Microglial MyD88 signaling regulates acute neuronal toxicity of LPS-stimulated microglia in vitro

Although the role of microglial activation in neural injury remains controversial, there is increasing evidence for a detrimental effect in the immature brain, which may occur in response to release of neurotoxic substances including pro-inflammatory cytokines. However, the signaling mechanisms involved in microglial-induced neuronal cell death are unclear. Microglia isolated from the brains of wild-type (WT) or MyD88 knockout (KO) mice were exposed to PBS or the TLR4-ligand LPS (100 ng/mL) for 2, 6, 14, or 24 h, and the microglia-conditioned medium (MCM) collected. Detection of multiple inflammatory molecules in MCM was performed using a mouse 22-plex cytokine microbead array kit. Primary neuronal cultures were supplemented with the 14 or 24 h MCM, and the degree of neuronal apoptosis examined after exposure for 24 h. Results showed a rapid and sustained elevation in multiple inflammatory mediators in the MCM of WT microglia exposed to LPS, which was largely inhibited in MyD88 KO microglia. There was a significant increase in apoptotic death measured at 24 h in cultured neurons exposed to CM from either 14 or 24 h LPS-stimulated WT microglia (p < .05 vs. WT control). By contrast, there was no increase in apoptotic death in cultured neurons exposed to CM from 14 or 24 h LPS-stimulated MyD88 KO microglia (p = .15 vs. MyD88 KO control). These data suggest that MyD88-dependent activation of microglia by LPS causes release of factors directly toxic to neurons.     

Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study

Dopamine (DA) and serotonin (5-HT) seem to be involved in several of the effects of ethanol (EtOH). Acetaldehyde (AcH), especially in the brain, induces effects that mimic those of EtOH. The purpose of this study was to investigate the effects of local perfusion of EtOH and AcH on extracellular DA and 5-HT in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Aldh2-KO mice were used as a model of aldehyde dehydrogenase 2 deficiency in humans to examine the effects of AcH. Mice were perfused with Ringer’s solution (control), EtOH (100, 200, or 500 mM) and AcH (100, 200, or 500 μM) into the dorsal striatum. Dialysate samples were collected every 5 min, and then analyzed with HPLC coupled to an ECD. We found that local perfusion with 500 mM EtOH increased extracellular levels of DA (p < 0.05) in both Aldh2-KO and WT mice, while 5-HT levels remain unchanged. EtOH at a dose of 200 mM also increased DA in WT mice, but this was limited to a 30–40-min time-point. In contrast, perfusion with 200 and 500 μM AcH decreased both DA and 5-HT (p < 0.05) in Aldh2-KO mice, but this decrease was not found in WT mice at any AcH dose, indicating an effect of AcH on DA and 5-HT levels. There were no genotype effects on the basal levels of DA and 5-HT. These results indicate that high EtOH can stimulate DA, whereas high AcH can depress both DA and 5-HT in the dorsal striatum of mice.     

Neuropathological and behavioral sequelae in IL-1R1 and IL-1Ra gene knockout mice after soman (GD) exposure

Soman (GD) exposure results in status epilepticus (SE) that leads to neurodegeneration, neuroinflammation, and behavioral consequences including learning and memory deficits. The neuroinflammatory response is characterized by the upregulation of the pro-inflammatory cytokine, interleukin-1 (IL-1), which mediates the expression of other neurotoxic cytokines induced after GD exposure. However, the specific role of IL-1 signaling has not been defined in terms of the consequences of GD-induced SE. Therefore, the purpose of this study was to regulate IL-1 signaling and study the behavioral deficits and neurodegeneration that occur after convulsion onset. Wild type (WT), IL-1 receptor (IL-1R1) knockout (KO), and IL-1 receptor antagonist (IL-1Ra) KO mice were exposed to a convulsive dose of GD, and behavior was evaluated up to 18 days later. Activity was studied using the Open Field, anxiety was assessed in the Zero Maze, and spatial learning and memory were evaluated with the Barnes Maze. The animals were euthanized at 24 hours and 18 days to determine neuropathology in the piriform cortex, amygdala, thalamus, and CA1, CA2/3, and CA4 regions of the hippocampus. Unlike the IL-1Ra KO, the IL-1R1 KO showed less neuropathology compared to WT at 24 hours, but moderate to severe injury was found in all strains at 18 days. Compared to their saline controls, the exposed WT mice were significantly more active in the Open Field, and the IL-1R1 KO strain showed reduced anxiety in the Zero Maze Test. Compared to WT mice, IL-1R1 and IL-1Ra KO mice had spatial learning and memory impairments in the Barnes Maze. Therefore, the IL-1 signaling pathway affects neurodegeneration and behavior after GD-induced convulsions.     

