Inger Lise Bogen

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters.

Studies of synapsin‐deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild‐type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double‐labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain.

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.

The importance of synapsin I and II for neurotransmitter levels and vesicular storage in cholinergic, glutamatergic and GABAergic nerve terminals.

The aim of this study was to examine the importance of the vesicle-associated synapsin I and II phosphoproteins for the accumulation of neurotransmitters in central cholinergic as compared to central glutamatergic and GABAergic nerve terminals. In brain homogenate samples from mice devoid of synapsin I and II, the levels of vesicular transporters for glutamate (VGLUT1-2) and GABA (VGAT) were decreased by 35-40% in striatum and cortex, while no change was apparent for the vesicular acetylcholine transporter (VAChT). The severe decrease in the levels of amino acid vesicular transporters caused only minor changes in the concentrations of the respective neurotransmitters in homogenates of the three selected brain areas from synapsin I- and II-deficient mice. However, when measured in a crude vesicular fraction, the concentrations of glutamate and GABA were decreased by 48-60% in synapsin-deficient mice, with a similar decrease in the levels of VGLUT1, VGLUT2 and VGAT. In comparison, the concentration of acetylcholine and the level of VAChT were not significantly different from wild-type in the vesicular fraction. No changes were seen in the activity of specific enzymes involved in the synthesis of acetylcholine, glutamate or GABA, however, immunoblotting indicated a decrease in the protein level of glutamic acid decarboxylase, isoform 65 (GAD(65)). In conclusion, the results indicate that neurotransmitter regulation in central cholinergic synapses may be less dependent on synapsin I and II compared to the marked alterations seen in the glutamatergic and GABAergic synapses.

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles.

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and 13C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. 13C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down‐regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.

Marine omega-3 polyunsaturated fatty acids induce sex-specific changes in reinforcer-controlled behaviour and neurotransmitter metabolism in a spontaneously hypertensive rat model of ADHD

BackgroundPrevious reports suggest that omega-3 (n-3) polyunsaturated fatty acids (PUFA) supplements may reduce ADHD-like behaviour. Our aim was to investigate potential effects of n-3 PUFA supplementation in an animal model of ADHD.MethodsWe used spontaneously hypertensive rats (SHR). SHR dams were given n-3 PUFA (EPA and DHA)-enriched feed (n-6/n-3 of 1:2.7) during pregnancy, with their offspring continuing on this diet until sacrificed. The SHR controls and Wistar Kyoto (WKY) control rats were given control-feed (n-6/n-3 of 7:1). During postnatal days (PND) 25–50, offspring were tested for reinforcement-dependent attention, impulsivity and hyperactivity as well as spontaneous locomotion. The animals were then sacrificed at PND 55–60 and their neostriata were analysed for monoamine and amino acid neurotransmitters with high performance liquid chromatography.Resultsn-3 PUFA supplementation significantly enhanced reinforcement-controlled attention and reduced lever-directed hyperactivity and impulsiveness in SHR males whereas the opposite or no effects were observed in females. Analysis of neostriata from the same animals showed significantly enhanced dopamine and serotonin turnover ratios in the male SHRs, whereas female SHRs showed no change, except for an intermediate increase in serotonin catabolism. In contrast, both male and female SHRs showed n-3 PUFA-induced reduction in non-reinforced spontaneous locomotion, and sex-independent changes in glycine levels and glutamate turnover.ConclusionsFeeding n-3 PUFAs to the ADHD model rats induced sex-specific changes in reinforcement-motivated behaviour and a sex-independent change in non-reinforcement-associated behaviour, which correlated with changes in presynaptic striatal monoamine and amino acid signalling, respectively. Thus, dietary n-3 PUFAs may partly ameliorate ADHD-like behaviour by reinforcement-induced mechanisms in males and partly via reinforcement-insensitive mechanisms in both sexes.

Differential development of vesicular glutamate transporters in brain: An in vitro study of cerebellar granule cells

The cerebellar granule cells have been extensively used for studies on metabolism, neurotransmission and neurotoxicology, since they can easily be grown in cultures. However, knowledge about the development of different proteins essential for synaptic transmission in these cells is lacking. This study has characterized the developmental profiles of the vesicular glutamate transporters (VGLUTs) and the synaptic vesicle proteins synapsins and synaptophysin in cerebellar granule cells and in co-cultures containing both granule cells and astrocytes. The protein levels of VGLUT2 decreased by approximately 70% from days 2 to 7 in vitro, whereas the levels of VGLUT1 increased by approximately 95%. Protein levels of synapsin I, synapsin IIIa and synaptophysin showed a developmental pattern similar to VGLUT1 while synapsin II and VGLUT3 were absent. The mRNA expressions of VGLUT1 and VGLUT2 were in accordance with the protein levels. The results indicate both that cerebellar granule cells are mature at approximately 7 days in vitro, and that the up-regulation of VGLUT1 and down-regulation of VGLUT2 in cerebellar granule cells are both independent of surrounding astrocytes and neuronal input. The results of this study are discussed in relation to general developmental profiles of VGLUTs in other brain regions.

Mechanisms of penitrem-induced cerebellar granule neuron death in vitro: Possible involvement of GABAA receptors and oxidative processes

The fungal neurotoxin penitrem A has previously been found to cause neurological disorders in animals and humans after ingestion of contaminated food and/or feed. It penetrates the blood-brain-barrier and causes cerebellar pathology in rats, including mild effects on granule neurons. The aim of the current study was to investigate the potential toxicity of penitrem A in rat cerebellar granule neurons in vitro, and to examine the involvement of the GABAA, AMPA and NMDA receptors, intracellular signalling pathways as well as the role of oxidative stress in penitrem A-induced neuronal death. Cerebellar granule cells were exposed to penitrem A, alone or together with different pharmacological agents, before cell survival was assessed with the MTT assay or formation of reactive oxygen species (ROS) was investigated with the DCF assay. Penitrem A caused a time- and concentration-dependent reduction in cell survival, as well as a concentration-dependent increase in ROS production. Co-incubation with diazepam, GABA, BAPTA-AM, vitamin E, SP600125 and cyclosporine A significantly reduced cell death. Our results show that penitrem A is toxic to cerebellar granule neurons in vitro. Further, ROS production and the GABAA receptor are likely to be involved in the induction of neuronal death following penitrem A exposure. A disruption of calcium homeostasis and activation of the JNK pathway may also play a role in penitrem A neurotoxicity.

Glutamatergic neurotransmission in the synapsin I and II double knock-out mouse

The synaptic vesicle-associated synapsin proteins may participate in synaptic transmission, but their exact functional role(s) here remain(s) uncertain. We here briefly describe the important characteristics of the synapsin proteins, and review recent studies on transgenic mice devoid of the gene products encoded by the synapsin I and II genes, where both neurochemical, cell biological and electrophysiological methods have been employed. We present evidence for synapsin effects on both neurotransmitter synthesis and homeostasis, as well as on synaptic vesicle development and functions. Moreover, we describe physiological analyses of excitatory glutamatergic hippocampal synapses where a novel synapsin-dependent delayed response enhancement (DRE) phase occurs, and demonstrate the postnatal developmental patterns of both frequency facilitations and DRE responses. Finally, we report synapsin I and II effects in distinct excitatory glutamatergic synapses in the hippocampus, and indicate that synapsin-dependent modulations of synaptic function may use distinct presynaptic response patterns in order to induce different classes of presynaptic plasticity.

Synapsins I and II Are Not Required for Insulin Secretion from Mouse Pancreatic beta-cells

Synapsins are a family of phosphoproteins that modulate the release of neurotransmitters from synaptic vesicles. The release of insulin from pancreatic β-cells has also been suggested to be regulated by synapsins. In this study, we have utilized a knock out mouse model with general disruptions of the synapsin I and II genes [synapsin double knockout (DKO)]. Stimulation with 20 mm glucose increased insulin secretion 9-fold in both wild-type (WT) and synapsin DKO islets, whereas secretion in the presence of 70 mm K(+) and 1 mm glucose was significantly enhanced in the synapsin DKO mice compared to WT. Exocytosis in single β-cells was investigated using patch clamp. The exocytotic response, measured by capacitance measurements and elicited by a depolarization protocol designed to visualize exocytosis of vesicles from the readily releasable pool and from the reserve pool, was of the same size in synapsin DKO and WT β-cells. The increase in membrane capacitance corresponding to readily releasable pool was approximately 50fF in both genotypes. We next investigated the voltage-dependent Ca(2+) influx. In both WT and synapsin DKO β-cells the Ca(2+) current peaked at 0 mV and measured peak current (I(p)) and net charge (Q) were of similar magnitude. Finally, ultrastructural data showed no variation in total number of granules (N(v)) or number of docked granules (N(s)) between the β-cells from synapsin DKO mice and WT control. We conclude that neither synapsin I nor synapsin II are directly involved in the regulation of glucose-stimulated insulin secretion and Ca(2)-dependent exocytosis in mouse pancreatic β-cells.

Low-Chlorinated Non-Dioxin-like Polychlorinated Biphenyls Present in Blood and Breast Milk Induce Higher Levels of Reactive Oxygen Species in Neutrophil Granulocytes than High-Chlorinated Congeners.

Despite their ban several decades ago, polychlorinated biphenyls (PCBs) still pose a health threat to human beings due to their persistent and accumulative nature and continued presence in the environment. Non‐dioxin‐like (NDL)‐PCBs have earlier been found to have effects on the immune system, including human neutrophil granulocytes. The aim of this study was to investigate the differences between ortho‐chlorinated NDL‐PCBs with a low or high degree of chlorination in their capability to induce the production of reactive oxygen species (ROS) in human neutrophil granulocytes in vitro. We used some of the congeners occurring at the highest levels in blood, breast milk and food: PCB 52 representing the low‐chlorinated congeners and PCB 180 the high‐chlorinated congeners. In addition, the extensively studied PCB 153 was included as a reference compound. ROS production was assessed with the luminol‐amplified chemiluminescence and DCF fluorescence assays. The involvement of intracellular signalling mechanisms was investigated using different pharmacological substances. At high concentrations (10–20 μM), PCB 52 induced more ROS than PCB 153 and PCB 180. The role of extracellular signal‐regulated kinase (ERK) 1/2 and/or ERK 5 signalling in PCB‐induced ROS production was implicated through the reduction in ROS in the presence of the specific inhibitor U0126, whereas reduced ROS production after the use of SB203580 and SP600125 indicated the involvement of the p38 mitogen‐activated protein kinase (MAPK) and c‐Jun amino‐terminal kinase (JNK) pathways, respectively. In addition, the calcineurin inhibitor FK‐506, the intracellular calcium chelator BAPTA‐AM and the antioxidant vitamin E reduced the levels of ROS. The intracellular signalling mechanisms involved in ROS production in human neutrophil granulocytes appeared to be similar for PCB 52, PCB 153 and PCB 180. Based on the results from the present and previous studies, we conclude that for abundant ortho‐chlorinated PCBs found in the blood, low‐chlorinated congeners induce higher production of ROS in neutrophil granulocytes than high‐chlorinated congeners. This could be relevant during acute exposure scenarios when high concentrations of PCBs are present.