Ilex kudingcha C.J. Tseng Mitigates Phenotypic Characteristics of Human Autism Spectrum Disorders in a Drosophila Melanogaster Rugose Mutant.

Autism spectrum disorders (ASD) have heterogeneous etiologies involving dysfunction of central nervous systems, for which no effective pan-specific treatments are available. Ilex kudingcha (IK) C.J. Tseng is a nootropic botanical used in Asia for neuroprotection and improvement of cognition. This study establishes that a chemically characterized extract from IK (IKE) mitigates behavioral traits in the Drosophila melanogaster rugose mutant, whose traits resemble human ASD, and examines possible mechanisms. IKE treatment significantly ameliorated deficits in social interaction, short-term memory, and locomotor activity in Drosophila rugose, and significantly increased synaptic bouton number of size more than 2 µm2 in the neuromuscular junctions (NMJs) of Drosophila rugose. To clarify mechanism(s) of IKE action, methylphenidate (MPH), a dopamine transporter inhibitor, was included as a reference drug in the behavioral assays: MPH significantly improved social interaction and short-term memory deficit in Drosophila rugose; administration of the dopamine D1 receptor antagonist SCH23390 and dopamine D2 receptor antagonist sulpiride reversed the ameliorative effects of both MPH and IKE on the social interaction deficits of Drosophila rugose. To extend analysis of IKE treatment to the vertebrate central nervous system, ASD-associated gene expression in mouse hippocampus was studied by RNA-seq: IKE treatment altered the expression of genes coding phosphoinositide 3-kinases/protein kinase B (PI3K-Akt), proteins in glutamatergic, dopaminergic, serotonergic, and GABAergic synapses, cAMP response element-binding protein (CREB), and RNA transporter proteins. These results provide a foundation for further analysis of IKE as a candidate for treatment of some forms of ASD.

Brain γ-Tocopherol Levels Are Associated with Presynaptic Protein Levels in Elderly Human Midfrontal Cortex.

BACKGROUND: Higher vitamin E intake has been widely related to lower risks of cognitive decline and dementia. Animal models suggest that this relationship might be (partially) explained by the protection of vitamin E against presynaptic protein oxidation. OBJECTIVE: In this cross-sectional study, we aimed to examine the associations between brain tocopherols and presynaptic protein levels in elderly humans. METHODS: We examined associations of α- and γ-tocopherol brain levels with presynaptic protein levels in 113 deceased participants (age 88.5±6.0 years, 45 (40%) female) from the prospective Memory and Aging project. Three distinct presynaptic proteins, a SNARE protein composite, a synaptotagmin synaptophysin composite and the protein-protein interaction between synaptosomal-associated protein 25 (SNAP-25), and syntaxin were measured in two cortical brain regions. Linear regression models assessed associations of brain tocopherols with presynaptic protein levels. RESULTS: Higher brain γ-tocopherol levels were associated with higher levels of the SNARE protein composite, complexin-I, complexin-II, the synaptotagmin synaptophysin composite, and septin-5 in the midfrontal cortex (B(SE) = 0.272 to 0.412 (0.084 to 0.091), p < 0.001 to 0.003). When additionally adjusted for global Alzheimer's disease pathology, cerebral infarcts, and Lewy body disease pathology, these associations remained largely similar. No associations were found between α-tocopherol and presynaptic protein levels. CONCLUSION: In this cross-sectional study, we found higher brain γ-tocopherol levels were associated with presynaptic protein levels in the midfrontal cortex. These results are consistent with a proposed role of vitamin E to maintain presynaptic protein levels.

Presynaptic glutamatergic transmission and feedback system of oxytocinergic neurons in the hypothalamus of a rat model of adjuvant arthritis.