Role of adaptor protein MyD88 in TLR-mediated preconditioning and neuroprotection after acute excitotoxicity

Excitotoxic cell death is a crucial mechanism through which neurodegeneration occurs in numerous pathologies of the central nervous system (CNS), such as Alzheimer’s disease, stroke and spinal cord injury. Toll-like receptors (TLRs) are strongly expressed on microglial cells and are key regulators of the innate immune response to neuronal damage. However, it is still unclear whether their stimulation is protective or harmful in excitotoxic contexts. In this study, we demonstrate that systemic administration of lipopolysaccharide (LPS) or Pam3CSK4 24 h prior to an intrastriatal injection of kainic acid (KA) significantly protected cortical neurons in the acute phase of injury. Protection could not be detected with the TLR3 ligand poly-IC. Histological analyses revealed that microglia of LPS and Pam3CSK4 pre-conditioned group were primed to react to injury and exhibited a stronger expression of Tnf and Tlr2 mRNA. We also found that mice deficient for MyD88, a critical adaptor protein for most TLR, were more vulnerable than WT mice to KA-induced excitotoxicity at early (12 h and 24 h) and late (10 days) time points. Finally, bone-marrow chimeric mice revealed that MyD88 signaling in CNS resident cells, but not in cells of hematopoietic origin, mediates the protective effect. This study unravels the potential of TLR2 and TLR4 agonists to induce a protective state of preconditioning against KA-mediated excitotoxicity and further highlights the beneficial role of cerebral MyD88 signaling in this context.     

Involvement of TLR4 in the long-term epigenetic changes,rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence

Studies in humans and experimental animals have demonstrated the vulnerability of the adolescent brain to actions of ethanol and the long-term consequences of binge drinking, including the behavioral and cognitive deficits that result from alcohol neurotoxicity, and increased risk to alcohol abuse and dependence. Although the mechanisms that participate in these effects are largely unknown, we have shown that ethanol by activating innate immune receptors, toll-like receptor 4 (TLR4), induces neuroinflammation, impairs myelin proteins and causes cognitive dysfunctions in adolescent mice. Since neuroimmune signaling is also involved in alcohol abuse, the aim of this study was to assess whether ethanol treatment in adolescence promotes the long-term synaptic and molecular events associated with alcohol abuse and addiction. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3 g/kg) for 2 weeks, we showed that binge-like ethanol treatment in adolescent mice promotes short- and long-term alterations in synaptic plasticity and epigenetic changes in the promoter region of bdnf and fosb, which increased their expression in the mPFC of young adult animals. These molecular events were associated with long-term rewarding and anxiogenic-related behavioral effects, along with increased alcohol preference. Our results further showed the participation of neuroimmune system activation and the TLR4 signaling response since deficient mice in TLR4 (TLR4-KO) are protected against molecular and behavioral alterations of ethanol in the adolescent brain. Our results highlight a new role of the neuroimmune function and open up new avenues to develop pharmacological treatments that can normalize the immune signaling responsible for long-term effects in adolescence, including alcohol abuse and related disorders.     

