment on the attenuation of MDMA-produced hyperactivity produced by the antagonist, ritanserin (0.0 - 10.0mg/kg). Results MDMA pretreatment produced widespread reductions in SERT binding densities 2 weeks following administration. Prior exposure to MDMA rendered rats tolerant to MDMA-produced hyperactivity when tested 2, but not 12, weeks following MDMA administration. Two weeks following MDMA pretreatment rats were also less responsive to the clomipramine-produced attenuation of MDMA-produced hyperactivity. MDMA pretreatment failed to alter M-CPP -produced hypolocomotion or increased emergence latency, but decreased the ability for DOI to induce WDS. Further, MDMA pretreated rats exhibited tolerance to RS102221 as shown by a rightward shift in the dose effect curve and complete tolerance to ritanserin. Conclusions Following MDMA pretreatment, the decreased SERT binding densities and inability of clomipramine to attenuate MDMA-produced effects might explain tolerance to the locomotor activating effects produced by MDMA. Functional recovery also occurred with extended abstinence from the drug, suggesting that MDMA produced transient serotonergic alterations. The results support the idea that the 5-HT2a and 5-HT2c receptors that modulate MDMA-produced hyperactivity are functionally distinct from the receptors that mediate m-CPP- and DOI-induced behavioural responses, as m-CPP-produced behaviours were resilient, yet RS102221-induced effects were reduced, by MDMA pretreatment. RS102221 is highly selective in comparison to ritanserin, yet there was only one dose that produced significant potentiation of MDMA-produced hyperactivity, whereas there were several effective ritanserin doses. This suggests that the 5-HT2a receptors had a greater role in modulating MDMA-produced hyperactivity. Additionally, 5-HT2a receptors might be more susceptible to MDMA-induced desensitisation than 5-HT2c receptors, as MDMA pretreated rats exhibited some tolerance to the potentiating effects of RS102221 but were unresponsive to any ritanserin dose. In conclusion, MDMA-induced locomotor tolerance was attributable to decreased SERT densities and function as well as desensitisation of 5-HT2a receptors that facilitate hyperactivity.</p>

A Behavioural Analysis of Serotonergic Functional Status Following MDMA Exposure

<p>Rationale +/- 3,4-Methylenedioxymethamphetamine (MDMA) produces effects on a number of neurochemical systems. Many studies have shown that repeated MDMA administration produces deficits in central serotonergic neurotransmission, which have been suggested to underlie some of the behavioural changes associated with use. Objectives The present studies sought to evaluate the functional statuses of the serotonin transporter (SERT) and the serotonin2c (5-HT2c) and serotonin2a (5-HT2a) receptors following treatment with MDMA to determine whether behavioural deficits could be attributed to alterations in these proteins. Methods Rats received a pretreatment regimen of MDMA (4 x 10mg/kg MDMA injections administered at 2h intervals) or the saline vehicle and, 2 weeks later, [3H] paroxetine binding was undertaken to assess densities of SERT. In other groups, dose-effect curves for MDMA-produced hyperactivity were determined. Additional groups were tested following a 12-week withdrawal period from MDMA in order to assess whether there was recovery of function. The functional status of the SERT was further examined by determining the effect of MDMA pretreatment on the reduction in MDMA-produced hyperactivity (0.0 - 10.0mg/kg) produced by the selective serotonin reuptake inhibitor, clomipramine (0.0 - 5.0mg/kg). The ability for the 5-HT2c receptor agonist, m-CPP (0.0 - 2.5mg/kg) to produce hypolocomotion or increased emergence latency or for the 5-HT2a receptor agonist, DOI (0.0 - 2.0mg/kg) to produce wetdog shakes (WDS) were examined in MDMA pretreated rats. The ability for the 5-HT2c receptors to modulate MDMA-produced hyperactivity was assessed by examining the effect of MDMA pretreatment on the potentiation of MDMA-produced hyperactivity produced by the selective antagonist, RS102221 (0.0 - 1.0mg/kg). Conversely, the modulatory abilities of the 5-HT2a receptors were assessed by examining the effect of MDMA pretreatment on the attenuation of MDMA-produced hyperactivity produced by the antagonist, ritanserin (0.0 - 10.0mg/kg). Results MDMA pretreatment produced widespread reductions in SERT binding densities 2 weeks following administration. Prior exposure to MDMA rendered rats tolerant to MDMA-produced hyperactivity when tested 2, but not 12, weeks following MDMA administration. Two weeks following MDMA pretreatment rats were also less responsive to the clomipramine-produced attenuation of MDMA-produced hyperactivity. MDMA pretreatment failed to alter M-CPP -produced hypolocomotion or increased emergence latency, but decreased the ability for DOI to induce WDS. Further, MDMA pretreated rats exhibited tolerance to RS102221 as shown by a rightward shift in the dose effect curve and complete tolerance to ritanserin. Conclusions Following MDMA pretreatment, the decreased SERT binding densities and inability of clomipramine to attenuate MDMA-produced effects might explain tolerance to the locomotor activating effects produced by MDMA. Functional recovery also occurred with extended abstinence from the drug, suggesting that MDMA produced transient serotonergic alterations. The results support the idea that the 5-HT2a and 5-HT2c receptors that modulate MDMA-produced hyperactivity are functionally distinct from the receptors that mediate m-CPP- and DOI-induced behavioural responses, as m-CPP-produced behaviours were resilient, yet RS102221-induced effects were reduced, by MDMA pretreatment. RS102221 is highly selective in comparison to ritanserin, yet there was only one dose that produced significant potentiation of MDMA-produced hyperactivity, whereas there were several effective ritanserin doses. This suggests that the 5-HT2a receptors had a greater role in modulating MDMA-produced hyperactivity. Additionally, 5-HT2a receptors might be more susceptible to MDMA-induced desensitisation than 5-HT2c receptors, as MDMA pretreated rats exhibited some tolerance to the potentiating effects of RS102221 but were unresponsive to any ritanserin dose. In conclusion, MDMA-induced locomotor tolerance was attributable to decreased SERT densities and function as well as desensitisation of 5-HT2a receptors that facilitate hyperactivity.</p>

