Effect of topiramate and dBcAMP on expression of the glutamate transporters GLAST and GLT-1 in astrocytes cultured separately, or together with neurons.

The mechanism of the antiepileptic drug topiramate is not fully understood, but interaction with the excitatory neurotransmission, e.g. glutamate receptors, is believed to be part of its anticonvulsant effect. The glutamate transporters GLAST and GLT-1 are responsible for the inactivation of glutamate as a neurotransmitter and it was therefore investigated if topiramate might affect the expression of GLAST and GLT-1 in astrocytes cultured separately or together with neurons. Since expression and membrane trafficking of glutamate transporters are affected by the protein kinase C system as well as by dBcAMP it was also investigated if these signalling pathways might play a role. In astrocyte cultures expressing mainly GLAST treatment with dBcAMP (0.25 mM) led to an increased expression of the total amount of GLAST as well as of its membrane association. The enhanced expression in the membrane was particularly pronounced for the oligomeric form of GLAST. No detectable effect on the expression of GLAST in astrocytes treated with topiramate in the presence and absence of protein kinase C activators or inhibitors was observed. Astrocytes co-cultured with neurons expressed both GLAST and GLT-1. In these cultures prolonged exposure to 30 muM topiramate (10 days) led to a statistically significant increase (P<0.025) in the membrane expression of GLAST. In case of GLT-1, culture in the presence of 30 microM topiramate for 1 and 10 days led to alterations in the total, cytoplamic and membrane expression of the oligomeric form of the transporter.

Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission.

The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways at transcriptional, translational and post-translational levels. This opens for a number of therapeutic strategies by which the efficacy of excitatory and inhibitory neurotransmission may be manipulated.

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions.

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation.

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.

Psychological assessment of blood related renal donors.

The impact of kidney donation on the psychological health of 31 living related donors was assessed by administering certain psychological tests before and after the operation (for donating the kidney). The results indicated a significant rise in the somatization subscale of the Middlesex Hospital Questionnaire (MHQ) from a mean of 1.61 to 3.23. There was no significant change in the other variables of these instruments or in the locus of control score. Only about one-fourth of the donors had prior knowledge of renal transplant. In almost all cases, the decision to donate had been voluntary and immediate, motivated by a concern for the recipient; there was virtually no second thoughts or regrets subsequently, which was apparently partly related to the opinions of other relatives who positively valued the act of donation.