Kisspeptin1 modulates odorant‐evoked fear response via two serotonin receptor subtypes (5‐HT1A and 5‐HT2) in zebrafish

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT_1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT₁ and 5‐HT₂ receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT_1A and 5‐HT₂ receptors.

     

Comparative effect of lurasidone and blonanserin on cortical glutamate,dopamine, and acetylcholine efflux: role of relative serotonin (5‐HT)2A and DA D2 antagonism and 5‐HT1A partial agonism

Atypical antipsychotic drugs (AAPDs) have been suggested to be more effective in improving cognitive impairment in schizophrenia than typical APDs, a conclusion supported by differences in receptor affinities and neurotransmitter efflux in the cortex and the hippocampus. More potent serotonin (5‐HT)_2A than dopamine (DA) D₂ receptors antagonism, and direct or indirect 5‐HT_1A agonism, characterize almost all AAPDs. Blonanserin, an AAPD, has slightly greater affinity for D₂ than 5‐HT_2A receptors. Using microdialysis and ultra performance liquid chromatography‐mass spectrometry/mass spectrometry, we compared the abilities of the typical APD, haloperidol, three AAPDs, blonanserin, lurasidone, and olanzapine, and a selective 5‐HT_1A partial agonist, tandospirone, and all, except haloperidol, were found to ameliorate the cognitive deficits produced by the N‐methyl‐d‐aspartate antagonist, phencyclidine, altering the efflux of neurotransmitters and metabolites in the rat cortex and nucleus accumbens. Blonanserin, lurasidone, olanzapine, and tandospirone, but not haloperidol, increased the efflux of cortical DA and its metabolites, homovanillic acid and 3,4‐dihydroxyphenylacetic acid. Olanzapine and lurasidone increased the efflux of acetylcholine; lurasidone increased glutamate as well. None of the compounds significantly altered the efflux of 5‐HT or its metabolite, 5‐hydroxyindole acetic acid, or GABA, serine, and glycine. The ability to increase cortical DA efflux was the only shared effect of the compounds which ameliorates the deficit in cognition in rodents following phencyclidine.

     

Effect of genetic and pharmacological blockade of GABA receptors on the 5‐HT2C receptor function during stress

Serotonin (5‐HT)_2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5‐HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5‐HT_2C receptor‐induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5‐HT turnover by a 5‐HT_2C receptor agonist (RO 60‐0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA‐A or GABA‐B receptors in mice. Neither the GABA‐B receptor antagonist phaclofen nor the specific genetic ablation of either GABA‐B1a or GABA‐B1b subunits altered the inhibitory effect of RO 60‐0175, although 5‐HT turnover was markedly decreased in GABA‐B1a knock‐out mice in both basal and stress conditions. In contrast, the 5‐HT_2C receptor‐mediated inhibition of 5‐HT turnover was reduced by the GABA‐A receptor antagonist bicuculline. However, a significant effect of 5‐HT_2C receptor activation persisted in mutant mice deficient in the α₃ subunit of GABA‐A receptors. It can be inferred that non‐α₃ subunit‐containing GABA‐A receptors, but not GABA‐B receptors, mediate the 5‐HT_2C‐induced inhibition of stress‐induced increase in hippocampal 5‐HT turnover in mice.

     

Drebrin depletion alters neurotransmitter receptor levels in protein complexes,dendritic spine morphogenesis and memory‐related synaptic plasticity in the mouse hippocampus

Drebrin an actin‐bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous drebrin knockout mice generated in our laboratory and related wild‐type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5‐hydroxytryptamine receptor 1A (5‐HT1_AR), and 5‐hydroxytryptamine receptor 7 (5‐HT7R) were significantly reduced in hippocampus of drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel‐based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory‐related hippocampal synaptic plasticity upon drebrin depletion. These findings provide unprecedented experimental support for a role of drebrin in the regulation of memory‐related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines.

     

Depolarization‐induced suppression of a glycinergic synapse in the superior olivary complex by endocannabinoids

The neuronal endocannabinoid system is known to depress synaptic inputs retrogradely in an activity‐dependent manner. This mechanism has been generally described for excitatory glutamatergic and inhibitory GABAergic synapses. Here, we report that neurones in the auditory brainstem of the Mongolian gerbil (Meriones unguiculatus) retrogradely regulate the strength of their inputs via the endocannabinoid system. By means of whole‐cell patch‐clamp recordings, we found that retrograde endocannabinoid signalling attenuates both glycinergic and glutamatergic post‐synaptic currents in the same types of neurones. Accordingly, we detected the cannabinoid receptor 1 in excitatory and inhibitory pre‐synapses as well as the endocannabinoid‐synthesising enzymes (diacylglycerol lipase α/β, DAGLα/β) post‐synaptically through immunohistochemical stainings. Our study was performed with animals aged 10–15 days, that is, in the time window around the onset of hearing. Therefore, we suggest that retrograde endocannabinoid signalling has a role in adapting inputs during the functionally important switch from spontaneously generated to sound‐related signals.

     

Epidermal fatty acid‐binding protein protects nerve growth factor‐differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABP's induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.

     

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non‐steroidal anti‐inflammatory drugs: role of mitochondrial calcium

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca²⁺ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca²⁺ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca²⁺ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca²⁺ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca²⁺ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca²⁺ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.