Epstein–Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis

Epstein–Barr virus-induced gene 3 (EBI3) associates with p28 and p35 to form the immunomodulatory cytokines IL-27 and IL-35, respectively. Infection of EBI3 ?/? mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality that was not associated with impaired ability to control viral replication but enhanced T cell and macrophage infiltration into the CNS. IFN-γ secretion from virus-specific CD4 + and CD8 + T cells isolated from infected EBI3 ?/? mice was augmented while IL-10 expression muted in comparison to infected WT mice. These data demonstrate a regulatory role for EBI3-associated cytokines in controlling host responses following CNS viral infection.     

TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0 g/kg) for 2 weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.     

Knockout of vascular early response gene worsens chronic stroke outcomes in neonatal mice

Vascular early response gene (Verge) is a novel immediate early gene that is highly expressed during developmental angiogenesis and after ischemic insults in adult brain. However, the role of Verge after neonatal injury is not known. In the present study, we investigated the hypothesis that Verge contributes to vascular remodeling and tissue repair after neonatal ischemic injury. The Rice–Vanucci model (RVM) was employed to induce neonatal stroke in both Verge knockout (KO) and wild-type (WT) postnatal day 10 (P10) mice. Histological and behavioral outcomes at acute (24 h), subacute (7 days) and chronic (30 days) phases were evaluated. Angiogenesis, neurogenesis, and glial scar formation were also examined in the ischemic brain. No significant differences in outcomes were found between WT and Verge mice at 24 h or 7 days after stroke. However genetic deletion of Verge led to pronounced cystic cavitation, decreased angiogenensis and glial scar formation in the ischemic hemisphere compared to WT mice at 30 days. Verge KO mice also had significantly worse functional outcomes at 30 days which was accompanied by decreased neurogenesis and angiogenesis in the ischemic hemisphere. Our study suggests that Verge plays an important role in the induction of neurogenesis and angiogenesis after ischemia, contributes to improved tissue repair, and enhances chronic functional recovery.     

Calorie restriction dose-dependently abates lipopolysaccharide-induced fever,sickness behavior,and circulating interleukin-6 while increasing corticosterone

In mice a 50% calorie restriction (CR) for 28 days attenuates sickness behavior after lipopolysaccharide (LPS) and these mice demonstrate a central anti-inflammatory bias. This study examined the dose-dependent effect of CR on sickness behavior (fever, anorexia, cachexia) and peripheral immune markers post-LPS. Male Sprague–Dawley rats fed ad libitum or CR by 50% for 14, 21, or 28 days were injected on day 15, 22, or 29 with 50 μg/kg of LPS or saline (1 mL/500 g). Changes in body temperature (T_b), locomotor activity, body weight, and food intake were determined. A separate cohort of rats was fed ad libitum or CR by 50% for 28 days and serum levels of corticosterone (CORT), interleukin 6 (IL-6), and IL-10 were determined at 0, 2, and 4 h post-LPS. The rats CR for 28 days demonstrated the largest attenuation of sickness behavior: no fever, limited reduction in locomotor activity, no anorexia, and reduced cachexia following LPS. Rats CR for 14 and 21 days demonstrated a partial attenuation of sickness behavior. Rats CR for 14 days demonstrated a larger increase in T_b, larger reduction in locomotor activity, and larger weight loss compared to rats CR for 21 days. Serum CORT was increased at 2 h post-LPS in ad libitum and CR groups; however it was two times larger in the CR animals. Levels of IL-6 were significantly attenuated at 2 h post-LPS in the CR animals. IL-10 levels were similar post-LPS. CR results in an enhanced anti-inflammatory response in the form of increased CORT and diminished pro-inflammatory signals.     

Effects of RFamide-related peptide-1 (RFRP-1) microinjections into the central nucleus of amygdala on passive avoidance learning in rats

The amygdaloid body (AMY) plays an important role in memory, learning and reward-related processes. RFRP-1 immunoreactive fibers and NPFF receptors were identified in the AMY, and previously we verified that RFRP-1 infused into the central nucleus of AMY (CeA) induced place preference. The aim of the present study was to examine the possible effects of RFRP-1 in the CeA on passive avoidance learning. Male Wistar rats were examined in two-compartment passive avoidance paradigm. Animals were shocked with 0.5 mA current and subsequently were microinjected bilaterally with 50 ng or 100 ng RFRP-1 in volume of 0.4 μl, or 20 ng NPFF receptor antagonist RF9 (ANT) alone, or antagonist 15 min before 50 ng RFRP-1 treatments into the CeA. Fifty nanogram dose of RFRP-1 significantly increased the step-through latency time, the 100 ng RFRP-1 and the ANT alone were ineffective. The effect of 50 ng RFRP-1 was eliminated by the ANT pretreatment. Our results suggest that intraamygdaloid RFRP-1 enhances learning processes and memory in aversive situations and this effect can specifically be prevented by ANT pretreatment.     

Effects of voluntary wheel running on LPS-induced sickness behavior in aged mice

Peripheral stimulation of the innate immune system with LPS causes exaggerated neuroinflammation and prolonged sickness behavior in aged mice. Regular moderate intensity exercise has been shown to exert anti-inflammatory effects that may protect against inappropriate neuroinflammation and sickness in aged mice. The purpose of this study was to test the hypothesis that voluntary wheel running would attenuate LPS-induced sickness behavior and proinflammatory cytokine gene expression in ∼22-month-old C57BL/6 J mice. Mice were housed with a running wheel (VWR), locked-wheel (Locked), or no wheel (Standard) for 10 weeks, after which they were intraperitoneally injected with LPS across a range of doses (0.02, 0.08, 0.16, 0.33 mg/kg). VWR mice ran on average 3.5 km/day and lost significantly more body weight and body fat, and increased their forced exercise tolerance compared to Locked and Shoebox mice. VWR had no effect on LPS-induced anorexia, adipsia, weight-loss, or reductions in locomotor activity at any LPS dose when compared to Locked and Shoebox groups. LPS induced sickness behavior in a dose-dependent fashion (0.33 > 0.02 mg/kg). Twenty-four hours post-injection (0.33 mg/kg LPS or Saline) we found a LPS-induced upregulation of whole brain TNFα, IL-1β, and IL-10 mRNA, and increased IL-1β and IL-6 in the spleen and liver; these effects were not attenuated by VWR. We conclude that VWR does not reduce LPS-induced exaggerated or prolonged sickness behavior in aged animals, or 24 h post-injection (0.33 mg/kg LPS or Saline) brain and peripheral proinflammatory cytokine gene expression. The necessity of the sickness response is critical for survival and may outweigh the subtle benefits of exercise training in aged animals.     

Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-β levels in male mice

The manipulation of the enteric microbiota with specific prebiotics and probiotics, has been shown to reduce the host’s inflammatory response, alter brain chemistry, and modulate anxiety behaviour in both rodents and humans. However, the neuro-immune and behavioural effects of prebiotics on sickness behaviour have not been explored. Here, adult male CD1 mice were fed with a specific mix of non-digestible galacto-oligosaccharides (Bimuno®, BGOS) for 3 weeks, before receiving a single injection of lipopolysaccharide (LPS), which induces sickness behaviour and anxiety. Locomotor and marble burying activities were assessed 4 h after LPS injection, and after 24 h, anxiety in the light–dark box was assessed. Cytokine expression, and key components of the serotonergic (5-Hydroxytryptamine, 5-HT) and glutamatergic system were evaluated in the frontal cortex to determine the impact of BGOS administration at a molecular level. BGOS-fed mice were less anxious in the light–dark box compared to controls 24 h after the LPS injection. Elevated cortical IL-1β concentrations in control mice 28 h after LPS were not observed in BGOS-fed animals. This significant BGOS × LPS interaction was also observed for 5HT2A receptors, but not for 5HT1A receptors, 5HT, 5HIAA, NMDA receptor subunits, or other cytokines. The intake of BGOS did not influence LPS-mediated reductions in marble burying behaviour, and its effect on locomotor activity was equivocal. Together, our data show that the prebiotic BGOS has an anxiolytic effect, which may be related to the modulation of cortical IL-1β and 5-HT2A receptor expression. Our data suggest a potential role for prebiotics in the treatment of neuropsychiatric disorders where anxiety and neuroinflammation are prominent clinical features.     