The neurohypophysial hormone oxytocin (OXT) is synthesized in the hypothalamic paraventricular and supraoptic nuclei. Recently, some studies have considered OXT to be important in sensory modulation and that the OXT protein is upregulated by acute and chronic nociception. However, the mechanism by which OXT is upregulated in neurons is unknown. In this study, we examined the resting membrane potentials and excitatory postsynaptic currents in OXT-ergic neurons in the paraventricular nucleus in adjuvant arthritis rat model, a model of chronic inflammation, using whole-cell patch-clamping. Transgenic rats expressing OXT and monomeric red fluorescent protein 1 (mRFP1) fusion protein to visualize the OXT-ergic neurons were used, and the OXT-mRFP1 transgenic rat model of adjuvant arthritis was developed by injection of heat-killed Mycobacterium butyricum. Furthermore, the feedback system of synthesized OXT was also examined using the OXT receptor antagonist L-368,899. We found that the resting membrane potentials and frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-monomeric red fluorescent protein 1 neurons in the paraventricular nucleus were significantly increased in adjuvant arthritis rats. Furthermore, L-368,899 dose-dependently increased the frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-ergic neurons. Following bath application of the GABAA receptor antagonist picrotoxin and the cannabinoid receptor 1 antagonist AM 251, L-368,899 still increased the frequency of miniature excitatory postsynaptic currents. However, following bath application of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, L-368,899 did not alter the miniature excitatory postsynaptic current frequency. Thus, it is suggested that OXT-ergic neuron activity is upregulated via an increase in glutamate release, and that the upregulated OXT neurons have a feedback system with released endogenous OXT. It is possible that nitric oxide, but not GABA, may contribute to the feedback system of OXT neurons in chronic inflammation.

Presynaptic dopamine deficit in minimally conscious state patients following traumatic brain injury.

Dopaminergic stimulation has been proposed as a treatment strategy for post-traumatic brain injured patients in minimally conscious state based on a clinical trial using amantadine, a weak dopamine transporter blocker. However, a specific contribution of dopaminergic neuromodulation in minimally conscious state is undemonstrated. In a phase 0 clinical trial, we evaluated 13 normal volunteers and seven post-traumatic minimally conscious state patients using 11C-raclopride PET to estimate dopamine 2-like receptors occupancy in the striatum and central thalamus before and after dopamine transporter blockade with dextroamphetamine. If a presynaptic deficit was observed, a third and a fourth 11C-raclopride PET were acquired to evaluate changes in dopamine release induced by l-DOPA and l-DOPA+dextroamphetamine. Permutation analysis showed a significant reduction of dopamine release in patients, demonstrating a presynaptic deficit in the striatum and central thalamus that could not be reversed by blocking the dopamine transporter. However, administration of the dopamine precursor l-DOPA reversed the presynaptic deficit by restoring the biosynthesis of dopamine from both ventral tegmentum and substantia nigra. The advantages of alternative pharmacodynamic approaches in post-traumatic minimally conscious state patients should be tested in clinical trials, as patients currently refractory to amantadine might benefit from them.

Effects of epoxygenases on the nonadrenergic noncholinergic relaxant responses induced by electrical field stimulation in rabbit corpus cavernosum.

We aimed to investigate the effects of epoxygenases on electrical field stimulation (EFS)-mediated nitric oxide (NO)-dependent and NO-independent nonadrenergic noncholinergic (NANC) relaxations in isolated rabbit corpus cavernosum. The tissues of 20 male adult albino rabbits (2.5-3 kg) were suspended in organ baths containing aerated Krebs solution, and isometric contractions were recorded. EFS-mediated NANC relaxations were obtained on phenylephrin (3 × 10-5  M)-contracted tissues in the presence of guanethidine (10-6  M) and atropine (10-6  M). Miconazole (10-9 -10-4  M), 17-octadecynoic acid (ODYA) (10-10 -10-5  M), 14,15-epoxyeicosatrienoic acid (EET) (10-11 -10-8  M), 11,12-EET (10-12 -3 × 10-8  M) and 20-hydroxyeicosatetraenoic acid (HETE) (10-11 -3 × 10-8  M) were added cumulatively (n = 5-7 for each set of experiments). For NO-independent relaxations, Nω -nitro-l-arginine methyl ester (l-NAME) (10-4  M) was added before a group of experiments. Depending on the concentration, miconazole, 17-ODYA, 14,15-EET, 11,12-EET, and 20-HETE significantly enhanced both NO-dependent and NO-independent EFS-mediated relaxations (p < 0.05). Epoxygenases showed similar effect on NO-dependent and NO-independent relaxant responses except 20-HETE which caused significantly more enhanced relaxation on NO-dependent responses (p < 0.05). No drug caused a significant relaxation response on tissues contracted with phenylephrine. Epoxygenases contribute to EFS-mediated NO-dependent and NO-independent NANC relaxations by presynaptic mechanisms, offering a new treatment alternative for erectile dysfunction which needs to be explored in further in vivo, molecular and clinical studies.

