Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.

Stroke is one of the leading causes of death worldwide. At present, the only available treatment is thrombolysis, which should be initiated no later than 4.5 hours after onset of symptoms. Several studies have shown that an attenuation of the inflammatory response in relation to stroke could widen the therapeutic window. However, the immune system has important functions following infarction, such as removal of dead cells and the subsequent astrocytosis as well as prevention of post-ischemic infection. Hence, detailed knowledge concerning the temporal profile of leukocyte infiltration is necessary in order to develop new and effective treatments.The purpose of this review is to determine the temporal profile of leukocyte (neutrophil granulocytes, macrophages and T-cells) infiltration following experimental stroke. We found that the number of neutrophil granulocytes peaks between day 1 and 3 after experimental stroke, with short occlusion times (30 and 60 minutes of middle cerebral artery occlusion (MCAO)) leading to a later peak in response (P <0.001). Macrophages/microglia were found to peak later than day 3 and stay in the infarcted area for longer time periods, whereas duration of occlusion had no influence on the temporal infiltration (P = 0.475). Studies on T-cell infiltration are few; however, a tendency towards infiltration peak at later time points (from day 4 onwards) was seen.This review provides a framework for the instigation of post-stroke anti-inflammatory treatment, which could prove beneficial and widen the therapeutic window compared to current treatment options.

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1deltag --> X3sigmag-) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

5-(4-Piperidyl)isoxazol-3-ol (4-PIOL, 10), a structural analog of 4-aminobutanoic acid (GABA, 1) and the GABAa agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 5), is a lowefficacy partial GABAa agonist. A number of compounds bioisosterically derived from 10, including 5-(4-piperidyl)isothiazol-3-ol (11), 3-(4-piperidyl)isoxazol-5-ol (12), 5-(l,2,3,6-tetrahydropyrid-4-yl)isoxazol-3-ol (13), and 5-(l,2,3,6-tetrahydropyrid-4-yl)isothiazol-3-ol ( 14), were synthesized and tested as GABAa receptor ligands. Whereas none of these compounds significantly affected GABAb receptor binding or GABA uptake, they showed affinities for GABAa receptor sites in the low-micromolar range. Using cultured cerebral cortical neurons and whole-cell patch-clamp techniques, the efficacies of these compounds relative to that of the full GABAa agonist, isoguvacine (8) (20 µ ), were determined. The relative efficacy of 11, which has a higher receptor affinity (IC50 = 1.3 ± 0.3 µ ) than 10 (IC50 = 9.3 ± 2.6 µ ), was comparable with that of 10 (30-35%). The tetrahydropyridine analog of 10, compound 13, showed a markedly lower receptor affinity (IC50 = 32 ± 10 µ ) and apparently a lower relative efficacy than 10. The corresponding unsaturated analog of 11, compound 14, showed a slightly weaker receptor affinity (IC50 = 4.0 ± 2.0 µ ) but a significantly higher relative efficacy (50-55%) than 11. The 5-isoxazolol isomer of 10, compound 12, showed a reduced receptor affinity (IC50 = 26 ± 7 µ ) and a very low relative efficacy. Substitution of propanoic or propenoic acid moieties for the acidic heterocyclic units of these compounds gave the monocyclic amino acids 15-18, which have very little or no affinity for GABAa receptor sites.