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

Abstract 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

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

Abstract 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 astrocytederived 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

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 are 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

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

<div><p>G protein-gated K⁺ channels (GIRK; Kir3), activated by Gβγ subunits derived from G_i/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (I_evoked) and neurotransmitter-independent basal (I_basal) GIRK activities are physiologically important, but mechanisms of I_basal and its relation to I_evoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that I_basal and I_evoked are interrelated: the extent of activation by neurotransmitter (activation index, R_a) is inversely related to I_basal. 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 Gβγ expressed in <i>Xenopus</i> 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 I_basal and I_evoked in <i>Xenopus</i> oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse I_basal-R_a correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gα_i/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gα_i/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 Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.</p></div

Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.

<p>(<b>A</b>) A representative whole-recording of GIRK current in a neuron. Switching from low-K⁺ extracellular solution to a high-K⁺ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited I_evoked. Arrows show the amplitudes of I_basal, I_evoked and I_total. Extent of activation, R_a, is defined as I_total/I_basal. (<b>B</b>) Inverse correlation between I_basal and R_a in oocytes and neurons. To allow direct comparison of I_basal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#sec019" target="_blank">Methods</a>). The correlation between R_a and I_basal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.</p

Measuring the surface density of GIRK1/2 and Gβγ in <i>Xenopus</i> oocytes.

<p><b>(A)</b> Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of <i>Xenopus</i> oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). <b>(B)</b> Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from I_βγ (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a>), shown for comparison. <b>(C)</b> Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). <b>(D)</b> Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: I_βγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm², or 22.8±3.3 YFP molecules/μm². The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. <b>(E)</b> Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. <b>(F)</b> Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.</p

Whole-cell currents of GIRK1/2, the calculated surface density and I_βγ/I_total in <i>Xenopus</i> oocytes.

<p>Data are shown as mean±SEM (except I_βγ/I_total), number of cells is shown in parentheses. Data for each entry were collected from at least 2 independent experiments. The Table summarizes separate sets of experiments: those where I_basal, I_evoked and I_total were measured (in each oocyte); and those where Gβγ was coexpressed and I_βγ was measured. In addition, in Set 2, I_basal was measured in each experiment in a separate group of oocytes not injected with Gβγ RNA. For the low density group in oocytes, there was ~30% difference (p = 0.017) for I_basal between the two sets of experiments, probably because of variability among oocyte batches. In intermediate and high density groups I_basal was not different (p>0.4) for both sets of experiments.</p><p>Whole-cell currents of GIRK1/2, the calculated surface density and I_βγ/I_total in <i>Xenopus</i> oocytes.</p

Single channel and whole-cell data reveal incomplete activation of GIRK1/2 by agonist compared to Gβγ.

<p><b>(A)</b> Activity of GIRK1/2 in a cell-attached patch of an oocyte expressing the channel, m2R and Gβγ, without an agonist in the pipette. Right panel shows a 2 minutes segment of record, with zoom (below) on a shorter segment. The amplitude distribution histogram of the same 2 min-segment is shown on the right. Red line shows a two-component Gaussian fit. I_single was determined as the difference between the fitted midpoints (μ) of the GIRK current peak on the right (μ2) and the left peak which corresponds to noise (μ1). <b>(B)</b> Activity of GIRK1/2 channels in a cell-attached patch of an oocyte expressing the channel and m2R and activated by 2 μM ACh present in the patch pipette. (Asterisks denote artifacts produced by capacity discharges of patch clamp headstage). The corresponding amplitude histogram of the 2 min-segment of the record is shown on the right. In A and B, GIRK1/2 was expressed at low densities (GIRK1, 10–50 pg RNA; GIRK2, 7–17 pg RNA) whereas RNAs of m2R (1–2 ng/oocyte) and Gβγ (5:1 ng/oocyte) were chosen to produce saturating concentrations of these proteins. Inward K⁺ currents are shown as upward deflections from zero level. In the traces shown, acquisition was at 20 KHz with 5 KHz analog filter. Very similar values of I_single were obtained with 2 KHz filtering (not shown). <b>(C)</b> Single channel currents (left plot) are identical with either ACh or Gβγ. <b>(D)</b> P_o is lower with ACh than with Gβγ (p = 0.029). Bars in C and D show mean±SEM, number of patches is shown above the bars. <b>(E)</b> Summary of whole-cell GIRK1/2 currents at three expression levels (densities). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a> for details. <b>(F)</b> Left panel shows the I_total/I_βγ ratios at three channel densities, calculated from data of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.t001" target="_blank">Table 1</a>. The right panel shows the fractional open probabilities of channels occupied by 0–4 Gβγ, same as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004598#pcbi.1004598.g002" target="_blank">Fig 2C</a> but in a simple graphic form. The red dotted lines are drawn to allow direct comparison of the experimental data from the left panel with the estimates of fractional P_o from the right panel.</p