Article Figures & Data
Figures
- Figure 1.
AP Ca signals in all dendritic compartments. A, 2PLSM image stack of a layer 2/3 pyramidal neuron, with different dendritic domains labeled. The inset shows a line scan (yellow dashed line) through the spine (S) and dendrite (D). B, Top, Current-clamp recording with brief current injection (arrow) to elicit an AP. Middle, A 500 Hz line scan through the spine (S) and dendrite (D) shows a green AP Ca signal ([Ca]bAP). Bottom, [Ca]bAP quantified as ΔG/Gsat, in spine (red) and dendrite (black). [Ca]bAP amplitude is calculated over a 20 ms window after the AP (gray box). C, Average [Ca]bAP in baseline conditions (red) and after wash-in of the VSCC mixture (black) in spines (left) and dendrites (right). SEM is represented by the shaded regions for baseline (pink) and after wash-in of the VSCC mixture (gray). D, [Ca]bAP amplitude versus radial distance from the soma for spines (red) and dendrites (black) in apical (left) and basal (right) dendrites. [Ca]bAP amplitude versus radial distance for spines (red line) and dendrites (black line) was fit with a single exponential.
- Figure 2.
Modulation of AP Ca signals in apical and basal spines and dendrites. A, Average [Ca]bAP for apical oblique spines (left) and dendrites (right) in baseline conditions (red) and after wash-in of 5 μm baclofen (black). B, Summary of changes in [Ca]bAP amplitude after wash-in of ACSF (blue) or 5 μm baclofen (green) in apical oblique, apical main, and apical tuft spines and dendrites. The box plots show median, interquartile range, and 10–90% range. The circles indicate individual experiments. The asterisks indicate significant (p < 0.05) difference from 100% or between ACSF and 5 μm baclofen. C, Average [Ca]bAP for proximal (<75 μm) basal spines (left) and dendrites (right) in baseline conditions (red) and after wash-in of 5 μm baclofen (black). D, Summary of changes in [Ca]bAP amplitude after wash-in of ACSF (blue) or 5 μm baclofen (green) in proximal and distal basal spines and dendrites. The asterisks indicate significant (p < 0.05) difference from 100% or between ACSF and 5 μm baclofen.
- Figure 3.
Modulation of excitability. A, Left, Average hyperpolarization in response to a −30 pA current injection for 300 ms in baseline conditions (red) and after wash-in of 5 μm baclofen (black). Right, Average AP elicited by brief current injection in baseline conditions (red) and after wash-in of 5 μm baclofen (black). The ADP was measured by taking the width at 10% amplitude (width10%) (dashed blue line). B, Summary of changes in input resistance (Rin), AP amplitude (Amp), half-width (width50%), and width10% after wash-in of ACSF (blue) or 5 μm baclofen (green). The asterisks indicate significant (p < 0.05) difference from 100% or between ACSF and 5 μm baclofen. C, Positive correlation between width10% and Rin for individual cells (circles) in baseline conditions (red) and after wash-in of 5 μm baclofen (black). The data were fit with a linear regression (blue line) with a Spearman's correlation coefficient (ρ) of 0.85. D, Left, No correlation between the change in [Ca]bAP and the change in Rin in either spines (red) or dendrites (black). Right, No correlation between the change in [Ca]bAP and the change in width10% in either spines (red) or dendrites (black).
- Figure 4.
Local modulation of AP Ca signals and excitability. A, 2PLSM image stack of a layer 2/3 pyramidal neuron with pressure ejection of Alexa 488 (green) from a bent glass pipette (asterisk) positioned adjacent to a dendritic segment in the apical tuft. B, Average [Ca]bAP for the apical tuft (top) and distal basal (bottom) spines (left) and dendrites (right) in baseline conditions (red) and after puffing of 15 μm baclofen (black). C, Summary of changes in [Ca]bAP amplitude after puffing of ACSF (blue) or 15 μm baclofen (green) in apical tuft and distal basal spines and dendrites. D, Top, Summary of changes in Rin after puffing of ACSF or 15 μm baclofen in apical and basal dendrites. Bottom, No correlation between the change in [Ca]bAP and the change in Rin after puffing of baclofen in either spines (red) or dendrites (black). E, Top, Summary of changes in width10% after puffing of ACSF or 15 μm baclofen in apical and basal dendrites. The asterisks indicate significant (p < 0.05) difference from 100% or between ACSF and 15 μm baclofen. Bottom, No correlation between the change in [Ca]bAP and the change in width10% after puffing of baclofen in either spines (red) or dendrites (black).
