Comparative assessment of the effects of DREADDs and endogenously expressed GPCRs in hippocampal astrocytes on synaptic activity and memory

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have proven themselves as one of the key in vivo techniques of modern neuroscience, allowing for unprecedented access to cellular manipulations in living animals. With respect to astrocyte research, DREADDs have become a popular method to examine the functional aspects of astrocyte activity, particularly G-protein coupled receptor (GPCR)-mediated intracellular calcium (Ca²⁺) and cyclic adenosine monophosphate (cAMP) dynamics. With this method it has become possible to directly link the physiological aspects of astrocytic function to cognitive processes such as memory. As a result, a multitude of studies have explored the impact of DREADD activation in astrocytes on synaptic activity and memory. However, the emergence of varying results prompts us to reconsider the degree to which DREADDs expressed in astrocytes accurately mimic endogenous GPCR activity. Here we compare the major downstream signaling mechanisms, synaptic, and behavioral effects of stimulating Gq-, Gs-, and Gi-DREADDs in hippocampal astrocytes of adult mice to those of endogenously expressed GPCRs.

Keywords: behavior; glia; signal transduction; signaling pathways; tripartite synapse.

FIGURE 1

Schematic of astrocytic G-protein coupled receptor (GPCR) signaling. Activation of membrane-bound astrocytic GPCRs triggers the exchange of GDP for GTP at the α subunit and dissociation of α and βγ subunits from the receptor (dotted arrows). Once released, α_s stimulates adenylyl cyclase activity leading to the production of cAMP from ATP (1). α_q binds PLC, catalyzing diacylglycerol (DAG) and IP₃ synthesis (2). IP₃ binds its receptor on the endoplasmic reticulum (ER) to release Ca²⁺ from internal stores (3). α_i inhibits adenylyl cyclase activity (4). The Gi βγ subunit can potentiate cAMP elevations mediated by Gs-GPCRs (5), and is speculated to enhance Gq-GPCR-mediated Ca²⁺ release from stores via the PLC-IP₃ pathway (6), although this has yet to be proven in astrocytes (*). Figure created with Biorender.com.

FIGURE 2

Schematic representation of intracellular (top), synaptic (middle), and behavioral (bottom) effects of endogenous Gq-, Gs-, and Gi-GPCR stimulation in astrocytes, and that of corresponding Gq-, Gs-, and Gi-DREADDs (hM3Dq, rM3Ds, and hM4Di). Astrocytic Gq-GPCRs/hM3Dq activation (left) evokes Ca²⁺ elevations via the PLC-IP₃ signaling pathway. This increases mEPSCs, SICs (conflicting evidence for hM3Dq), LTP and PAP plasticity at synapses. Only one study illustrates memory-related behavioral effects of Gq-GPCR activation in astrocytes – in pathological condition (AD) Gq-GPCR activation mediated memory impairment, while hM3Dq activations mediate memory enhancement under physiological conditions. Astrocytic Gs-GPCR and rM3Ds activation (centre) evokes cAMP elevations through stimulation of adenylyl cyclase (AC), and rM3Ds activations evoke Ca²⁺ elevations through unknown mechanisms. We speculate that astrocytic A2AR activation impairs LTP. Astrocytic βAR activation is shown to mediate morphological plasticity, possibly including the PAPs, and lactate release, thereby stimulating LTP at synapses. These synaptic effects have not been shown for the Gs-DREADD hM3Ds, illustrating a major gap in the literature. Behavioral effects of astrocytic A2AR and rM3Ds activation is on memory extinction, however βAR stimulation evokes memory enhancement. Astrocytic Gi-GPCR and hM4D activation (right) evokes reductions in cAMP levels by inhibition of AC. However, the endogenous Gi-GPCR mGluR3 can also elevate cAMP levels by action of the βγ subunit, through potentiation of Gs-coupled elevations. Stimulation of Gi-GPCRs evokes Ca²⁺ elevations via the PLC signaling pathway. Corresponding synaptic effects of cAMP- and Ca²⁺-elevations include synaptic potentiation and depression, whereby cAMP accumulation leads to activation of presynaptic adenosine 1 receptors (A₁R) and LTP disruption. Downregulated cAMP by astrocytic Gi-GPCRs mediate post-synaptic NMDAR activation and AMPAR internalization, thereby evoking LTD. Together these intracellular and synaptic effects, evoked by lowering astrocytic cAMP-levels, have a negative effect on memory retention. Astrocytic Gi-DREADD (hM4Di) activation is also shown to evoke cAMP reductions and thereby inhibition of fear learning. In addition, other studies reported that astrocytic hM4Di activation can also evoke transient Ca²⁺ elevations that return to levels below baseline thereafter. These Ca²⁺ effects are shown to increase frequency of SICs, firing rates (FR) and LTP in nearby neurons, potentially having a positive effect on memory. The final effect of Gi-DREADD signaling on memory therefore seems to depend on the net outcome of cAMP and Ca²⁺ effects. Figure created with www.biorender.com.