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.

Secretory vesicle trafficking in awake and anaesthetized mice: differential speeds in axons versus synapses.

KEY POINTS: Despite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain. DCVs are transported at similar average speeds in the anaesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher. Microtubule plus (+)-end extension imaging visualized microtubular growth at 0.12 µm/s and revealed that DCVs were transported faster in the anterograde direction. DCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release. Our work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveals novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality. ABSTRACT: Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamocortical projections of anaesthetized and awake mice. We found an average speed of 1 µm/s, maximal speeds of up to 5 µm/s and a pausing fraction of ∼11%. Directionality of transport differed between anaesthetized and awake mice. In vivo microtubule +-end extension imaging using MACF18-GFP revealed microtubular growth at 0.12 µm/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (∼2.1 µm/s) than retrograde transport (∼1.4 µm/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.

Striatal Reinnervation Process after Acute Methamphetamine-Induced Dopaminergic Degeneration in Mice.

Methamphetamine (METH), an amphetamine derivate, may increase the risk of developing Parkinson's disease (PD). Human and animal studies have shown that METH produces persistent dopaminergic neurotoxicity in the nigrostriatal pathway, despite initial partial recovery. To determine the processes leading to early compensation, we studied the detailed morphology and distribution of tyrosine hydroxylase immunoreactive fibers (TH-ir) classified by their thickness (types I-IV) before and after METH. Applying three established neurotoxic regimens of METH: single high dose (1 × 30 mg/kg), multiple lower doses (3 × 5 mg/kg) or (3 × 10 mg/kg), we show that METH primarily damages type I fibers (the thinner ones), and to a much lesser extend types II-IV fibers including sterile axons. The striatal TH terminal partial recovery process, consisting of a progressive regrowth increases in types II, III, and IV fibers, demonstrated by co-localization of GAP-43, a sprouting marker, was observed 3 days post-METH treatment. In addition, we demonstrate the presence of growth-cone-like TH-ir structures, indicative of new terminal generation as well as improvement in motor functions after 3 days. A temporal relationship was observed between decreases in TH-expression and increases in silver staining, a marker of degeneration. Striatal regeneration was associated with an increase in astroglia and decrease in microglia expression, suggesting a possible role for the neuroimmune system in regenerative processes. Identification of regenerative compensatory mechanisms in response to neurotoxic agents could point to novel mechanisms in countering the neurotoxicity and/or enhancing the regenerative processes.

Prostaglandin E2 modulates presynaptic regulation of GnRH neurons via EP4 receptors in accordance with estrogen milieu.

