Activity-dependent modulation of synapse-regulating genes in astrocytes.

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.

Astrocytes, neurons, synapses: a tripartite view on cortical circuit development.

In the mammalian cerebral cortex neurons are arranged in specific layers and form connections both within the cortex and with other brain regions, thus forming a complex mesh of specialized synaptic connections comprising distinct circuits. The correct establishment of these connections during development is crucial for the proper function of the brain. Astrocytes, a major type of glial cell, are important regulators of synapse formation and function during development. While neurogenesis precedes astrogenesis in the cortex, neuronal synapses only begin to form after astrocytes have been generated, concurrent with neuronal branching and process elaboration. Here we provide a combined overview of the developmental processes of synapse and circuit formation in the rodent cortex, emphasizing the timeline of both neuronal and astrocytic development and maturation. We further discuss the role of astrocytes at the synapse, focusing on astrocyte-synapse contact and the role of synapse-related proteins in promoting formation of distinct cortical circuits.

Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.

The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gßγ.

G protein-gated K+ channels (GIRK; Kir3), activated by Gßγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gßγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gßγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gßγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gßγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gßγ, consistent with recruitment to the cell surface of Gßγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gßγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gßγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gßγ and Gα.

The role of neuronal versus astrocyte-derived heparan sulfate proteoglycans in brain development and injury.

Astrocytes modulate many aspects of neuronal function, including synapse formation and the response to injury. Heparan sulfate proteoglycans (HSPGs) mediate some of the effects of astrocytes on synaptic function, and participate in the astrocyte-mediated brain injury response. HSPGs are a highly conserved class of proteoglycans, with variable heparan sulfate (HS) chains that play a major role in determining the function of these proteins, such as binding to growth factors and receptors. Expression of both the core proteins and their HS chains can vary depending on cellular origin, thus the functional impact of HSPGs may be determined by the cell type in which they are expressed. In the brain, HSPGs are expressed by both neurons and astrocytes; however, the specific contribution of neuronal HSPGs compared with astrocyte-derived HSPGs to development and the injury response is largely unknown. The present review examines the current evidence regarding the roles of HSPGs in the brain, describes the cellular origins of HSPGs, and interrogates the roles of HSPGs from astrocytes and neurons in synaptogenesis and injury. The importance of considering cell-type-specific expression of HSPGs when studying brain function is discussed.

Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3ß (GSK3ß) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gßγ subunits. Li(+) did not impair direct activation of GIRK channels by Gßγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gßγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

Further characterization of regulation of Ca(V)2.2 by stargazin.

Stargazin, a transmembrane protein expressed in the nervous system, shares similarities with the γ1 subunit of skeletal muscle calcium channels. It was thus termed γ2 subunit of neuronal calcium channels. Stargazin downregulates the expression of Ca(V)2 channels, however, its functional modulation of these channels remains debated. We have reported that Stargazin modulates Ca(V)2.2 channel by a Gßγ-dependent mechanism and suggested that Stargazin is not a true subunit of this channel, since all its effects on channel function are dependent on the presence of Gßγ. Moreover, Stargazin also modulated the GIRK channel in a Gßγ-dependent fashion. Here we report that Gßγ-dependent modulation by Stargazin of the biophysical properties of Ca(V)2.2 is unrelated to its negative effect on channel expression and current amplitude. Finally, we suggest that this Gßγ dependent modulation of Stargazin may have physiological relevance, since it was still present when we used Ca²(+) as charge carrier, instead of Ba²(+).

Stargazin modulates neuronal voltage-dependent Ca(2+) channel Ca(v)2.2 by a Gbetagamma-dependent mechanism.

Loss of neuronal protein stargazin (gamma(2)) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel gamma(1) subunit, stargazin and other family members (gamma(3-8)) were classified as gamma subunits of neuronal voltage-gated calcium channels (such as Ca(V)2.1-Ca(V)2.3). Here, we report that stargazin interferes with G protein modulation of Ca(V)2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gbetagamma-induced inhibition of Ca(V)2.2 channel currents, caused either by coexpression of the Gbetagamma dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gbetagamma overcame the effects of stargazin. High affinity Gbetagamma scavenger proteins m-cbetaARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cbetaARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the Ca(V)beta(3) subunit. However, in some cases, coexpression of Ca(V)beta(3) blunted the modulation by stargazin. Finally, the Gbetagamma-opposing action of stargazin was not unique to Ca(V)2.2, as stargazin also inhibited the Gbetagamma-mediated activation of the G protein-activated K(+) channel. Purified cytosolic C-terminal part of stargazin bound Gbetagamma in vitro. Our results suggest that the regulation by stargazin of biophysical properties of Ca(V)2.2 are not exerted by direct modulation of the channel but via a Gbetagamma-dependent mechanism.

A single low dose of tetrahydrocannabinol induces long-term cognitive deficits.

Delta(9)-Tetrahydrocannabinol (THC) was shown to exert either neuroprotective or neurotoxic effects. Based on our in vitro studies and on pharmacokinetic considerations, we have recently presented a hypothesis that explains this dual activity of THC. This explanation is based on the assumption that extremely low doses of cannabinoids are neurotoxic. The present study verifies this assumption and shows that a single injection of 0.001 mg/kg THC (3-4 orders of magnitude lower than conventional doses) significantly affected the performance of mice in the Morris water maze test 3 weeks later. The THC-injected mice showed both longer escape latencies and lower scores in the probe tests compared to their matched controls, indicating the induction of cognitive deficits.