Comparative analysis of optogenetic actuators in cultured astrocytes.

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, D-serine and L-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca(2+) ([Ca(2+)]i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca(2+) permeability (Ca(2+) translocating channelrhodopsin, CatCh). We show here that [Ca(2+)]i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca(2+) from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca(2+)]i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoß2AR (Gs-coupled) resulted in astrocytic [Ca(2+)]i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca(2+)]i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.

Purinergic signalling in the rostral ventro-lateral medulla controls sympathetic drive and contributes to the progression of heart failure following myocardial infarction in rats.

Heart failure may lead to hypoperfusion and hypooxygenation of tissues and this is often exacerbated by central and obstructive sleep apnoeas associated with recurrent episodes of systemic hypoxia which triggers release of ATP within the CNS circuits controlling sympathetic outflow. Using in vitro and in vivo models we tested two hypotheses: (1) activated brainstem astroglia release ATP and via release of ATP activate sympathoexcitatory neurones of the rostral ventrolateral medulla (RVLM); and (2) ATP actions in the RVLM contribute to sympathoexcitation, progression of left ventricular (LV) remodelling and development heart failure secondary to myocardial infarction. In vitro, optogenetic activation of RVLM astrocytes transduced to express light-sensitive channelrhodopsin-2 activated sympathoexcitatory RVLM neurones in ATP-dependent manner. In anaesthetised rats in vivo, similar optogenetic activation of RVLM astrocytes increased sympathetic renal nerve activity, arterial blood pressure and heart rate. To interfere with ATP-mediated signalling by promoting its extracellular breakdown, we developed a lentiviral vector to express an ectonucleotidase--transmembrane prostatic acid phosphatase (TMPAP) on the cellular membranes. In rats with myocardial infarction-induced heart failure, expression of TMPAP bilaterally in the RVLM led to lower plasma noradrenaline concentration, maintained left ventricular end diastolic pressure, attenuated decline in dP/dT (max) and shifted the LV pressure-volume relationship curve to the left. These results show that activated RVLM astrocytes are capable of increasing sympathetic activity via release of ATP while facilitated breakdown of ATP in the RVLM attenuates the progression of LV remodelling and heart failure secondary to myocardial infarction.

Astrocytes control breathing through pH-dependent release of ATP.

Astrocytes provide structural and metabolic support for neuronal networks, but direct evidence demonstrating their active role in complex behaviors is limited. Central respiratory chemosensitivity is an essential mechanism that, via regulation of breathing, maintains constant levels of blood and brain pH and partial pressure of CO2. We found that astrocytes of the brainstem chemoreceptor areas are highly chemosensitive. They responded to physiological decreases in pH with vigorous elevations in intracellular Ca2+ and release of adenosine triphosphate (ATP). ATP propagated astrocytic Ca2+ excitation, activated chemoreceptor neurons, and induced adaptive increases in breathing. Mimicking pH-evoked Ca2+ responses by means of optogenetic stimulation of astrocytes expressing channelrhodopsin-2 activated chemoreceptor neurons via an ATP-dependent mechanism and triggered robust respiratory responses in vivo. This demonstrates a potentially crucial role for brain glial cells in mediating a fundamental physiological reflex.