Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity.

GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg(2+) model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity

Suppressor T-cell activity in responder x nonresponder (C57BL/10 x DBA/1)F(1) spleen cells responsive to l-glutamic acid(60)-L-alanine(30)-L-tyrosine (10)

The ability of spleen cells from (responder X nonresponder)F(1) mice immunized with various GAT-Mφ, GAT-MBSA, and soluble GAT to develop IgG GAT-specific PFC responses in vitro after stimulation with responder and nonresponder parental and F(1) GAT-Mφ, was investigated. F(1) spleen cells from mice immunized with F(1) GAT-Mφ or GAT-MBSA developed secondary responses to responder and nonresponder parental and F(1) GAT- Mφ, but not to unrelated third party GAT-Mφ. Spleen cells from F(1) mice immunized with either parental GAT-Mφ developed secondary responses to F(1) GAT-Mφ and only the parental GAT-Mφ used for immunization in vivo. Soluble GAT-primed F(1) spleen cells responded to F(1) and responder parental, but not nonresponder parental, GAT-Mφ. Simultaneous immunization in vivo with the various GAT-Mφ or GAT-MBSA plus soluble GAT modulated the response pattern of these F(1) spleen cells such that they developed secondary responses only to F(1) and parental responder GAT-Mφ regardless of the response pattern observed after immunization with the various GAT-Mφ or GAT-MBSA alone. These observations demonstrate the critical importance of the physical state of the GAT used for immunization in determining the subsequent response pattern of immune F(1) spleen cells to the parental and F(1) GAT-Mφ. Further, suppressor T cells, capable of inhibiting primary responses to GAT by virgin F(1) spleen cells stimulated by nonresponder parental GAT-Mφ, were demonstrated in spleens of F(1) mice immunized with soluble GAT, but not those primed with F(1) GAT-Mφ. Because responder parental mice develop both helper and suppressor T cells after immunization with GAT-Mφ, and soluble GAT preferentially stimulates suppressor T cells whereas GAT-Mφ stimulate helper T cells in nonresponder parental mice, these observations suggest that distinct subsets of T cells exist in F(1) mice which behave phenotypically as responder and nonresponder parental T cells after immunization with soluble GAT and GAT- Mφ

Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cells.

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT-1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals

Increased expression of GABA transporters, GAT-1 and GAT-3, in the deafferented superior colliculus of the rat.

GABA transporters (GATs) play a critical role in the translemmal transport of GABA in neurons and glial cells. Two major brain GATs, GAT-1 and GAT-3, are found in astrocytes in the adult brain. Astroglia demonstrate morphological and molecular changes in response to brain injury and deafferentation. The present study was designed to determine whether the expression of GATs changes after nerve deafferentation using the rat superior colliculus (SC) as a model. The immunoreactivity for GAT-1 and GAT-3, as well as GABA and glutamic acid decarboxylase (GAD)-65 and GAD-67, was studied in the SC of control rats and rats with unilateral optic nerve transections. Immunolabeling for both GAT-1 and GAT-3 was increased in the neuropil of the denervated SC as compared to that for the SC of control rats or for the unaffected SC of experimental rats. In contrast, immunoreactivity for GABA, GAD-65 and GAD-67 was not altered. The change in the immunolabeling of GAT-1 and GAT-3 was detectable at 1 day postlesion and became more evident between 3-30 days postlesion. At the electron microscopic level, immunoreactivity for both GAT-1 and GAT-3 in the unaffected SC was localized to astrocytic processes, whereas GAT-1 immunolabeling was also present in synaptic terminals. In the deafferented SC, immunolabeling for both GATs was elevated in the somata and processes of hypertrophied astrocytes as compared to that in the unaffected SC, whereas GAT-1 labeling in neuronal profiles was largely unchanged. A substantial increase of GAT-1 and GAT-3 in astrocytes following optic nerve transection suggests that these cells play a role in modulating GABA's action in the deafferented SC

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : V. STIMULATION OF SUPPRESSOR T CELLS IN NONRESPONDER MICE BY THE TERPOLYMERL-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 (GAT)

In recent studies we have found that GAT not only fails to elicit a GAT-specific response in nonresponder mice but also specifically decreases the ability of nonresponder mice to develop a GAT-specific PFC response to a subsequent challenge with GAT bound to the immunogenic carrier, MBSA. Studies presented in this paper demonstrate that B cells from nonresponder, DBA/1 mice rendered unresponsive by GAT in vivo can respond in vitro to GAT-MBSA if exogenous, carrier-primed T cells are added to the cultures. The unresponsiveness was shown to be the result of impaired carrier-specific helper T-cell function in the spleen cells of GAT-primed mice. Spleen cells from GAT-primed mice specifically suppressed the GAT-specific PFC response of spleen cells from normal DBA/1 mice incubated with GAT-MBSA. This suppression was prevented by pretreatment of GAT-primed spleen cells with anti-θ serum plus C or X irradiation. Identification of the suppressor cells as T cells was confirmed by the demonstration that suppressor cells were confined to the fraction of the column-purified lymphocytes which contained θ-positive cells and a few non-Ig-bearing cells. The significance of these data to our understanding of Ir-gene regulation of the immune response is discussed

A functional role for both GABA transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus

Tonic γ-aminobutyric acid (GABA)(A) receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased

Intra- and interspecific polymorphisms ofLeishmania donovani andL. tropica minicircle DNA

A pair of degenerate polymerase chain reaction (PCR) primers (LEI-1, TCG GAT CC[C,T] [G,C]TG GGT AGG GGC GT; LEI-2, ACG GAT CC[G,C] [G,C][A,C]C TAT [A,T]TT ACA CC) defining a 0.15-kb segment ofLeishmania minicircle DNA was constructed. These primers amplified not only inter- but also intraspecifically polymorphic sequences. Individual sequences revealed a higher intraspecific than interspecific divergence. It is concluded that individual sequences are of limited relevance for species determination. In contrast, when a data base of 19 different sequences was analyzed in a dendrographic plot, an accurate species differentiation was feasible

Alteration of GABA transporter expression in the rat cerebral cortex following needle puncture and colchicine injection.

In the adult cerebral cortex, GABA transporters (GATs) are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that (1) the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated with the injections; (2) increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by transcellular signaling across the corpus callosum; and (3) the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities