Revisión bibliográfica y análisis documental.

The data presented in this thesis, in addition to Fiacco et al (2007) and Agulhon et al (2010), at first glance appear to directly contradict the current state of the literature and the theory of gliotransmission. This perception is incorrect, as the data presented herein merely suggest that the mechanism by which gliotransmission is thought to occur needs to be reexamined, specifically in regards to the necessity of IP3R-mediated intracellular Ca2+ increases in astrocytes. This is supported by the current evidence for gliotransmission that has used either non-selective or non- physiological stimuli to elicit Ca2+ increases in astrocytes. These methods may produce experimental artifacts which can be interpreted as positive data for gliotransmission. For example, non-physiological Ca2+ increases induced by

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uncaging of IP3 do not travel through the cell with the same spatial profile as

endogenous GPCR activation but can trigger the release of gliotransmitters (Fiacco and McCarthy, 2004). Further, neuronal synaptic activity shows no modulation upon selective activation of astrocyte intracellular Ca2+ increases in the MrgA1 mouse model. However, experiments using uncaging of IP3 in MrgA1 astrocytes displayed increases in the frequency of AMPA receptor-mediated sEPSCs, demonstrating that gliotransmission can occur from astrocytes in this model in a stimulus-dependent manner (Fiacco et al., 2007).

We propose that if gliotransmission does occur under physiological

conditions, IP3R-mediated Ca2+ increases are not the predominant mechanism by which Ca2+ triggers release of gliotransmitters. Recently, spontaneous near membrane Ca2+ events, not associated with Gq-GPCR activation, were observed using a membrane tethered genetic Ca2+ indicator protein in cultured astrocytes (Shigetomi et al., 2010). These authors argue that based on their findings, that large global Ca2+ increases triggered by bath application of Gq-GCPR agonists are

fundamentally different than spontaneous and neuronal activity evoked increases and may therefore represent non-physiological responses. This is inconsistent with the fact that nearly all reports of gliotransmission involve global Ca2+ increases in astrocytes upon either uncaging of Ca2+ or IP3, or activation of Gq-GPCRs. Our findings contradict the concept that Gq-GCPR, IP3R-meditated Ca2+ increases in astrocytes cause gliotransmission. Activation of the MrgA1 receptor causes global Ca2+ increases in astrocytes similar to pharmacological activation of commonly used Gq-GPCR agonists without effecting synaptic transmission (Fiacco et al., 2007;

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Agulhon et al., 2010). Blockade of global intracellular Ca2+ increases in the IP3R2 KO mouse model has no affect on basal synaptic transmission or long term

potentiation (Petravicz et al., 2008; Agulhon et al., 2010). These findings represent a significant conflict in the theory of gliotransmission that remains unresolved.

While the focus of Ca2+ signaling in astrocytes has been on IP3R-mediated increases, there are alternative mechanisms. Calcium permeable AMPA receptors represent a mechanism for Ca2+ entry into glial cells. These receptors are

expressed on several glial cell types including NG2 cells (Ge et al., 2006; Hamilton et al., 2009), oligodendrocytes (Butt, 2006), and Bergmann glia (Bellamy, 2006). Astrocytes in the hippocampus of juvenile mice are known to express functional Ca2+-permeable AMPA receptors (Porter and McCarthy, 1995b; Seifert and Steinhauser, 1995). AMPA receptors on astrocytes during development show an increase in expression of receptor subunits of relatively low Ca2+-permeability but still maintain appreciable levels of Ca2+ permeable behavior (Seifert et al., 2003). Further evidence for AMPA receptor expression on astrocytes was obtained via functional imaging using the sodium sensitive dye SFBI-AM in hippocampal

astrocytes in situ (ages P12-P21). This study reveals significant intracellular sodium transients in astrocytes in response to neuronal afferent stimulation (Langer and Rose, 2009). Sodium signals using SFBI-AM were recorded in SR101 positive astrocytes with passive membrane properties indicative of protoplasmic

hippocampal astrocytes. While the majority of the sodium signal in these cells was blocked by the glutamate transporter TBOA, roughly 20% of the signal was

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attributed to AMPA receptors. It remains to be determined if the AMPA receptor component of the signal is from Ca2+-permeable or impermeable receptors.

There exists potential for a novel mechanism by which astrocytes may

increase intracellular Ca2+ levels. Electrogenic uptake of both GABA and glutamate from the synaptic cleft has been extensively demonstrated in astrocytes (Bergles and Jahr, 1997, , 1998; Diamond and Jahr, 2000). Intracellular sodium increases as a result of the electrogenic requirements for transporter uptake of GABA or

glutamate have been demonstrated by functional imaging studies in astrocytes of juvenile and young adult mice (Doengi et al., 2009; Langer and Rose, 2009).

Doengi et al. (2009) proposed that GABA transport by astrocytes caused elevations in intracellular sodium levels in olfactory bulb astrocytes, thereby reducing the activity of the sodium/calcium exchanger. This led to an increase in intracellular Ca2+ sufficient to activate Ca2+-induced Ca2+ release (CICR) from IP3 receptors. While this mechanism was shown only in juvenile animals, Langer and Rose (2009) used both juvenile and young adult mice (P12-P22) when showing significant

increases in glutamate transporter mediated sodium concentrations in hippocampal astrocytes. This raises the possibility of this release mechanism occurring in mature astrocytes, which express sodium/calcium exchangers (Blaustein et al., 2002; Minelli et al., 2007). Evidence for a sodium/calcium exchanger modulation of Ca2+ signals exists in cultured astrocytes in response to potassium depolarization (Paluzzi et al., 2007), osmotic swelling (Rojas et al., 2008), AMPA receptor activation (Smith et al., 2000) and in response to glutamate transporter activity (Rojas et al., 2007). Finally, astrocytes in the visual cortex of ferrets display finely tuned Ca2+ increases that

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mapped in tight correlation to neuronal responses induced by orientation and spatial frequency stimuli (Schummers et al., 2008). These activity-dependent Ca2+

increases in astrocyte were blocked by the glutamate transporter antagonist TBOA, implicating glutamate transport as a necessary component to the Ca2+ increases. These findings fit with the concept that transporter generated Ca2+ signals involving sodium and the sodium/calcium exchanger could underlie a physiological

mechanism for intracellular Ca2+ increases both independent of and involving IP3R- mediated Ca2+ release. Further, it would be possible to generate small, localized microdomains of Ca2+ signals via a mechanism depending on the level of transporter activity. Langer and Rose (2009) demonstrated activity-dependent scaling of

transporter mediated sodium increases in patch-loaded hippocampal astrocytes that could potentially lead to localized Ca2+ increases through a glutamate transporter- sodium/calcium exchanger mechanism.

While both of the mechanisms mentioned above present alternative Ca2+ sources that could be involved in regulated exocytosis, there exists no data in astrocytes in situ or in vivo to support this. However, it is evident that studies into alternative mechanisms of intracellular Ca2+ increases in astrocytes will be crucial to understanding the role of astrocyte Ca2+ signaling in physiology and pathology.