Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss‐of‐function mutations in α2 Na+,K+ ATPase (α2 NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2‐knockin mice with reduced expression of α2 NKA. The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K+ clearance by cortical astrocytes during neuronal activity and reduced density of GLT‐1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2‐knockin mice, demonstrating key physiological roles of α2 NKA and supporting tight coupling with GLT‐1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild‐type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2‐knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine.

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γ‐Aminobutyric acid 1 (GAT‐1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT‐1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT‐2 and GAT‐3 levels in KOs were unchanged and that GAT‐3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl‐evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABAAR‐mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post‐synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABAAR‐mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT‐1 KO mice GAT‐3 is not redistributed and GADs are adaptively changed and indicate that GAT‐1 has a prominent role in both tonic and phasic GABAAR‐mediated inhibition, in particular during sustained neuronal activity.

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In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 downregulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, by performing a quantitative analysis of the degree of co-localization of synaptotagmin (SYT) 1 and 2, synaptic vesicle protein 2 (SV2) A and B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ terminals and synaptic vesicles (SVs) in rat cerebral cortex. Co-localization studies showed that VGLUT1 puncta had high levels of SV2A and B and of Rab3c, intermediate levels of SYT1, and low levels of SYT2 and Rab3c; VGLUT2 puncta exhibited intermediate levels of all presynaptic proteins studied; whereas vesicular GABA transporter (VGAT) puncta had high levels of SV2A and SYT2, intermediate levels of SYT1, Rab3a, and Rab3c, and low levels of SV2B. Since SV2B is reportedly expressed by glutamatergic neurons and we observed SV2B expression in VGAT puncta, we performed electron microscopic studies and found SV2B positive axon terminals forming symmetric synapses. Immunoisolation studies showed that the expression levels of the protein isoforms varied in the three populations of SVs. Expression of SYT1 was highest in VGLUT1–SVs, while SYT2 expression was similar in the three SV groups. Expression of SV2A was similarly high in all three SV populations, except for SV2B levels that were very low in VGAT SVs. Finally, Rab3a levels were similar in the three SV groups, while Rab3c levels were highest in VGLUT1–SVs. These quantitative results extend our previous studies on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of the respective release machineries can be generated by the differential complement of SV proteins involved in distinct stages of the release process.

20 ('Have you ever had delirium tremens or seizures, severe shaking, heard voices or seen things that were not there after heavy drinking?'). None of these analyses yielded a significant association with the NR2B genotype. We performed additional subdivisions of our sample with respect to sex and psychiatric comorbidity, including affective disorders, anxiety disorders and substance abuse other than alcohol dependence. No significant association with the NR2B genotype was observed (Cochran-Armitage test, data not shown).In conclusion, our results show no increased risk for alcohol dependence associated with a C2873T-SNP genotype of the NR2B subunit, and show no significant association with alcohol-dependent phenotypes presumed to carry a high genetic load. Although the NR2B subunit remains an important candidate gene for alcoholism, our results suggest that the C2873T polymorphism is not a promising lead to be followed up in larger patient samples. A systematic assessment of all the informative genetic variations of the NR2B subunit for an association with alcohol dependence is warranted.This work was supported by the programme 'Addictive Behaviour: Improving Healthcare through Interdisciplinary Research' (01EB0110/Landesprojekt) to GS and SWe the National genome research network projects 'Genetics of Alcoholism' (FKZ 01GS0117) to GS and 'Generation of cDNA/Sequencing' to SWi. SIR -Glutamate (Glu), the main excitatory neurotransmitter in the cerebral cortex, has been implicated in several neuropsychiatric disorders, including schizophrenia. 1 The effects of Glu are determined, among others, by the action of high-affinity Glu transporters (GluTs). 2 To date, five GluTs have been characterized: GLT-1, GLAST, EAAC1, EAAT4, and EAAT5. 2,3 Clozapine, a highly effective antipsychotic, 4 increases the extracellular levels of Glu in the rat frontal cortex. 5,6 Recently, we showed that clozapine reduces cortical expression of GLT-1 and decreases total Glu uptake by 60% in the rat frontal cortex, 6 suggesting that the clozapine-induced increase in Glu levels depends on GLT-1 down-regulation. In the neocortex, EAAT4 and EAAT5 expression is poor or absent 3 and therefore their contribution to total cortical Glu uptake is extremely small, whereas GLAST and EAAC1 are robustly expressed 7,8 and mediate a sizeable fraction of total Glu uptake. 9 Whether the expression of GLAST and EAAC1 is altered by clozapine is unknown.Thus, we used immunocytochemical and Western blotting methods to investigate whether clozapine

In monoamine-releasing terminals, neurotransmitter transporters – in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space – are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy.