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Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines.

Bloodgood BL, Giessel AJ, Sabatini BL - PLoS Biol. (2009)

Bottom Line: We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale.Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine.These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.

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Synaptic AMPAR activation determines the early but not late phase of Ca entry into the spine.(A) uEPSPs (left) and Δ[Ca]spine (right) evoked in control conditions (black) or in the presence of the NMDAR antagonists CPP and MK801 (red). (B) uEPSP evoked Δ[Ca]spine measured in the presence of NMDAR antagonists (as in Panel A). Δ[Ca]spine is fit by a single exponential with τ = 42 ms (black dashed line), which is used as the deconvolution kernel throughout. (C) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the AMPAR antagonist NBQX (red). (D) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of 5.0 µM CTZ to accentuate AMPAR opening (red). (E) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the SK channel antagonist apamin (red). (F and G) The amplitude of the peak (F) and prolonged phase of iCa (G) plotted as a function of uEPSP amplitude for control conditions, AMPAR blockade (NBQX), AMPAR enhancement (low CTZ and CTZ), and SK channel blockade (apamin).
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pbio-1000190-g003: Synaptic AMPAR activation determines the early but not late phase of Ca entry into the spine.(A) uEPSPs (left) and Δ[Ca]spine (right) evoked in control conditions (black) or in the presence of the NMDAR antagonists CPP and MK801 (red). (B) uEPSP evoked Δ[Ca]spine measured in the presence of NMDAR antagonists (as in Panel A). Δ[Ca]spine is fit by a single exponential with τ = 42 ms (black dashed line), which is used as the deconvolution kernel throughout. (C) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the AMPAR antagonist NBQX (red). (D) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of 5.0 µM CTZ to accentuate AMPAR opening (red). (E) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the SK channel antagonist apamin (red). (F and G) The amplitude of the peak (F) and prolonged phase of iCa (G) plotted as a function of uEPSP amplitude for control conditions, AMPAR blockade (NBQX), AMPAR enhancement (low CTZ and CTZ), and SK channel blockade (apamin).

Mentions: In order to determine if the depolarization reached in the spine during synaptic activity shapes synaptic signals, we examined if the opening of AMPARs, which provide the bulk of current influx that produces the EPSP, secondarily alters the magnitude or kinetics of Ca current into the spine (iCa) (Figure 3). Previous studies have reported variable effects of blocking AMPARs on the peak of synaptically evoked Ca transients [9],[24],[25]. However, in fluorescence imaging of Ca transients, the time course of iCa is obscured by the presence of the exogenous Ca indicator, which slows the clearance of Ca from the spine. In the absence of exogenous buffer, the low (∼25) endogenous Ca buffer capacity of the apical spines of CA1 pyramidal neurons allows spine head Ca to closely follow the kinetics of opening of Ca sources [26]. To determine the time course of iCa, we corrected for the kinetics of Ca handling by performing a deconvolution with the impulse response of Ca handling of the spine (see methods) [26],[27]. The impulse response was estimated from the decay of the synaptically evoked fluorescence transient measured in the presence of NMDAR antagonists, which is generated by Ca influx that is impulse-like relative to the decay kinetics of Δ[Ca]spine. Blockade of NMDARs with CPP/MK801 had no significant effect on uEPSP amplitudes (1.04±0.19 mV, n = 18, and 1.23±0.23 mV, n = 17, control and CPP/MK801, respectively) but reduced the early phase (20–50 ms post-uncaging) of Δ[Ca]spine ∼60% (early ΔG/Gsat: 9.48%±1.12% and 4.09%±0.62% in control and CPP/MK801; p<0.05) while nearly eliminating the later portion (50–120 ms post-uncaging) (late ΔG/Gsat: 8.48%±1.3% and 0.90%±0.16%; p<0.05) (Figure 3A). These results confirm the dominant role of Ca influx through NMDARs in generating synaptically evoked Ca transients and, in particular, in mediating the prolonged phases of synaptic Ca influx. The remaining spine Ca transient was well described by a single exponential decay with a time constant of ∼42 ms, which was used as the Ca impulse response in deconvolution analysis below.


Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines.