     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

The high‐affinity D2/D3 agonist D512 protects PC12 cells from 6‐OHDA‐induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease

In this study, in vitro and in vivo experiments were carried out with the high‐affinity multifunctional D2/D3 agonist D‐512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre‐treatment with D‐512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6‐hydroxydopamine administration in a dose‐dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre‐treatment with 0.5 mg/kg D‐512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D‐512 may constitute a novel viable therapy for Parkinson's disease.

     

Striatal serotonin 2C receptors decrease nigrostriatal dopamine release by increasing GABA‐A receptor tone in the substantia nigra

Drugs acting at the serotonin‐2C (5‐HT2C) receptor subtype have shown promise as therapeutics in multiple syndromes including obesity, depression, and Parkinson's disease. While it is established that 5‐HT2C receptor stimulation inhibits DA release, the neural circuits and the localization of the relevant 5‐HT2C receptors remain unknown. This study used dual‐probe in vivo microdialysis to investigate the relative contributions of 5‐HT2C receptors localized in the rat substantia nigra (SN) and caudate‐putamen (CP) in the control of nigrostriatal DA release. Systemic administration (3.0 mg/kg) of the 5‐HT2C receptor selective agonist Ro 60‐0175 [(αS)‐6‐Chloro‐5‐fluoro‐α‐methyl‐1H‐indole‐1‐ethanamine fumarate] decreased, whereas intrastriatal infusions of the selective 5‐HT2C antagonist SB 242084 [6‐Chloro‐2,3‐dihydro‐5‐methyl‐N‐[6‐[(2‐methyl‐3‐pyridinyl)oxy]‐3‐pyridinyl]‐1H‐indole‐1‐carboxyamide; 1.0 μM] increased, basal DA in the CP. Depending on the site within the SN pars reticulata (SNpr), infusions of SB 242084 had more modest but significant effects. Moreover, infusions of the GABA‐A receptor agonist muscimol (10 μM) into the SNpr completely reversed the increases in striatal DA release produced by intrastriatal infusions of SB 242084. These findings suggest a role for 5‐HT2C receptors regulating striatal DA release that is highly localized. 5‐HT2C receptors localized in the striatum may represent a primary site of action that is mediated by the actions on GABAergic activity in the SN.

     

S‐nitrosoglutathione prevents blood–brain barrier disruption associated with increased matrix metalloproteinase‐9 activity in experimental diabetes

Hyperglycemia is known to induce microvascular complications, thereby altering blood–brain barrier (BBB) permeability. This study investigated the role of matrix metalloproteinases (MMPs) and their endogenous inhibitors in increased BBB permeability and evaluated the protective effect of S‐nitrosoglutathione (GSNO) in diabetes. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days and GSNO was administered orally (100 μg/kg body weight) daily for 8 weeks after the induction of diabetes. A significant decline in cognitive functions was observed in diabetic mice assessed by Morris water maze test. Increased permeability to different molecular size tracers accompanied by edema and ion imbalance was observed in cortex and hippocampus of diabetic mice. Furthermore, activity of both pro and active MMP‐9 was found to be significantly elevated in diabetic animals. Increased in situ gelatinase activity was observed in tissue sections and isolated microvessels from diabetic mice brain. The increase in activity of MMP‐9 was attributed to increased mRNA and protein expression in diabetic mice. In addition, a significant decrease in mRNA and protein expression of tissue inhibitor of matrix metalloproteinase‐1 was also observed in diabetic animals. However, GSNO supplementation to diabetic animals was able to abridge MMP‐9 activation as well as tissue inhibitor of matrix metalloproteinase‐1 levels, restoring BBB integrity and also improving learning and memory. Our findings clearly suggest that GSNO could prevent hyperglycemia‐induced disruption of BBB by suppressing MMP‐9 activity.

     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

Brain serotonin synthesis in MDMA (ecstasy) polydrug users: an alpha‐[11C]methyl‐l‐tryptophan study

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) use may have long‐term neurotoxic effects. In this study, positron emission tomography with the tracer alpha‐[¹¹C]methyl‐l ‐tryptophan (¹¹C‐AMT) was used to compare human brain serotonin (5‐HT) synthesis capacity in 17 currently drug‐free MDMA polydrug users with that in 18 healthy matched controls. Gender differences and associations between regional ¹¹C‐AMT trapping and characteristics of MDMA use were also examined. MDMA polydrug users exhibited lower normalized ¹¹C‐AMT trapping in pre‐frontal, orbitofrontal, and parietal regions, relative to controls. These differences were more widespread in males than in females. Increased normalized ¹¹C‐AMT trapping in MDMA users was also observed, mainly in the brainstem and in frontal and temporal areas. Normalized ¹¹C‐AMT trapping in the brainstem and pre‐frontal regions correlated positively and negatively, respectively, with greater lifetime accumulated MDMA use, longer durations of MDMA use, and shorter time elapsed since the last MDMA use. Although the possibility of pre‐existing 5‐HT alterations pre‐disposing people to use MDMA cannot be ruled out, regionally decreased 5‐HT synthesis capacity in the forebrain could be interpreted as neurotoxicity of MDMA on distal (frontal) brain regions. On the other hand, increased 5‐HT synthesis capacity in the raphe and adjacent areas could be due to compensatory mechanisms.

     

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of [³H]hemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (K_D) and maximum number of binding sites (B_max); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.