Histamine responses of large neostriatal interneurons in histamine H1 and H2 receptor knock-out mice

Histamine (HA) is an important neuro-modulator, contributing to a variety of physiological responses in the mammalian central nervous system (CNS). However there is little information about the cell/signaling mechanism underlying its role. In the present study, we characterized HA responses in single large neostriatal neurons acutely dissociated from wild type (WT) and HA receptor knock-out (KO) mice, with a particular emphasis on identifying the role of HA receptor subtypes. HA (10 μM) and a selective H₂ receptor agonist dimaprit (1 μM) both evoked an inward current in H₁-KO mice, and HA and a selective H₁ receptor agonist HTMT (10 μM) both evoked an inward current in H₂-KO mice. In the H₁ and H₂ double (H_1/2) KO mice, there was no response to either the application of HA or the selective H₁, H₂ receptor agonists. Hence we have confirmed that the targeted genes were indeed absent in these KO mice and that both receptor subtypes contribute to HA's excitatory actions. Furthermore the HA-induced inward currents were mediated by a decrease in current through K⁺ channels. In addition, we observed the effects of methamphetamine (METH) on the locomotor activity of WT and HA receptor KO mice, and found that METH-induced behavioral sensitization is evident in H_1/2-KO mice, but not in H₁- or H₂-KO mice. These observations suggest that suppressive roles of HA on methamphetamine-induced behavioral sensitization would be mediated through both H₁ and H₂ receptors in the CNS including neostriatum.     

Zinc gluconate toxicity in wild-type vs. MT1/2-deficient mice

Previous studies have suggested that oral zinc supplementation can help reduce the duration of the common cold; however, the use of intranasal (IN) zinc is strongly associated with anosmia, or the loss of the sense of smell, in humans. Prior studies from this lab showed that upregulation of metallothioneins (MT) is a rapid and robust response to zinc gluconate (ZG). Therefore, we assessed the role of MT in the recovery of nasal epithelial damage resulting from IN zinc administration. The main studies in this investigation used a high dose of ZG (170 mM) to ensure ablation of the olfactory mucosa, so that the progression of histological and functional recovery could be assessed. In vivo studies using wild-type, MT1/2 knockout mice (MT KO), and heterozygotes administered ZG by IN instillation showed profound loss of the olfactory mucosa in the nasal cavity. Recovery was monitored, and a lower percentage of the MT KO mice were able to smell 28 d after treatment; however, no significant difference was observed in the rate of cell proliferation in the basal layer of the olfactory epithelium between MT KO and wild-type mice. A lower concentration of ZG (33 mM), equivalent to that found in homeopathic IN ZG preparations, also caused olfactory epithelial toxicity in mice. These studies suggest that the use of zinc in drug formulations intended for IN administration in humans must be carefully evaluated for their potential to cause olfactory functional deficits.     

Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy

The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague–Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n = 12)] or phosphate buffered saline [PBS (n = 7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21 days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21 days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p < 0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.     

Primary murine microglia are resistant to nitric oxide inhibition of indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that is activated by proinflammatory cytokines, including interferon-γ (IFNγ), and metabolizes tryptophan along the kynurenine pathway. Activation of murine macrophages induces not only IDO but also nitric oxide synthase (iNOS), and the ensuing production of nitric oxide (NO) inhibits IDO. To determine the sensitivity of primary cultures of murine microglia to NO, microglia were stimulated with recombinant murine IFNγ (1 ng/ml) and lipopolysaccharide (LPS) (10 ng/ml). This combination of IFNγ + LPS synergized to produce maximal amounts of nitrite as early as 16 h. Steady-state mRNAs for both iNOS and IDO were significantly increased by IFNγ + LPS at 4 h post-treatment, followed by an increase in IDO enzymatic activity at 24 h. Murine microglia (>95% CD11b⁺) were pretreated with the iNOS inhibitor, L-NIL hydrochloride, at a dose (30 μM) that completely abrogated production of nitrite. L-NIL had no effect on IDO mRNA at 4 h or IDO enzymatic activity at 24 h following stimulation with IFNγ + LPS. These data establish that IDO regulation in murine microglia is not restrained by NO, thereby permitting the accumulation of kynurenine and its downstream metabolites in the central nervous system.     