Bloodgood BL, Giessel AJ, Sabatini BL - PLoS Biol. (2009)

Synaptic AMPAR activation determines the early but not late phase of Ca entry into the spine.(A) uEPSPs (left) and Δ[Ca]spine (right) evoked in control conditions (black) or in the presence of the NMDAR antagonists CPP and MK801 (red). (B) uEPSP evoked Δ[Ca]spine measured in the presence of NMDAR antagonists (as in Panel A). Δ[Ca]spine is fit by a single exponential with τ = 42 ms (black dashed line), which is used as the deconvolution kernel throughout. (C) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the AMPAR antagonist NBQX (red). (D) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of 5.0 µM CTZ to accentuate AMPAR opening (red). (E) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the SK channel antagonist apamin (red). (F and G) The amplitude of the peak (F) and prolonged phase of iCa (G) plotted as a function of uEPSP amplitude for control conditions, AMPAR blockade (NBQX), AMPAR enhancement (low CTZ and CTZ), and SK channel blockade (apamin).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734993&req=5

pbio-1000190-g003: Synaptic AMPAR activation determines the early but not late phase of Ca entry into the spine.(A) uEPSPs (left) and Δ[Ca]spine (right) evoked in control conditions (black) or in the presence of the NMDAR antagonists CPP and MK801 (red). (B) uEPSP evoked Δ[Ca]spine measured in the presence of NMDAR antagonists (as in Panel A). Δ[Ca]spine is fit by a single exponential with τ = 42 ms (black dashed line), which is used as the deconvolution kernel throughout. (C) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the AMPAR antagonist NBQX (red). (D) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of 5.0 µM CTZ to accentuate AMPAR opening (red). (E) uEPSPs (left), Δ[Ca]spine (middle), and iCa (right) in control conditions (black) and in the presence of the SK channel antagonist apamin (red). (F and G) The amplitude of the peak (F) and prolonged phase of iCa (G) plotted as a function of uEPSP amplitude for control conditions, AMPAR blockade (NBQX), AMPAR enhancement (low CTZ and CTZ), and SK channel blockade (apamin).
Mentions: In order to determine if the depolarization reached in the spine during synaptic activity shapes synaptic signals, we examined if the opening of AMPARs, which provide the bulk of current influx that produces the EPSP, secondarily alters the magnitude or kinetics of Ca current into the spine (iCa) (Figure 3). Previous studies have reported variable effects of blocking AMPARs on the peak of synaptically evoked Ca transients [9],[24],[25]. However, in fluorescence imaging of Ca transients, the time course of iCa is obscured by the presence of the exogenous Ca indicator, which slows the clearance of Ca from the spine. In the absence of exogenous buffer, the low (∼25) endogenous Ca buffer capacity of the apical spines of CA1 pyramidal neurons allows spine head Ca to closely follow the kinetics of opening of Ca sources [26]. To determine the time course of iCa, we corrected for the kinetics of Ca handling by performing a deconvolution with the impulse response of Ca handling of the spine (see methods) [26],[27]. The impulse response was estimated from the decay of the synaptically evoked fluorescence transient measured in the presence of NMDAR antagonists, which is generated by Ca influx that is impulse-like relative to the decay kinetics of Δ[Ca]spine. Blockade of NMDARs with CPP/MK801 had no significant effect on uEPSP amplitudes (1.04±0.19 mV, n = 18, and 1.23±0.23 mV, n = 17, control and CPP/MK801, respectively) but reduced the early phase (20–50 ms post-uncaging) of Δ[Ca]spine ∼60% (early ΔG/Gsat: 9.48%±1.12% and 4.09%±0.62% in control and CPP/MK801; p<0.05) while nearly eliminating the later portion (50–120 ms post-uncaging) (late ΔG/Gsat: 8.48%±1.3% and 0.90%±0.16%; p<0.05) (Figure 3A). These results confirm the dominant role of Ca influx through NMDARs in generating synaptically evoked Ca transients and, in particular, in mediating the prolonged phases of synaptic Ca influx. The remaining spine Ca transient was well described by a single exponential decay with a time constant of ∼42 ms, which was used as the Ca impulse response in deconvolution analysis below.

Bottom Line: We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale.Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine.These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.

Show MeSH
Related in: MedlinePlus