Prostaglandin E2 (PGE2) promotes gonadotropin secretion by regulating the activity of neurons that release gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, the mechanisms of action of PGE2 at these neurons have yet to be fully explored. We examined the effects of PGE2 on the generation of miniature excitatory postsynaptic currents (mEPSCs) at GnRH neurons as measured by whole-cell, patch-clamp recordings. GnRH neurons were identified in slices prepared from the preoptic areas of female GnRH-EGFP rats. Exposure to PGE2 significantly increased the frequency, but not the amplitude, of the mEPSCs generated on the day of proestrus, but neither frequency nor amplitude was altered on day 1 of diestrus. These data suggest that the action of PGE2 on mEPSC frequency varies depending on the stage of estrous. An estrogen-dependence of PGE2's action was further supported by the increased frequency, but not amplitude, of mEPSCs generated at GnRH neurons prepared from estrogen-primed ovariectomized rats. Conversely, PGE2 had no effect on mEPSC frequency or amplitude at GnRH neurons in cholesterol-treated rats. Subsequent experiments to identify candidate receptors for PG2E's action revealed that exposure to a PGE2 receptor 4 (EP4) agonist, but not EP1 or EP2 agonists, mimicked the effects achieved by PGE2 exposure. These effects of mEPSCs could be reversed using an EP4 antagonist, illustrating the specificity of the effect. Collectively, these data demonstrate that PGE2 can alter excitatory synaptic neurotransmission at GnRH neurons via EP4 signaling at presynaptic site(s) in an estrogen-dependent fashion during proestrus.

GABAergic Agonists Modulate the Glutamate Release from Frontal Cortex Synaptosomes of Rats with Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that mimics many of the clinical and pathological features of multiple sclerosis. We have previously described a significant diminution in the GABAergic regulation of glutamate release from synaptosomes of EAE rats isolated during the acute stage of the disease. In order to explore the possible metabolic pathways responsible for this alteration, in this work we evaluate the direct effect of different GABAergic agonists on the glutamate release and concomitant synapsin I phosphorylation in synaptosomes from the frontal cortex of control and EAE animals. The results show that GABA as well as the GABA receptor agonists Muscimol (GABAA agonist) and Baclofen (GABAB agonist) caused a decrease in glutamate release in control rats paralleled by a similar reduction in synapsin I phosphorylation. Meanwhile synaptosomes from EAE animals are responsive only to Baclofen with respect to nontreated EAE synaptosomes, since glutamate release from the synaptosomes treated with Muscimol was similar to that observed in EAE rat synaptosomes which was already reduced as consequence of the disease. In the case of the benzodiazepines Diazepam and Clonazepam (GABAA allosteric agonists), both of them induced a reduction in glutamate release in synaptosomes from the CFA rats, effect that was only observed in synaptosomes of EAE rats treated with Clonazepam. In all cases both benzodiazepines showed a higher effect on synapsin I phosphorylation than in glutamate release. These results indicate that the extent of GABAergic modulation of presynaptic terminals depends on the type of agonist employed and this regulation is altered in the frontal cortex during the acute phase of EAE with respect to control animals.

Modifications of excitatory and inhibitory transmission in rat hippocampal pyramidal neurons by acute lithium treatment.

The acute effects of high-dose Li(+) treatment on glutamatergic and GABAergic transmissions were studied in the "synaptic bouton" preparation of isolated rat hippocampal pyramidal neurons by using focal electrical stimulation. Both action potential-dependent glutamatergic excitatory and GABAergic inhibitory postsynaptic currents (eEPSC and eIPSC, respectively) were dose-dependently inhibited in the external media containing 30-150 mM Li(+), but the sensitivity for Li(+) was greater tendency for eEPSCs than for eIPSCs. When the effects of Li(+) on glutamate or GABAA receptor-mediated whole-cell responses (IGlu and IGABA) elicited by an exogenous application of glutamate or GABA were examined in the postsynaptic soma membrane of CA3 neurons, Li(+) slightly inhibited both IGlu and IGABA at the 150 mM Li(+) concentration. Present results suggest that acute treatment with high concentrations of Li(+) acts preferentially on presynaptic terminals, and that the Li(+)-induced inhibition may be greater for excitatory than for inhibitory transmission.

New insights into the acute actions from a high dosage of fluoxetine on neuronal and cardiac function: Drosophila, crayfish and rodent models.

