Exploring the role of astrocytes in dopaminergic circuits

This PhD project investigated the role of astrocyte in the Ventral Tegmental Area (VTA), one of the main sources of dopamine (DA) in the mammalian brain, and in the hippocampal spatial encoding circuit, one of the VTA DA target regions. In the VTA, we characterized the astrocyte Ca²⁺ responses to the burst firing of a single DA neuron and to norepinephrine (NE) bath-applications. Upon the burst firing of a DA neuron and the ensuing somatodendritic release of DA and endocannabinoids (eCBs), the activation of astrocyte DA D2-type receptor and eCB CB1R triggers in these glial cells intracellular Ca2+ elevations. Astrocyte activation, coupled with another signal, possibly NO, released during DA neuron bursts, leads to a burst-induced long-term potentiation (bLTP) of the excitatory transmission onto adjacent DA neurons. At the basis of this potentiation is the pre-synaptic activation of the mGluR1 receptor that mediates a sustained increase in glutamate release probability. Despite the impairment of bLTP in young IP3R2-/- mice, in which the burst-induced Ca2+ elevations in astrocytes are completely abolished, bLTP is still present in adult IP3R2-/- mice. The overexpression of hPMCA, a plasmatic Ca2+ pump, in VTA astrocytes of adult IP3R2-/- mice resulted in the abolition of the bLTP. This result suggests that astrocyte Ca2+ responses to DA neuron bursts are preserved, at least in part, in adult IP3R2-/- mice. One of the aims of my doctoral research was to evaluate the Ca2+ response of VTA astrocytes following the burst firing activity of a DA neuron in IP3R2+/+ and IP3R2-/- adult mice. Using genetically encoded Ca2+ indicators and two-photon Ca²⁺ imaging in brain slices from both IP3R2+/+ and IP3R2⁻/⁻ mice, we provided further evidence that the Ca2+ signalling of VTA astrocytes plays a key role in the mechanism of bLTP, even in adult IP3R2-/- mice. Regarding NE, we explored whether VTA astrocytes respond to this neuromodulator associated with stress. Confocal and two-photon Ca²⁺ imaging experiments revealed that NE induces robust, dose-dependent Ca²⁺ responses in VTA astrocytes at both soma and fine processes. Using a pharmacological approach we, then, observed that these Ca2+ responses are mainly mediated by α₁-adrenergic receptors. In IP3R2⁻/⁻ mice the NE-induced Ca²⁺ responses were significantly reduced, compared to IP3R2+/+ mice, but still present even at the soma. Employing a pharmacological approach, our data indicate that IP3R1 and/or IP3R3 receptors contribute to the residual Ca²⁺ response in IP3R2-/- mice. In Hippocampus, we explored the role of astrocytes in spatial memory of reward locations and its impairment in Alzheimer's disease (AD), using the PS2APP as an AD mouse model. Behavioural assessments revealed that while both WT and PS2APP mice learned to perform the spatial tasks, the AD model exhibited long-term spatial memory deficits at 8 months of age. Two-photon Ca²⁺ imaging experiments demonstrated a progressive reduction in hippocampal astrocyte Ca²⁺ responses to DA starting at 6 months in B6.152H mice, preceding observable memory impairments. Moreover, in order to measuring the spatial representation of reward locations, as percentage of neuronal place field at the reward locations, we combined our spatial memory task with Ca2+ imaging of hippocampal neurons in freely moving animals. Preliminary results show that at 3 months of age, both WT and B6152H mice show a similar percentage of place field concomitant to the reward locations. This result suggests that at 3 months of age the spatial memory of the reward location is not yet affected by AD progression.