Intrapericardial Delivery of APA-Microcapsules as Promising Stem Cell Therapy Carriers in an Experimental Acute Myocardial Infarction Model

The administration of cardiosphere-derived cells (CDCs) after acute myocardial infarction (AMI) is very promising. CDC encapsulation in alginate-poly-l-lysine-alginate (APA) could increase cell survival and adherence. The intrapericardial (IP) approach potentially achieves high concentrations of the therapeutic agent in the infarcted area. We aimed to evaluate IP therapy using a saline vehicle as a control (CON), a dose of 30 × 106 CDCs (CDCs) or APA microcapsules containing 30 × 106 CDCs (APA-CDCs) at 72 h in a porcine AMI model. Magnetic resonance imaging (MRI) was used to determine the left ventricular ejection fraction (LVEF), infarct size (IS), and indexed end diastolic and systolic volumes (EDVi; ESVi) pre- and 10 weeks post-injection. Programmed electrical stimulation (PES) was performed to test arrhythmia inducibility before euthanasia. Histopathological analysis was carried out afterwards. The IP infusion was successful in all animals. At 10 weeks, MRI revealed significantly higher LVEF in the APA-CDC group compared with CON. No significant differences were observed among groups in IS, EDVi, ESVi, PES and histopathological analyses. In conclusion, the IP injection of CDCs (microencapsulated or not) was feasible and safe 72 h post-AMI in the porcine model. Moreover, CDCs APA encapsulation could have a beneficial effect on cardiac function, reflected by a higher LVEF at 10 weeks.

Self-Administration of entactogen psychostimulants dysregulates GABA and Kappa Opioid Receptor signaling in the central nucleus of the amygdala of female Wistar rats

Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABAA receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-hour sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature postsynaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats) however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (postsynaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1mM U50488) and KOR antagonism (200nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.

Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

Chronic Antidiabetic Actions of Leptin: Evidence from Parabiosis Studies for a CNS-Derived Circulating Antidiabetic Factor

We used parabiosis to determine whether the central nervous system (CNS)-mediated antidiabetic effects of leptin are mediated by release of a brain-derived circulating factor(s). Parabiosis was surgically induced at 4 weeks of age and an intracerebroventricular (ICV) cannula was placed in the lateral cerebral ventricles at 12 weeks of age for ICV infusion of leptin or saline vehicle. Ten days after surgery, food intake, body weight and blood glucose were measured for 5 consecutive days and insulin-deficiency diabetes was induced in all rats by a single streptozotocin (STZ) injection (40 mg/kg). Five days after STZ injection, leptin or vehicle was infused ICV for 7 days, followed by 5-day recovery period. STZ increased blood glucose and food intake. Chronic ICV leptin infusion restored normoglycemia in leptin-infused rats while reducing blood glucose by ~27% in conjoined vehicle-infused rats. This glucose reduction was caused mainly by decreased hepatic gluconeogenesis. Chronic ICV leptin infusion also reduced net cumulative food intake and increased GLUT4 expression in skeletal muscle in leptin/vehicle compared to vehicle/vehicle conjoined rats. These results indicate that leptin’s CNS-mediated antidiabetic effects are mediated, in part, by release into the systemic circulation of a leptin-stimulated factor(s) that enhances glucose utilization and reduces liver gluconeogenesis.