Frequency modulation of synchronized Ca2+ spikes in cultured hippocampal networks through G-protein-coupled receptors

Abstract

Synchronized spontaneous Ca2+ spikes in networked neurons represent periodic burst firing of action potentials, which are believed to play a major role in the development and plasticity of neuronal circuitry. How these network activities are shaped and modulated by extrinsic factors during development, however, remains to be studied. Here we report that synchronized Ca2+ spikes among cultured hippocampal neurons can be modulated by two small factors that act on G-protein-coupled receptors (GPCRs): the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) and the chemokine SDF-1 (stromal cell-derived factor-1). PACAP effectively increases the frequency of the synchronized Ca2+ spikes when applied acutely; the PACAP potentiation of Ca2+ spikes requires the activation of the PACAP-specific PAC1 GPCRs and is mediated by the activation of cAMP signaling pathway. SDF-1, on the other hand, significantly reduces the frequency of these Ca2+ spikes through the activation of its specific GPCR CXCR4; the inhibitory action of SDF-1 is mediated by the inhibition of cAMP pathway through the Gi component of GPCRs. Taken together, these results demonstrate that synchronized neuronal network activity can be effectively modulated by physiologically and developmentally relevant small factors that act on GPCRs to target the cAMP pathway. Such modulation of neuronal activity through GPCRs may represent a significant mechanism that underlies the neuronal plasticity during neural development and functioning.

Figure 1.

Synchronized spontaneous Ca²⁺ spikes in cultured hippocampal neurons. a, Representative recordings of synchronized spontaneous Ca²⁺ spikes from hippocampal neurons 2 weeks in culture. Traces indicate relative changes of fluo-3 intensity (ΔF/F₀) over time in neurons from three microscopic fields randomly selected from the same culture dish. Each trace represents ΔF/F₀ of an individual neuron acquired every 2 sec (see Materials and Methods). b, Blockade of spontaneous synchronized Ca²⁺ spikes by TTX. The fluorescent image on the left shows four hippocampal neurons loaded with fluo-3. Scale bar, 5μm. Traces on the right depict Ca²⁺ spikes in these cells under control conditions, after application of control medium, and subsequent application of TTX (1 μm final).

Figure 2.

Increase in the frequency of synchronized Ca²⁺ spike firing induced by PACAP. a, Traces show synchronized Ca²⁺ spikes in three neurons randomly selected from a group of synchronically firing cells before and after bath addition of PACAP (10 nm). The time gap (//) is 5 min. b, Changes of the synchronized Ca²⁺ spike frequency 5–6 min after bath application of a different molecule or molecules. The change of the Ca²⁺ spike frequency was quantified by normalizing the spike number from a 2 min period (5–6 min) after bath application to the spike number in the control period and presented as the percentage. Data are presented as the mean ± SD from many cells (PACAP: n = 24; PACAP6–38: n = 21; PACAP + PACAP6–38: n = 22; 10 nm VIP: n = 22; 1 μm VIP: n = 23, PACAP6–38). Asterisks indicate significance against the control (p < 0.005, t test).

Figure 3.

Increased frequency of firing of hippocampal neurons by PACAP, as recorded by the whole-cell patch-clamp technique. a, Effects of PACAP on EPSCs in a hippocampal neuron from a 1 week culture. The trace represents the spontaneous EPSCs of a hippocampal neuron from a 1 week culture before and after bath application of 10 nm PACAP. The shape of each EPSC is depicted by the expanded traces. To determine the changes of the EPSC number over time, we counted the numbers of EPSCs every 1 min before and after the addition of PACAP (b). c, The increase in the number of EPSCs after PACAP addition was quantified by normalizing EPSC number from a 2 min period to that of the control period. Data represent mean ± SD.

Figure 4.

cAMP dependence of PACAP enhancement of Ca²⁺ spike frequency. a, Modulation of the frequency of spontaneous Ca²⁺ spikes by manipulation of cAMP signaling pathway. Traces show representative recordings of synchronized Ca²⁺ spikes in three hippocampal neurons randomly selected from the imaging field under baseline and after bath application of KT5720 (1 μm). b, Effects of different PAK antagonists and agonist on the frequency of the synchronized Ca²⁺ spikes. As described previously, the modulation was quantified by normalizing the number of spikes in 5–6 min after bath application to that of the control period. Data represent mean ± SD from populations of cells (1μm KT5720: n = 9; 200 μm Rp-cAMP: n = 21; 10 nm PACAP + Rp-cAMP: n = 18; 15 μm forskolin: n = 20). *p < 0.005 to the control group treated by control medium (t test). c, Traces indicate the Ca²⁺ spikes in three hippocampal neurons in the presence of 1 μm KT 5720 before and after PACAP application (10 nm). d, Bar graph summarizes the blockade of PACAP effects on Ca²⁺ spike frequency by KT5720 or Rp-cAMP.

Figure 5.

Inhibition of synchronized Ca²⁺ spikes by SDF-1. a, Representative recordings of synchronized Ca²⁺spikes in three hippocampal neurons randomly selected in the imaging field before and after addition of SDF-1(100nm). The time gap (//) indicates ∼5 min. b,The decrease of the Ca²⁺ spike frequency by SDF-1 is quantified by normalizing the number of spikes from 5–6 min after SDF-1 application to the control period. Data represent mean ± SD from populations of cells (100 nm SDF-1:n=35; 50 nm SDF-1,n=15). For PTX experiments, hippocampal cultures were pretreated with 200 ng/ml PTX for 16 hr before Ca²⁺ imaging (n = 11). *p < 0.005 to the control values treated by control medium (t test).

Figure 6.

cAMP dependence of SDF-1 inhibition of synchronized spontaneous Ca²⁺ spikes. a, Representative recordings of synchronized Ca²⁺ spike in three hippocampal neurons under baseline control conditions, in the presence of SDF-1 (100 nm), and after subsequent addition of 500 μm dibutyryl cAMP. b, The bar graph depicts the normalized frequency changes of control (saline application), SDF-1 addition, and after subsequent addition of db-cAMP. Data represent mean ± SD from 13 neurons of three different dishes. *p < 0.005 to the control values treated by control medium (t test).