The commonly used mood altering drug fluoxetine (Prozac) in humans has a low occurrence in reports of harmful effects from overdose; however, individuals with altered metabolism of the drug and accidental overdose have led to critical conditions and even death. We addressed direct actions of high concentrations on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine's action as a selective 5-HT reuptake inhibitor. There appears to be action in blocking action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events in the presynaptic terminals at NMJs of both Drosophila and crayfish. In rodent neurons, cytoplasmic Ca(2+) rises by fluoxetine and is thapsigargin dependent. The Drosophila larval heart showed a dose dependent effect in cardiac arrest. Acute paralytic behavior in crayfish occurred at a systemic concentration of 2mM. A high percentage of death as well as slowed development occurred in Drosophila larvae consuming food containing 100µM fluoxetine. The release of Ca(2+) from the endoplasmic reticulum in neurons and the cardiac tissue as well as blockage of voltage-gated Na(+) channels in neurons could explain the effects on the whole animal as well as the isolated tissues. The use of various animal models in demonstrating the potential mechanisms for the toxic effects with high doses of fluoxetine maybe beneficial for acute treatments in humans. Future studies in determining how fluoxetine is internalized in cells and if there are subtle effects of these mentioned mechanisms presented with chronic therapeutic doses are of general interest.

Orexins/hypocretins modulate the activity of NPY-positive and -negative neurons in the rat intergeniculate leaflet via OX1 and OX2 receptors.

Orexins/hypocretins (OXA and OXB) are two hypothalamic peptides involved in the regulation of many physiological processes including the sleep-wake cycle, food intake and arousal. The orexinergic system of the lateral hypothalamus is considered a non-specific peptidergic system, and its nerve fibers innervate numerous brain areas. Among many targets of orexinergic neurons is the intergeniculate leaflet (IGL) of the thalamus - a small but important structure of the mammalian biological clock. In rats, the IGL consists of GABAergic cells which also synthesize different neuropeptides. One group of neurons produces neuropeptide Y (NPY) and sends its axons to the master biological clock known as the suprachiasmatic nuclei. Another neuronal group produces enkephalin and is known to connect contralateral IGLs. This study evaluated the effects of orexins on identified IGL neurons revealing that 58% of the recorded neurons were sensitive to OXA (200nM) and OXB (200nM) administration. Both NPY-positive and -negative neurons were depolarized by these neuropeptides. Experiments using selective orexin receptor antagonists (SB-334867, 10µM and TCS-OX2-29, 10µM) suggested that both orexin receptors participate in the recorded OXA effects. In addition, IGL neurons were either directly depolarized by OXA or their activity was altered by changes in presynaptic inputs. We observed an increase of GABA release onto the investigated IGL neuron after OXA application, consistent with a presynaptic localization of the orexin receptors. An increase in miniature excitatory postsynaptic current frequency was not observed within the IGL. Our findings reinforce the connection between circadian clock physiology and the orexinergic system.

Undaria pinnatifida Promotes Spinogenesis and Synaptogenesis and Potentiates Functional Presynaptic Plasticity in Hippocampal Neurons.

Reductions in neurotrophic factors are implicated in synaptic dysfunction in the central nervous system, but exogenous neurotrophic factors with potential effects on neuritic regeneration and synaptic reconstruction could offer therapeutic and preventive strategies for treating memory-related neurological disorders. In an earlier effort to identify natural neurotrophic agents, we found that the ethanol extract of the edible marine alga Undaria pinnatifida (UPE) had promising effects on the neuritogenesis of cultured hippocampal neurons. Here, we further investigated the ability of UPE to promote spinogenesis and synaptogenesis in primary cultures of hippocampal neurons. It was found that UPE triggered significant increase in numbers of dendritic filopodia and spines, promoted the formation of excitatory and inhibitory synapses, and potentiated synaptic transmission by increasing the sizes of reserve vesicle pools at presynaptic terminals. These findings indicate a substantial role for UPE in the morphological and functional maturation of neurons and suggest that UPE is a possible therapeutic preventative measure and treatment for neurodegenerative diseases, such as those involving cognitive disorders and memory impairments.

Glutamate release machinery is altered in the frontal cortex of rats with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.

Impaired synaptic vesicle recycling contributes to presynaptic dysfunction in lipoprotein lipase-deficient mice.