- Figure 5.
GABA uncaging. A, Left, 2PLSM image stack of a layer 2/3 pyramidal neuron, displaying uncaging locations (asterisks) at the soma and at 10 μm intervals away. Right, Uncaging-evoked sIPSPs evoked at 10 μm intervals away from the soma. sIPSPs are color-coded to corresponding uncaging locations in 2PLSM image. B, sIPSP amplitude normalized to the response at the soma, as a function of distance from the soma. The vertical bars are the SEM. Points were fit with a single exponential (blue dashed line). C, Average sIPSP evoked by uncaging at the soma in baseline conditions (red) and after wash-in of 2 μm CGP-55845 (black).
- Figure 6.
Rapid local modulation of AP Ca signals and membrane potential. A, Average [Ca]bAP for apical oblique (top) and proximal basal (bottom) spines (left) and dendrites (right) with no uncaging (red) and 200 ms after uncaging of RuBi-GABA (black). B, Summary of changes in [Ca]bAP amplitude with uncaging (green) or uncaging in the presence of 2 μm CGP-55845 (blue) in apical oblique and proximal basal spines and dendrites. The asterisks indicate significant (p < 0.05) difference from 100% or between recording conditions. C, Summary of sIPSP amplitude elicited by uncaging (green) or uncaging in the presence of 2 μm CGP-55845 (blue) in apical and basal dendrites. The asterisks indicate significant (p < 0.05) difference from 100% or between uncaging conditions. D, No correlation between the uncaging-evoked change in [Ca]bAP and sIPSP amplitude in spines (red) or dendrites (black).
- Figure 7.
Direct modulation of VSCCs. A, Average [Ca]step for apical oblique (top) and proximal basal (middle) spines (left) and dendrites (right) in baseline conditions (red) and after wash-in of 5 μm baclofen (black). Bottom, Schematic of the voltage step used to elicit the [Ca]step. B, Summary of changes in [Ca]step amplitude after wash-in of ACSF (blue) or 5 μm baclofen (green) in apical oblique and proximal basal spines and dendrites. The asterisks indicate significant (p < 0.05) difference from 100% or between ACSF and 5 μm baclofen. C, Left, Summary of changes in Rin after wash-in of ACSF (blue) or 5 μm baclofen with either Cs-gluconate (green) or K-gluconate (yellow) internal. Right, Change in holding current in these different recording conditions. The asterisks indicate significant (p < 0.05) difference from 100%, or between different conditions.
- Figure 8.
Modulation of multiple VSCC subtypes. A, Average [Ca]step in baseline conditions (red) and after wash-in of the VSCC mixture (black) in spines (left) and dendrites (right). B, Summary of changes in [Ca]step after wash-in of ACSF, mibefradil (Mib), nimodipine (Nim), SNX-482, ω-conotoxin-MVIIC, or the VSCC mixture, in proximal basal spines (blue) and dendrites (green). The asterisks indicate significant (p < 0.05) difference from 100%, and the number signs (#) indicate significant (p < 0.05) difference from ACSF. C, Average [Ca]step in proximal basal spines (left) and dendrites (right) in the presence of the mixture (red) and after wash-in of 5 μm baclofen (black). D, Summary of changes in [Ca]step in proximal basal spines (blue) and dendrites (green) after wash-in of 5 μm baclofen in the presence of ACSF, mibefradil, nimodipine, SNX-482, ω-conotoxin-MVIIC, or VSCC mixture. The ranges for 90–100% block (top gray box) and 0–10% block (bottom gray box) were calculated using Equation 1 (see Materials and Methods), except for the spine in MVIIC, because MVIIC did not significantly influence the [Ca]step in B. All conditions are significant (p < 0.05) from 100%, and the asterisks have been omitted for clarity. The number signs (#) indicate significant (p < 0.05) difference from ACSF.
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