Mu-opioid receptor knockout mice are more sensitive to chlordiazepoxide-induced anxiolytic behavior

We have previously demonstrated benzodiazepine binding in the cortex and hippocampus of mu-opioid receptor knockout (KO) mice. It is known that benzodiazepine receptors are involved in regulating anxiety-like behaviors. Thus, the present study was designed to examine whether there are changes in anxiety-like behavior in mice lacking mu-opioid receptors. To produce anxiolytic activity (less anxiety), the prototype benzodiazepine receptor agonist chlordiazepoxide (CDP, 5 mg/kg) was intraperitoneally administered in wild type (WT) and mu-opioid receptor KO mice. We found that compared to WT mice, mu-opioid receptor KO mice showed enhanced anxiolytic activity to CDP, including increased number of entries into open arm, increased percentage of the time spent in open arms, and decreased percentage of the time spent in enclosed arms in the elevated plus-maze test. We also assessed protein expression of the gamma-aminobutyric acid (GABA) synthetic enzyme (glutamic acid decarboxylase; GAD). Western blotting data indicated that neither the lack of mu-opioid receptors nor CDP treatment altered cortical or hippocampal GAD₆₅ or GAD₆₇ protein expression. These data indicate that compared with WT, mu-opioid receptor KO mice experienced less anxiety and exhibited enhanced anxiolytic activity to CDP treatment, and these effects were not dependent on GAD₆₅ or GAD₆₇ protein expression. Our previous and present data suggest that the anxiolytic activity displayed in mu-opioid receptor KO mice is associated with upregulation of the benzodiazepine receptor system.     

Retinoic acid prevents synaptic deficiencies induced by alcohol exposure during gastrulation in zebrafish embryos

In this study, we examined the effects of alcohol exposure during gastrulation on zebrafish embryos, specifically focusing on excitatory synaptic activity associated with neurons (Mauthner cells) that are born during gastrulation. Furthermore, we determined whether co-treatment of alcohol and retinoic acid (RA) could prevent the effects of alcohol exposure during gastrulation. We exposed zebrafish embryos to ethanol (150 mM), RA (1 nM), or a combination of RA (1 nM) plus ethanol (150 mM) for 5.5 h from 5.25 h post fertilization (hpf) to 10.75 hpf (gastrulation). Ethanol treatment resulted in altered hatching rates, survivability and body lengths. Immunohistochemical analysis of Mauthner cells (M-cells) suggested that ethanol treatment resulted in smaller M-cell bodies and thinner axons, while electrophysiological recordings of AMPA miniature excitatory postsynaptic currents (mEPSCs) associated with M-cells showed that ethanol treated animals had a significantly reduced mEPSC frequency. Other mEPSC parameters such as amplitude, rise times and decay kinetics were not altered by exposure to alcohol. Locomotor studies showed that ethanol treatment resulted in altered C-bend escape responses. For instance, the C-bends of alcohol-treated fish were larger than control embryos. Thus, ethanol treatment during gastrulation altered a range of features in embryonic zebrafish. Importantly, co-treatment with RA prevented all of the effects of ethanol including survivability, body length, M-cell morphology, AMPA mEPSC frequency and escape response movements. Together these findings show that ethanol exposure during the brief period of gastrulation has a significant effect on neuronal morphology and activity, and that this can be prevented with RA co-treatment.