Lipoprotein lipase (LPL) is expressed at high levels in hippocampal neurons, although its function is unclear. We previously reported that LPL-deficient mice have learning and memory impairment and fewer synaptic vesicles in hippocampal neurons, but properties of synaptic activity in LPL-deficient neurons remain unexplored. In this study, we found reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) and readily releasable pool (RRP) size in LPL-deficient neurons, which led to presynaptic dysfunction and plasticity impairment without altering postsynaptic activity. We demonstrated that synaptic vesicle recycling, which is known to play an important role in maintaining the RRP size in active synapses, is impaired in LPL-deficient neurons. Moreover, lipid assay revealed deficient docosahexaenoic acid (DHA) and arachidonic acid (AA) in the hippocampus of LPL-deficient mice; exogenous DHA or AA supplement partially restored synaptic vesicle recycling capability. These results suggest that impaired synaptic vesicle recycling results from deficient DHA and AA and contributes to the presynaptic dysfunction and plasticity impairment in LPL-deficient neurons.

Artemisia santolinifolia enhances glutamatergic neurotransmission in the nucleus of the solitary tract.

Artemisia extracts have been used as remedies for a variety of maladies related to metabolic and gastrointestinal control. Because the vagal afferent-nucleus of the solitary tract (NST) synapse regulates the same homeostatic functions affected by Artemisia, it is possible that these extracts may have activity at the synaptic level in the NST. Therefore, we evaluated how extracts of three common medicinal Artemisia species, Artemisia santolinifolia (SANT), Artemisia scoparia (SCO), and Artemisia dracunculus L (PMI-5011), modulate the excitability of the glutamatergic vagal afferent-NST synapse. Our in vitro live cell calcium imaging data from prelabeled vagal afferent terminals show that SANT extract is a positive modulator of vagal afferent calcium levels, as the extract significantly increased the calcium signal relative to the time control. Neither SCO nor PMI-5011 extract altered the vagal calcium signals compared to the time control. Furthermore, whole cell voltage-clamp recordings from NST neurons corroborated the vagal terminal calcium data in that SANT extract also significantly increased miniature excitatory postsynaptic current (mEPSC) frequency in NST neurons. These data suggest that SANT extract could be a pharmacologically significant mediator of glutamatergic neurotransmission within the CNS.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Omega-3 fatty acids alter behavioral and oxidative stress parameters in animals subjected to fenproporex administration.

Studies have consistently reported the participation of oxidative stress in bipolar disorder (BD). Evidences indicate that omega-3 (ω3) fatty acids play several important roles in brain development and functioning. Moreover, preclinical and clinical evidence suggests roles for ω3 fatty acids in BD. Considering these evidences, the present study aimed to investigate the effects of ω3 fatty acids on locomotor behavior and oxidative stress parameters (TBARS and protein carbonyl content) in brain of rats subjected to an animal model of mania induced by fenproporex. The fenproporex treatment increased locomotor behavior in saline-treated rats under reversion and prevention model, and ω3 fatty acids prevented fenproporex-related hyperactivity. Moreover, fenproporex increased protein carbonyls in the prefrontal cortex and cerebral cortex, and the administration of ω3 fatty acids reversed this effect. Lipid peroxidation products also are increased in prefrontal cortex, striatum, hippocampus and cerebral after fenproporex administration, but ω3 fatty acids reversed this damage only in the hippocampus. On the other hand, in the prevention model, fenproporex increased carbonyl content only in the cerebral cortex, and administration of ω3 fatty acids prevented this damage. Additionally, the administration of fenproporex resulted in a marked increased of TBARS in the prefrontal cortex, hippocampus, striatum and cerebral cortex, and prevent this damage in the prefrontal cortex, hippocampus and striatum. In conclusion, we are able to demonstrate that fenproporex-induced hyperlocomotion and damage through oxidative stress were prevented by ω3 fatty acids. Thus, the ω3 fatty acids may be important adjuvant therapy of bipolar disorder.