Validación de la hipótesis

Despite decades of neuroscience research, many neurological disorders remain poorly understood. Interest in the role of glia in both normal and pathological states is increasing, particularly as evidence is presented for astrocyte modulation of neuronal activity. Recent research has provided evidence that astrocytes may have a greater function beyond providing structural and metabolic support for neurons. First, astrocytes account for approximately half of the brain’s volume and are found in every part of the CNS. Many neuronal processes and synapses are surrounded by astrocytes; a single astrocyte can envelop as many as 80,000 synapses (Bushong et al., 2002). Second, astrocytes and neurons express a similar

complement of receptors for neuroligands (Porter and McCarthy, 1996) and astrocytes are able to respond to numerous stimuli with an increase in intracellular calcium (Araque et al., 2001), which is generally accepted to be a form of glial excitability (Hansson and Ronnback, 2003). Third, astrocytes also appear to release gliotransmitters, including glutamate and ATP. For example, a calcium increase in astrocytes evokes an inward current in adjacent neurons that is dependent on glutamate release (Parpura et al., 1994; Carmignoto et al., 1997; Araque et al., 1998; Pasti et al., 2001) and uncaging IP3 in a single astrocyte causes a

glutamate-dependent increase in AMPA mEPSC frequency in a neighboring neuron (Fiacco and McCarthy, 2004). Yet little is actually known about what role astrocytes may play in listening to and modulating neuronal communication under physiological conditions.

Because an astrocyte-specific receptor and ligand have yet to be discovered, astrocytic responses cannot be isolated pharmacologically. The Ro1 transgenic mouse line was created to manipulate signaling events specifically in astrocytes in order to investigate the role of astrocyte GPCR signaling in modulating behavior and neuronal activity.

Targeting a Gi-coupled receptor to astrocytes. The tetracycline transactivator (tTA)

system was used to conditionally express Ro1 in astrocytes on a kappa opioid receptor knockout background. Ro1 is a kappa opioid receptor modified by replacing the second extracellular loop with the second extracellular loop of the delta opioid receptor. Ro1 has a greatly reduced affinity for endogenous ligands but is still activated by the highly selective small molecule drug spiradoline (Coward et al., 1998; Redfern et al., 1999). To restrict Ro1 expression to astrocytes, transgenic (tg) mice expressing the tetracycline transactivator (tTA) under the control of hGFAP, an astrocyte-specific tissue promoter, were crossed with a second line of mice carrying the Ro1 and LacZ genes under the control of the tet operon (tetO) promoter. Timing and level of Ro1 expression can be controlled with doxycycline, thus any potential developmental side effects of Ro1 expression could be eliminated by maintaining mice on doxycycline until maturity. Using spiradoline to activate Ro1 would allow the study of astrocytic Gi-coupled signaling on animal behavior and neuronal activity.

Ro1 expression and cellular localization. Immunoprecipitation assays and βgal staining demonstrated that Ro1 is expressed only in double tg mice (positive for both tTA and Ro1) and only when the animals are maintained in the absence of dox, confirming that Ro1 expression is indeed regulated by doxycycline without any apparent “leakiness”. Cellular localization of Ro1 was determined using immunocytochemistry; the localization of Ro1

immunostaining to GFAP positive cells and its absence in NeuN positive cells confirmed that Ro1 expression is being driven by the GFAP promoter. Because spiradoline, the synthetic ligand for Ro1, activates the kappa opioid receptor, the Ro1 genotype was moved onto a KOR knockout background to ensure that the only response to spiradoline is mediated by Ro1.

Gi signaling and Gi modulation of behavior. G protein coupled receptors (GPCRs) are a

class of seven transmembrane spanning receptors that are important mediators of intracellular signaling. The cytoplasmic tail of a GPCR interacts with the heterotrimeric G proteins, which are composed of α, β and γ subunits. In an inactive state, the α subunit binds GDP. Activation of the GPCR causes a conformational change in Gα, allowing GDP to be replaced with GTP and releasing the Gβγ dimer. There are multiple families of Gα that couple to different effector molecules. Gαi is so named for its inhibition of adenylate cyclase and

subsequent reduction of cAMP formation. A number of studies suggest that Gi-coupled

receptors in the CNS, including dopamine, serotonin, opioid and muscarinic acetylcholine receptors have roles in regulating both behavior and neuronal activity in normal and

pathological states. The focus of many of these studies has been on neuronal receptors even though astrocytes also express many of the same receptors and there is indirect evidence that activation of astrocytic receptors may play a role in the observed effects. The Ro1 mice were designed to allow us to directly test if Gi signaling in astrocytes alone can modulate behavior

or neuronal activity.

Alterations in Gi protein levels and activity have been implicated in multiple

pathological states, including depression and panic disorders (Joseph et al., 1993; Saitoh et al., 1993; Gurguis et al., 1999c; Gurguis et al., 1999e; Gurguis et al., 1999d; Gurguis et al.,

1999b; Gurguis et al., 1999a). There is a significant reduction in stimulated cAMP formation in frontal cortex tissue from human suicide victims (Valdizan et al., 2003) and both increases (Garcia-Sevilla et al., 1999) and decreases (Pacheco et al., 1996) in Gi protein levels have

been found in the brains of depressed subjects. As these studies were done on brain tissue lysates, neuronal and astrocytic components cannot be determined. Dopamine D2 receptors signal primarily via Gi pathways. Neuroleptic antipsychotic drugs, which are primarily

dopamine D2 receptor antagonists, are used to treat depression and panic disorders. Since nearly 1/3 of all D2 receptors in the cerebral cortex are located on astrocytes and astrocytic processes rich in D2 receptors surround cortical interneurons lacking D2 receptors (Khan et al., 2001), astrocytes seem to be ideally placed to mediate some of the therapeutic effects. Furthermore, the D2 agonist quinpirole causes an increase in intracellular calcium in cultured cortical astrocytes that can be blocked with the D2 antagonist raclopride (Khan et al., 2001); increases in astrocytic calcium are correlated with changes in neuronal activity and release of gliotransmitters (Pasti et al., 1997; Newman and Zahs, 1998; Parri et al., 2001). It is possible that activity at astrocytic dopamine receptors accounts for at least part of the effect of anti- depressant and antipsychotic drugs, and therefore defects in astrocytic Gi signaling could

have a critical role in these disorders.